Stimulus-induced patterns of bioelectric activity in human neocortical tissue recorded by a voltage sensitive dye.

Stimulus-induced pattern of bioelectric activity in human neocortical tissue was investigated by use of the voltage sensitive dye RH795 and a fast optical recording system. During control conditions stimulation of layer I evoked activity predominantly in supragranular layers showing a spatial extent of up to 3000 microm along layer III. Stimulation in white matter evoked distinct activity in infragranular layers with a spatial extent of up to 3000 microm measured along layer V. The mean amplitude of optical signals close to the stimulated sites in layer I and white matter determined 25 ms following the stimulus, decreased by 50% at a lateral distance of approximately 900 microm and 1200 microm, respectively. Velocity of spread along the vertical stimulation axis reached 0.24 m/s in the supragranular layers (layers I to III) and then decreased to 0.09 m/s following layer I activation; stimulation of white matter induced a velocity of spread in layer V of 0.38 m/s, which slowed down to 0.12 m/s when passing the lower border of lamina IV. The horizontal velocities of spread determined from the stimulation site to a lateral distance of 500 microm reached 0.26-0.28 m/s and 0.28-0.35 m/s for layer I and white matter stimulation, respectively. At larger distances velocity of spread decreased. Increased excitability (Mg(2+)-free solution) had no significant effect on the spatio-temporal distribution of evoked activity as compared with control conditions. There were also no obvious differences between the results obtained in slices, which generated spontaneously sharp waves and those which were not spontaneously active. About 30% of the slices (n=7) displayed a greatly different response pattern, which seemed not to be related in a simple way to the stimulation as was the case in the majority of the investigated slices. The activity pattern of those slices appeared atypical in regard to their deviations of the vertical and horizontal extent of activity, to their reduced spatial extent of activity during increased excitability, to their layer-related distribution of activity, and to the appearance of afterdischarges.Concluding, in 30% of the human temporal lobe slices atypical activity pattern occurred which obviously reflect intrinsic epileptiform properties of the resected tissue. The majority of slices showed stereotyped activity pattern without evidence for increased excitability.

Chloride distribution in the CA1 region of newborn and adult hippocampus by light microscopic histochemistry.

GABA, the main inhibitory neurotransmitter in the central nervous system, exerts its effect by rendering the postsynaptic GABAA receptors permeable to chloride ions. Thus, depolarizing or excitatory effects of GABA, experienced in early postnatal life or in certain regions and/or conditions of the adult brain, is thought to be associated with a reversed transmembrane chloride gradient. However, there is only limited direct information about the correlation of the actual excitatory versus inhibitory effects of GABA and the local chloride distribution. Precipitation of chloride with silver is a potential way to immobilize and visualize chloride ions in biological tissue. We examined the applicability of light microscopic histochemistry, based on trapping tissue chloride with silver ions during freeze-substitution or aldehyde fixation, to visualize the chloride distribution in hippocampal slices. The freeze-substitution procedure yielded better chloride retention while with aldehyde fixation tissue preservation was more appropriate. Both methods were qualitative only, had limited applicability to the superficial 20-30 microns of slices, but were able to demonstrate a reduced extracellular-to-intracellular chloride gradient in the CA1 pyramidal neurons of the newborn hippocampus as compared to adult animals. In the 4-aminopyridine model of epilepsy, redistribution of chloride from extracellular to intracellular space could also be demonstrated.

Transcriptional regulation of caspases in experimental pneumococcal meningitis.

Apoptosis and necrosis in brain account for neurological sequelae in survivors of bacterial meningitis. In meningitis, several mechanisms may trigger death pathways leading to activation of transcription factors regulating caspases mRNA synthesis. Therefore, we used a multiprobe RNA protection assay (RPA) to examine the expression of 9 caspase-mRNA in the course of experimental Streptococcus pneumoniae meningitis in mouse brain. Caspase-6, -7 and -11 mRNA were elevated 6 hours after infection. 12 hours after infection caspases-1, -2, -8 and -12 mRNA rose. Caspase-14 mRNA was elevated 18 h and caspase-3 mRNA 24 h after infection. In situ hybridization detected caspases-3, -8, -11 and -12 mRNA in neurons of the hippocampal formation and neocortex. Development of sepsis was paralleled by increased transcription of caspases mRNA in the spleen. In TNFalpha-deficient mice all caspases examined were less upregulated, in TNF-receptor 1/2 knockout mice caspases-1, -2, -7, -11 and -14 mRNA were increased compared to infected control animals. In caspase-1 deficient mice, caspases-11, and -12 mRNA levels did not rise in meningitis indicating the necessity of caspase-1 activating these caspases. Hippocampal formations of newborn mice incubated with heat-inactivated S. pneumoniae R6 showed upregulation of caspase-1, -3, -11 and -12 mRNA. These observations suggest a tightly regulated caspases network at the transcriptional level in addition to the known cascade at the protein level.

Organotypic hippocampal cultures. A model of brain tissue damage in Streptococcus pneumoniae meningitis.

Hippocampal slices of newborn rats were exposed to either heat-inactivated Streptococcus pneumoniae R6 (hiR6) equivalent to 10(6) and 10(8) CFU/ml, lipoteichoic acid (LTA) (0.3 microg/ml and 30 microg/ml), peptidoglycans (PG) (0.3, 30, 50 and 100 microg/ml), pneumococcal DNA (pDNA) (0.3 and 30 microg/ml) or medium only (control). Cell injury was examined by Nissl staining, Annexin V and NeuN immunohistochemistry, and quantified by propidium iodide (PI) uptake and by determining neuron-specific enolase (NSE) concentration in the culture medium. Necrotic and apoptotic cell damage occurred in all treatment groups. Overall damage (Nissl and PI staining) was most prominent after hiR6 (10(8) CFU/ml), followed by LTA (30 microg/ml), pDNA (30 microg/ml), and not detectable after PG (30 microg/ml) exposure. PG (100 microg/ml) induced severe damage. Apoptotic cells were most frequent after exposure to LTA and hiR6. Damage in the neuronal cell layers (NeuN, NSE) was most severe after treatment with hiR6 (10(8) CFU/ml), followed by PG (100 microg/ml), pDNA (30 microg/ml), and LTA (30 microg/ml).

Optical monitoring of neuronal activity during spontaneous sharp waves in chronically epileptic human neocortical tissue.

Functional changes in neuronal circuitry reflected in spontaneously occurring synchronous sharp field potentials (SSFP) have been reported to occur in human brain suffering from chronic epileptogenicity but not in primary nonepileptic tissue from peritumoral resectates. Voltage sensitive dyes and fast imaging were used to visualize spontaneously occurring rhythmic depolarizations correlated to SSFP in chronically epileptic human neocortical slices obtained during epilepsy surgery. Localized and spatially inhomogeneous neuronal depolarizations were found to underlie spontaneous SSFP, which remained unchanged and spatially restricted to foci <750 micrometer diam even under epileptogenic (low-Mg(2+)) conditions. In cases where ictaform paroxysmal activity occurred in low-Mg(2+) medium, neuronal depolarizations were wide-spread but still spatially inhomogeneous, and the events were preferentially initiated at distinct foci. The findings suggest that small neuronal networks are able to establish and maintain synchronous rhythmic and epileptiform activity.

Spatio-temporal distribution of epileptiform activity in slices from human neocortex: recordings with voltage-sensitive dyes.

The spatio-temporal distribution of epileptiform activity was investigated in slices from human temporal neocortex resected during epilepsy surgery. Activity was recorded by use of a voltage-sensitive dye and an optical recording system. Epileptiform activity was induced with 10 microM bicuculline and electrical stimulation of layer I. In 10 slices from six patients investigated, epileptiform activity spread across most of the slice. Largest amplitudes were located in layer II/III. Epileptiform activity was characterized by long-lasting potentials with slow rising phases and a low velocity of spread in the horizontal direction (0.044 m/s). This spatio-temporal pattern of epileptiform activity in human slices was similar to that found previously in neocortical slices from guinea pigs with bicuculline. In four of nine human slices investigated under control bath conditions (in non-epileptogenic medium), the spatio-temporal activity patterns were similar to those of guinea pigs in non-epileptogenic medium. In the remaining five human slices, however, the spread in the horizontal direction was significantly larger (4188 microm) in non-epileptogenic medium than that found in slices from guinea pigs (2171 microm). Activity in human slices showing such 'wide spread' in control bath conditions occasionally had characteristic features of epileptiform activity. Further work will have to clarify whether these epileptiform features reflect intrinsic epileptiform properties in human tissue slices.

Intracellular calcium redistribution accompanies changes in total tissue Na+, K+ and water during the first two hours of in vitro incubation of hippocampal slices.

Changes of total tissue water, Ca, Na and K contents were monitored in whole transverse hippocampal slices of the guinea-pig during the first 2 h of in vitro incubation. A brief, 75% increase in tissue Ca was noted during the initial 15 min of maintenance, in contrast to a permanent increase of sodium and water contents, coupled to simultaneous decrease of potassium level. The rate of tissue Na, K and water changes comprised a rapid phase at the first 10-20 min, parallel with the increase of the tissue Ca content, and a slow phase during the rest of the incubation period. Development of specific morphological alterations, representative of ischemic/hypoxic lesions and a translocation of calcium from cytoplasm to mitochondria and endoplasmic reticulum during slice maintenance, was also detected by electron microscopy. A two-step mechanism might explain the development of a new steady-state total calcium content of slices. in which the cellular Ca2+ uptake at the beginning of incubation, likely triggered by hypoxic/ ischemic trauma of slice preparation, is followed by a balanced Ca2+ influx, extrusion and sequestration (predominantly into mitochondria and endoplasmic reticulum) during maintenance.

Changes in paired-pulse facilitation correlate with induction of long-term potentiation in area CA1 of rat hippocampal slices.

The phenomenon of long-term potentiation is widely used as an experimental model of memory. An approach that has been used to study its underlying mechanisms is to analyse its interaction with presynaptic paired-pulse facilitation. Several studies found no evidence for an interaction in the CA1 hippocampal area, whereas other data, for example from quantal analysis, suggested that presynaptic mechanisms contribute to the maintenance of long-term potentiation. In the present study, initial slopes of field potentials in area CA1 were measured in rat hippocampal slices. "Conventional" long-term potentiation was induced by high-frequency (100 Hz) afferent tetanization of the testing input. "Associative" long-term potentiation was induced by combining lower frequency (40 Hz) tetanization of a testing input with high-frequency tetanization of a second input. The paired-pulse facilitation ratio decreased in the majority of experiments in which long-term potentiation was induced conventionally, but it decreased, increased or did not change after inducing associative potentiation. Decreases in the paired-pulse facilitation correlated inversely with the initial (pre-tetanic) facilitation ratio. A more detailed regression analysis suggests that this correlation results from two other correlations: (i) that between changes in paired-pulse facilitation and the magnitude of long-term potentiation, and (ii) that between initial paired-pulse facilitation and the magnitude of long-term potentiation. The first correlation prevailed during the initial 10 min following tetanization, while the second prevailed 40-60 min later. A post-tetanic decrease in paired-pulse facilitation is evidence for an involvement of presynaptic mechanisms in the maintenance of long-term potentiation. The lack of significant changes in some studies could be due to the inclusion in the analyses of experiments with long-term potentiation of small magnitude, in which changes in paired-pulse facilitation ratios would have been inconsistent. The present study suggests that the early (10-20 min) and late (40-50 min) phases of long-term potentiation were mediated by different mechanisms, with a mixture of these mechanisms during the intermediate period. On the basis of the present and previous studies, the following scheme of involvement of several mechanisms in long-term potentiation maintenance is proposed. The early phase includes two major mechanisms: an increase in the probability of transmitter release, leading to an apparent increase in the number of effective release sites, and an increase in efficacy of one transmitter quantum, probably due to an increased number of postsynaptic receptors. The later phase of long-term potentiation is attributed to an increase in the number of transmitter zones, presumably due to structural modifications.

Spatiotemporal distribution of intracellular calcium transients during epileptiform activity in guinea pig hippocampal slices.

Calcium ions are known to play an important role in epileptogenesis. Although there is clear evidence for increased neuronal calcium influx during epileptiform potentials, direct measurements of the corresponding intracellular calcium transients are rare and the origin of calcium influx is not known. Therefore the spatial and temporal distribution of intracellular calcium transients during epileptiform activity in guinea pig hippocampal slices was monitored with the use of the indicator Calcium-Green and a fast optical recording method. Two models of epilepsy (bicuculline and low Mg2+) were compared. In both models, single epileptiform events were evoked by electrical stimulation of the Schaffer collaterals in CA1 or of stratum pyramidale in area CA3. Intracellular calcium transients during epileptiform activity were approximately 5 times larger than during control stimulation. Calcium transients during epileptiform activity were present across at least the entire CA1 area, whereas presynaptic calcium transients from stimulated fibers were only seen at a distance up to 1 mm from the stimulation site. DL-2-amino-5-phosphonovaleric acid (APV), a specific antagonist of the N-methyl-D-aspartate (NMDA) receptor, abolished low-Mg2+ epileptiform activity and reduced bicuculline-induced epileptiform activity; it reduced calcium transients following stimulation of CA1 by only 29% (bicuculline) and 38% (low Mg2+). For comparison, calcium transients during control stimulation were 78% (bicuculline) and 69% (low Mg2+) smaller than epileptiform calcium transients. At a distance from the stimulation site, calcium transients and their NMDA-receptor-dependent components were largest in stratum pyramidale in the bicuculline model and in stratum oriens in the low-Mg2+ model. In both models, minimal onset latencies of calcium influx shifted with increasing distance to the stimulation electrode from stratum radiatum to stratum oriens. APV reduced the extent of spread of calcium transients in the low-Mg2+ model. In the bicuculline model, the spatial extent of spread of epileptiform calcium transients was not affected by application of APV; however, the mean velocity of spread was reduced from 0.20 to 0.12 m/s. In conclusion, the large size of calcium transients and of their NMDA-receptor-dependent components in stratum pyramidale or stratum oriens as well as shortest onset latencies of calcium transients at these sites suggest an important role of cell somata, basal dendrites, and possibly local circuit excitatory interactions for the generation and spread of epileptiform activity.

Involvement of silent synapses in the induction of long-term potentiation and long-term depression in neocortical and hippocampal neurons.

Changes in the latency of small excitatory postsynaptic potentials were observed in association with induction of long-term modifications of synaptic transmission in slices of rat neocortex and guinea-pig hippocampus. After potentiation response latency decreased in 3/10 cases in the neocortex and in 6/24 cases in the hippocampus, and increased after depression in 4/8 cases in the neocortex. These latency changes could not be attributed to changes in presynaptic fibre excitability, monosynaptic inhibition, release kinetics or activation kinetics of postsynaptic ion channels. We conclude therefore that potentiation led to the activation of previously silent synapses of fast-conducting afferents and depression to the inactivation of previously functional synapses. Thus, neocortical and hippocampal synapses can be in a non-functional state, and regimes that induce long-term potentiation and depression not only change the efficacy of synapses but also alter their functional state.

Partial suppression of GABAA-mediated inhibition induces spatially restricted epileptiform activity in guinea pig neocortical slices.

The spatio-temporal distribution of evoked activity in guinea pig neocortical slices was investigated during partial suppression of gamma-aminobutyric acid (GABA)A-mediated synaptic inhibition with different concentrations of bicuculline. Activity patterns were recorded by use of a voltage-sensitive dye and a fast optical recording technique. At non-epileptogenic concentrations of bicuculline (0.6-2.5 microM), evoked potentials were of longer duration and larger amplitude, but the spatial extent of spread in the horizontal direction was unaffected. At threshold epileptogenic concentrations of bicuculline (1.25-5 microM), spatially restricted epileptiform activity developed at a distance from the stimulation site which was clearly separated from potentials with non-epileptic characteristics close to the stimulation site. It is concluded that, under moderate disinhibition, stimulus-evoked activity has a suppressive effect on spread and development of epileptiform activity, probably through synchronous activation of still-functioning inhibitory circuits.

Epileptiform activity in the guinea-pig neocortical slice spreads preferentially along supragranular layers--recordings with voltage-sensitive dyes.

The spread of epileptiform activity was monitored in guinea-pig neocortical slices by the use of a voltage-sensitive dye (RH795) and a fast optical recording technique. Epileptiform activity induced by bicuculline methiodide (10-20 microM) and single-pulse stimulation spread from the stimulation site in layer I or in the white matter across most of the slice. Different lesions were made in the slice in order to specify the neuronal connections used for spread in the horizontal direction. In the slice, intracortical connections are necessary for the spread of epileptiform activity, as shown by vertical cuts through all cortical layers but sparing the white matter. Horizontal connections were interrupted by cuts parallel to the axis of pyramidal neurons through either supragranular or infragranular layers. Vertical connections were interrupted by cuts perpendicular to the axis of pyramidal neurons separating supragranular and infragranular layers. Spread of epileptiform activity in the horizontal direction was not hindered by horizontal cuts. Vertical cuts through infragranular layers also did not hinder the spread of epileptiform activity. In contrast, vertical cuts through supragranular layers either abolished completely (nine slices) or delayed significantly (ten slices) the spread of epileptiform activity. The mean delay at the supragranular lesion was 44 ms in layer III and 30 ms in layer V; at the infragranular lesion the mean delay was 2 ms in layer III and 6 ms in layer V. Also, with horizontal cuts, in three out of five slices the velocity of spread was significantly lower in infragranular as compared to supragranular layers. It is concluded that both supra- and infragranular layers if isolated possess the ability to initiate and propagate epileptiform activity independently. However, in the intact slice the influence of the supragranular networks on initiation and propagation of epileptiform activity appears to dominate.

Neurophysiological analysis of long-term potentiation in mammalian brain.

Long-term potentiation (LTP) is a persistent increase in postsynaptic response following a high-frequency presynaptic activation. Characteristic LTP features, including input specificity and associativity, make it a popular model to study memory mechanisms. Mechanisms of LTP induction and maintenance are briefly reviewed. Increased intracellular Ca2+ concentration is shown to be critical for LTP induction. This increase is believed to be based on Ca2+ influx secondary to activation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Existence of other sources of Ca2+ increase and other critical factors is now becoming evident. They include voltage-dependent Ca2+ channels, Ca2+ intracellular stores, metabotropic glutamate receptors, 'modulatory' transmitters. An example of an involvement of voltage-dependent Ca2+ channels is potentiation induced by intracellular depolarizing pulses. LTP can be divided into decremental earlier (E-LTP) and non-decremental late (L-LTP) phases which explains some inconsistencies in studies of LTP mechanisms. E-LTP is suggested to be based on a transient increase in presynaptic release probabilities. A hypothesis is considered which explains L-LTP by suggesting that Ca2+ activates structural changes leading to an increase in the synaptic gap resistance. This enhances positive synaptic electrical feedback and augments release probability. The hypothesis predicts specific morphological changes, synchronous transmitter release of two or several quanta in some central synapses and the amplification of such synchronization following LTP induction. Data are discussed which maintain these predictions.

Interaction between paired-pulse facilitation and long-term potentiation in area CA1 of guinea-pig hippocampal slices: application of quantal analysis.

The aim of the study was to further specify mechanisms of maintenance of hippocampal long-term potentiation. Previous analysis of excitatory postsynaptic potentials showed increases in quantal content (mean number of neurotransmitter quanta released by every testing pulse) with smaller increases in quantal size (effect of one transmitter quantum) following long-term potentiation induction. Here we recorded intracellularly excitatory postsynaptic potentials from CA1 pyramidal neurons of guinea-pig hippocampal slices after minimal paired-pulse stimulation of monosynaptic inputs. Statistical parameters underlying excitatory postsynaptic potential fluctuations were estimated by a deconvolution procedure using a quantal model. The parameters of excitatory postsynaptic potentials following paired-pulse stimulation were studied before and after induction of long-term potentiation. Under both conditions, paired-pulse facilitation was found to be accompanied by increases in quantal content and quantal size. During long-term potentiation, paired-pulse facilitation of amplitude and quantal content was lower. The respective changes in the paired-pulse facilitation ratios correlated with long-term potentiation magnitude. In contrast, the paired-pulse facilitation of quantal size did not change significantly following long-term potentiation induction. The results are compatible with the existence of two separate mechanisms of long-term potentiation maintenance. They support the suggestion that changes in quantal content are mainly due to presynaptic mechanisms which are shared by long-term potentiation and paired-pulse facilitation. The mechanisms underlying changes in quantal size are of a different nature for long-term potentiation and paired-pulse facilitation. For long-term potentiation they might be located postsynaptically.

Calcium accumulation by dendritic mitochondria declines along the apical dendrites of pyramidal neurons in area CA1 of guinea pig hippocampal slices.

An electron microscopic histochemical study was performed in stratum radiatum of area CA1 of guinea pig hippocampal slices in order to determine the spatial distribution of a dendritic mitochondrial subpopulation which accumulated calcium during in vitro incubation. A distribution gradient was found along the course of apical dendrites exhibiting the highest density values at the base of the dendrites and decaying to baseline values at about 50 microns distal from the cell body layer. The pronounced calcium accumulation by mitochondria in the proximal apical dendrites was markedly but not completely reduced by blocking L-type Ca-channels. These results (i) support the observation of a clustered distribution of L-type Ca-channels at the base of apical dendrites, (ii) designate these voltage dependent Ca2+ channels as one of the possible routes for calcium influx caused by hypoxia/ischemia induced during slice preparation, and (iii) emphasize the role of mitochondrial calcium sequestering under ischemic/hypoxic conditions.

Spread of epileptiform potentials in the neocortical slice: recordings with voltage-sensitive dyes.

The spread of epileptiform potentials in guinea pig neocortical slices was investigated by use of voltage sensitive dyes and a fast optical recording technique. Epileptiform activity was induced in a perfusion medium containing 10-20 microM bicuculline-methiodide and by single pulse stimulation of layer I or the white matter. The location of minimal and maximal amplitudes, the shape of the potentials at specific sites and the velocity of spread were independent from the specific stimulation site. The expression of epileptiform activity appeared to depend on specific, possibly geometrical, properties of the tissue.

Immunohistochemical detection of raf protein kinase in cerebral cortical areas of adult guinea pigs and rats.

The raf protooncogenes are the cellular counterparts of the v-raf oncogene expressed by a murine sarcoma virus. The raf protooncogenes encode cytoplasmic serine/threonine-specific protein kinases which can be activated from different growth factor receptors by phosphorylation. The mRNAs of raf protooncogenes are found in a large variety of normal adult tissues, including the central nervous system. As concerns the distribution and localization of their protein products (the raf kinases), very few data are to be found in the literature. This is the first detailed description of their light microscopic localization in neocortical and allocortical areas of rodents. Preembedding immunohistochemical studies were performed on vibratome sections from the brains of adult guinea pigs and albino rats. The localizations of two isoenzymes, raf-1 kinase and B-raf kinase, were studied with the help of isoenzyme-specific polyclonal antibodies. Both of the antibodies detected raf protein-like immunoreactivity in many neurons and scattered glial cells of the sensory neocortex, and the cingular, pyriform, perirhinal and entorhinal allocortical areas. Pyramidal and non-pyramidal cells of Ammon's horn, granule cells of the dentate fascia and the large neurons in the hilar region were immunoreactive, too. The findings indicated that B-raf protein kinase and raf-1 kinase are present almost ubiquitously in the neurons of the investigated cortical structures. The intensity of staining obtained with serial dilutions of the antibodies indicated that the cytoplasmic concentration of B-raf kinase is tended to be higher than that of raf-1 kinase. The present findings suggested that the raf kinases are localized in postsynaptic structures, mainly in dendrites and cell bodies. Their cytosolic localization and their ability to undergo intracellular translocation during activation and phosphorylation raise the possibility that they play a pivotal role in the intracellular signaling of neurons.

The contribution of intracortical connections to horizontal spread of activity in the neocortex as revealed by voltage sensitive dyes and a fast optical recording method.

Coronal slices from guinea-pig visual neocortex were stained with voltage-sensitive fluorescence dyes RH414 or RH795. Activity was evoked by electrical stimulation of either the 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 94 microns. The distribution and spread of activity in the horizontal direction was analysed. Following stimulation of the white matter or layer I, two regions of activity were differentiated in the medio-lateral direction: a central region (approximately 1 mm wide) of high-amplitude activity close to the stimulation electrode and, distant from the stimulation electrode, peripheral regions of low-amplitude activity. Central and peripheral regions differed in their rates of decline, their relative extent with stimulation of different sites and within different layers. The total extent of non-synaptic evoked activity did not exceed that of the central region of high-amplitude activity. Along the extent of non-synaptic activity, onset latencies of potentials were almost constant. Thus, activity of high amplitude in the central region was likely mediated by simultaneous activation of distributed afferent fibres. In contrast, no non-synaptic activity was found in peripheral regions. Therefore it is suggested that this low-amplitude activity was mediated without direct afferent activation but via long-distance intracortical horizontal pathways. These pathways are known to terminate in layer III, and accordingly latencies of responses in the periphery were shortest in upper cortical layers, whereas in the central region, latencies increased from lower to upper cortical layers.

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.

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.