Laminar analysis of initiation and spread of epileptiform discharges in three in vitro models.

Overexcitation of neuronal networks in some forebrain structures and pathological synchronization of neuronal activity play crucial role in epileptic seizures. Seizure activity can be elicited experimentally by different convulsants. Because of various distribution of excitatory and inhibitory connections in the neocortex there might be laminar differences in seizure sensitivity. Current source density (CSD) analysis or immunocytochemical c-Fos localization offer suitable tools to localize increased activation of neurons during seizure. In the present experiments, interictal epileptiform activity elicited by 4-aminopiridine, bicuculline or Mg(2+)-free solution was recorded with a 16-channel multielectrode assembly in different layers of the somatosensory cortex, and CSDs were calculated. Parallel c-Fos immunocytochemistry was applied. Each convulsant elicited characteristic activation pattern. 4-aminopiridine induced relatively short discharges, which were associated with a huge sink in layer V, the sink and source pattern was relatively simple. Mg(2+)-free solution elicited the longest discharges, sinks appeared typically in the supragranular layers II and III than quickly distributed toward layers V and VI. Bicuculline induced rather similar seizure pattern as Mg(2+)-free solution, but the amplitudes of field potentials were larger, while the durations shorter. The peak of c-Fos activation, however, was not parallel with the largest electrical activation. Larger amount of stained cells appeared in layers II and III in 4-aminopiridine and bicuculline, respectively. In Mg(2+)-free solution the highest c-Fos activity was detected in upper layer VI. Long-lasting cellular effects do not always correspond to the largest electrical responses, which are primarily determined by the activation of asymmetrical pyramidal neurons. Interneurons, which possess more symmetric process arborisation, play less important role in the generation of field potentials, although they may be intensively activated during seizure.

GABAergic Interneurons are the Major Postsynaptic Targets of Median Raphe Afferents in the Rat Dentate Gyrus.

The termination pattern of median raphe axons was studied in the rat dentate gyrus using Phaseolus vulgaris leucoagglutinin as an anterograde tracer, in combination with postembedding immunostaining for gamma-amino-butyric acid (GABA), and pre-embedding immunostaining for calbindin D28k, parvalbumin and GABA. Postembedding immunogold staining for GABA revealed that the majority (73.7%) of anterogradely labelled median raphe boutons make synaptic contacts with GABA-immunoreactive postsynaptic targets, mainly with dendritic shafts and perikarya. Pre-embedding immunocytochemical double staining for the anterograde tracer and GABA confirmed the electron microscopic results and showed that varicose median raphe axons establish multiple contacts with fusiform interneurons in the hilus and different types of basket cells in the granule cell layer. Some of the innervated cells were shown to contain calbindin D28k, whereas GABAergic interneurons containing another calcium-binding protein, parvalbumin, were never seen to receive multiple contacts from axons of raphe origin. Our results suggest that serotonergic median raphe fibres influence the firing of dentate granule cells via local inhibitory interneurons. The mechanism of using these interneurons with extensive local connections as monosynaptic targets may explain the great efficacy of this pathway in the control of hippocampal electrical activity.

Visualization of non-synaptic release sites in the myenteric plexus of the snail Helix pomatia.

Following conventional glutaraldehyde-osmium tetroxide fixation, a rich myenteric plexus was detected in the gastrointestinal tract of the snail Helix pomatia. Although hundreds of nerve processes were observed in the extensive myoneural neuropil, true synaptic specializations were not recognized in them. In the absence of synaptic specializations, tannic acid-Ringer incubation was applied to visualize the non-synaptic release sites in the enteric nerve plexus. After incubation for 1 h, a great number of exocytosis profiles were recorded at nerve-muscle contacts, at axoglial connections and in the myoneural neuropil. The frequency of occurrence of exocytosis profiles was the same for adrenergic and peptidergic fibres. In some gut wall areas, a dense staining of both basal lamina and collagen fibres was observed. Invagination of dense basal lamina into the omega-shaped profiles of the axolemma led to "false exocytosis" profiles. A detailed morphological analysis is needed to distinguish false exocytosis profiles from the true transmitter-releasing loci.

Calretinin is present in non-pyramidal cells of the rat hippocampus--III. Their inputs from the median raphe and medial septal nuclei.

The subcortical innervation of a recently described subpopulation of non-pyramidal neurons, containing the calcium binding protein, calretinin, was investigated in the rat hippocampus using the anterograde tracer Phaseolus vulgaris-leucoagglutinin and double immunocytochemistry for calretinin and serotonin at the light and electron microscopic levels. Our results show that the GABAergic component of the septohippocampal pathway and the serotonergic raphe afferents establish multiple synaptic contacts with the calretinin-immunoreactive interneurons. The majority of the targets of both pathways were spine-free calretinin neurons known to innervate the dendritic region of the principal cells, but the GABAergic septal pathway was found to terminate also on the spiny neurons of stratum lucidum of the CA3 region and in the dentate hilus. The present results demonstrate that the serotonergic raphe-hippocampal and the GABAergic septohippocampal pathways are able to modulate dendritic inhibition of principal cells via calretinin-containing GABAergic interneurons.

Enteric neuromuscular junctions: comparison of ultrastructural features in different phylogenetic groups.

The enteric neuromuscular junctions of snail (Helix pomatia), locust (Locusta migratoria migratorioides), cockroach (Periplaneta americana), carp (Cyprinus carpio) and tench (Tinca tinca) were studied by means of different light and electron microscopic methods. The nitroblue tetrazolium staining revealed that the myenteric plexuses of the above species are composed of nerve cells, a network of varicose nerves and nerve bundles. Instead of highly organized ganglia, single neurons or small groups of 2-4 cells are characteristic of the invertebrates and fish studied. Catecholaminergic fluorescence induced by glyoxylic acid was detected in the muscular layer of the entire alimentary tract in snail and the hindgut of tench. Fluorescent nerves and perikarya were frequent in the snail gut, while only nerves and no perikarya were found in tench. A close contact between enteric muscles and nerves is the most common form of enteric neuromuscular junction in both the smooth (i.e. the molluscan and fish gut) and the striated (i.e. the insect gut) musculature. The striated musculature (i.e. the insect gut, the oesophagus of carp, and the oesophagus, stomach and the midgut of tench) also receives a synaptic input. Cytochemical evidence is provided of the cholinergic character of fish motor endplates. The ultrastructural appearance and vesicle population of certain nerve terminals suggest a universal role of aminergic and peptidergic control in gut motility.

Quantification of immunogold labelling reveals enrichment of glutamate in mossy and parallel fibre terminals in cat cerebellum.

The glutamate immunoreactivity of different cell populations was compared quantitatively in the cerebellar cortex of cat, using an antiserum raised against glutamate coupled to bovine serum albumin by glutaraldehyde. Neuronal and glial processes were identified on serial electron microscopic sections which were processed by a postembedding immunogold procedure. The surface density of colloidal gold particles was used for statistical comparison of the relative levels of glutamate in cell populations, or in different parts of the same population. The terminals of mossy and parallel fibres had significantly higher levels of glutamate immunoreactivity than Golgi cell terminals, granule cell dendritic digits, Purkinje cell dendrites or dendritic spines. Golgi cell terminals were identified by their position and GABA immunoreactivity as revealed by immunogold in serial sections. The dendritic digits of the putative glutamatergic granule cells had significantly higher glutamate immunoreactivity than did Purkinje cell dendrites and dendritic spines. Glial cell processes in the molecular layer had lower level of glutamate immunoreactivity than any of the neuronal processes. The results demonstrate that the highest levels of glutamate immunoreactivity occur in mossy and parallel fibre presynaptic terminals that are known to have an excitatory effect. This supports previous suggestions that glutamate may be a transmitter at these synapses. The measurement of the levels of putative amino acid transmitters in identified neuronal populations, or in different parts of the same population, could have wide applications in studies on the chemical neuroanatomy of the nervous system.

Synaptic effects of identified interneurons innervating both interneurons and pyramidal cells in the rat hippocampus.

GABAergic interneurons sculpt the activity of principal cells and are themselves governed by GABAergic inputs. To determine directly some of the sources and mechanisms of this GABAergic innervation, we have used dual intracellular recordings with biocytin-filled microelectrodes and investigated synaptic interactions between pairs of interneurons in area CA1 of the adult rat hippocampus. Of four synaptically-coupled interneuron-to-interneuron cell pairs, three presynaptic cells were identified as basket cells, preferentially innervating somata and proximal dendrites of pyramidal cells, but one differing from the other two in the laminar distribution of its dendritic and axonal fields. The fourth presynaptic interneuron was located at the border between strata lacunosum moleculare and radiatum, with axon ramifying within stratum radiatum. Action potentials evoked in all four presynaptic interneurons were found to elicit fast hyperpolarizing inhibitory postsynaptic potentials (mean amplitude 0.35 +/- 0.10 mV at a membrane potential of -59 +/- 2.8 mV) in other simultaneously recorded interneurons (n=4). In addition, three of the presynaptic interneurons were also shown to produce similar postsynaptic responses in subsequently recorded pyramidal cells (n=4). Electron microscopic evaluation revealed one of the presynaptic basket cells to form 12 synaptic junctions with the perisomatic domain (seven somatic synapses and five synapses onto proximal dendritic shafts) of the postsynaptic interneuron in addition to innervating the same compartments of randomly-selected local pyramidal cells (50% somatic and 50% proximal dendritic synapses, n=12). In addition, light microscopic analysis also indicated autaptic self-innervation in basket (12 of 12) and bistratified cells (six of six). Electron microscopic investigation of one basket cell confirmed six autaptic junctions made by five of its boutons. Together, these data demonstrate that several distinct types of interneuron have divergent output to both principal cells and local interneurons of the same (basket cells) or different type. The fast synaptic effects, probably mediated by GABA in both postsynaptic interneurons and principal cells are similar. These additional sources of GABA identified here in the input to GABAergic cells could contribute to the differential temporal patterning of distinct GABAergic synaptic networks.

Kappa opioid receptors are expressed by interneurons in the CA1 area of the rat hippocampus: a correlated light and electron microscopic immunocytochemical study.

A local GABA-system is known to have a mediatory function between several afferents and the principal cells of the hippocampus. This study examines the distribution and fine structure of kappa opioid receptor-immunoreactive elements in the CA1 subfield and reveals some new aspects concerning the structural basis of opioid-GABA interaction in the rat hippocampal formation. Kappa receptors were visualized immunocytochemically with a previously produced and characterized monoclonal antibody, the mAb KA8 (Maderspach, K., Németh, K., Simon, J., Benyhe, S., Szûcs, M., Wollemann, M., 1991. A monoclonal antibody recognizing kappa-, but not mu- and delta-opioid receptors. J. Neurochem. 56, 1897-1904). The antibody selectively recognizes the kappa opioid receptor with preference to the kappa(2) subtype. Neuronal cell bodies, proximal dendrites and occasionally glial processes surrounding neuronal perikarya were labelled in the CA1 area. The immunopositive cells were present mainly in the stratum oriens, followed by the stratum pyramidale in a rostrocaudally increasing number. Their shape was fusiform, or multipolar. Occasionally kappa receptor-immunoreactive boutons surrounding weakly immunopositive somata were also observed. Electron microscopy of immunopositive neurons showed that the DAB labelling was intensive in the perinuclear cytoplasm. The widths and electron densities of the postsynaptic densities of some axosomatic synapses were remarkably increased. Similar increase of postsynaptic densities were observable at some axodendritic and axospinous synapses. On the basis of their location and fine structural properties the labelled cells are suggested to be GABAergic inhibitory interneurons, probably belonging to the somatostatinergic sub-population. The axons of these inhibitory interneurons are known to arborize in the stratum lacunosum-moleculare where the entorhinal afferents terminate. A modulatory effect of opioids on the entorhinal input, mediated by somatostatinergic interneurons is suggested

Localization and quantitative distribution of biogenic monoamines in the intestinal tract of locust, snail and carp.

The localization and quantitative distribution of the biogenic monoamines of the intestinal tract has been studied in Locusta migratoria, Helix pomatia and Cyprinus carpio with morphological and biochemical methods. Electron microscopically dense-core vesicles of aminergic character were found in the varicose nerve fibres located in the intestinal muscles of all three animal species. Intensive green fluorescence characteristic of catecholamines was detectable in both the varicose nerve fibres and perikarya. Using a fluorimetric method the quantity of biogenic monoamines was measured in the gastrointestinal tract of all three species. The amount of adrenaline and noradrenaline was rather low: 0.03-0.29 ?g/g. The dopamine content reached the value of 6 ?g/g in the locust gut, and a significant amount of serotonin was present in the intestinal tract of all three species: 1.00-2.59 ?g/g. Compared to the adrenaline and noradrenaline serotonin proved to be higher by more than one order in each case. On the basis of morphological and biochemical results the authors suggest that biogenic monoamines are involved in the regulation of the gut muscle functioning both in the form of transmitters as well as neurohormones.

Enkephalinergic nerve terminals target inhibitory interneurons in the rat hippocampus.

Using correlated light and electron microscopic preembedding enkephalin immunocytochemistry combined with post-embedding GABA immunogold staining, we found morphological evidence of a direct connection between the enkephalinergic and GABAergic systems in the rat hippocampus. Enkephalin-immunoreactive boutons were found to be presynaptic to GABA-immunoreactive postsynaptic profiles, establishing type 2 symmetrical synapses on GABA-positive cell bodies and dendritic shafts in strata radiatum and lacunosum moleculare of the CA1 region. Thirty-six percent of all studied postsynaptic targets (n = 40) were non-pyramidal, including all somatic (n = 7) and 47% of the dendritic (n = 13) postsynaptic targets. The remaining 64% consisted of pyramidal dendritic shafts and spines. These results support previous physiological experiments suggesting that the opioidergic system takes part in disinhibitory processes in the hippocampal formation.

Glial fibrillary acidic protein (GFAP)-immunoreactivity is reduced by castration in the interpeduncular nucleus of male rats.

The interpeduncular nucleus of adult male rats was investigated for glial fibrillary acidic protein immunoreactivity. In intact animals the nucleus had an outstandingly intense immunostaining, particularly at its periphery, including the rostral, lateral, dorsomedial and dorsolateral subnuclei where, in addition to neuropil astrocytes, a substantial amount of perivascualr glia was found. Four weeks after castration, immunostaining decreased markedly in the core region of the nucleus corresponding to the caudal and medial subnuclei, and to a much lesser extent at the periphery. The immunoreactivity in pericapillary astrocytes proved to be insensitive to castration. Testosterone, if administered after castration prevented or restituted the loss of immunoreactivity. Beyond 4 months after castration, the effect of testosterone gradually declined. It is concluded that testosterone stimulates the expression of glial fibrillary acidic protein immunoreactivity in the interpeduncular nucleus. Our findings support the argument that gonadal steroids can influence astrocytes also in non-endocrine areas of the brain.

Pre- or postembedding immunocytochemistry: which to choose for the localization of glial fibrillary acidic protein (GFAP)?

Immunostaining for glial fibrillary acidic protein was performed in the hippocampus and cerebellum of adult rats in order to compare the distributions of immunolabelling after pre- and postembedding procedures. The reactions of protoplasmic astrocytes and pericapillary astrocyte processes were investigated at the electron microscopic level. After the pre-embedding reaction, visualized with 3,3'-diaminobenzidine tetrahydrochloride, a granular precipitate was observed to decorate the rough endoplasmic reticulum of the astrocyte cell bodies and a precipitate filled the cytoplasm in the astrocyte processes. In studies with the postembedding procedure, immunogold particles were observed to be attached exclusively to the intermediate filaments of the astrocytic cytoskeleton both in the cell body and in the processes. It is concluded that the diaminobenzidine precipitate diffuses in the cytosol, pre-embedding immunocytochemistry is therefore, suitable for the overall labelling of astrocytes, whereas the postembedding procedure reveals the exact intracytoplasmic localization of glial fibrillary acidic protein. This explains the similar pre-embedding immunostaining patterns of astrocytes with markedly different amounts of glial filaments (e.g. protoplasmic, fibrous and reactive) and stresses the importance of the use of the postembedding method when an exact intracellular localization is required. The results also suggest that, in spite of claims of soluble cytoplasmic pools of this protein, the glial filaments are the exclusive domains of immunoreactive glial fibrillary acidic protein.

Ovarian cycle-related changes of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus.

The interpeduncular nucleus (IPN) of female rats was studied across the estrous cycle to observe whether the expression of the astroglial marker, glial fibrillary acidic protein (GFAP) reacts to hormonal changes in an area not belonging to the 'endocrine brain'. A marked reduction of immunoreactive GFAP was observed in estrus as compared to the immunoreactivities in met- and proestrus. This finding is consistent with earlier observations in the endocrine hypothalamus, but also proves that gonadal steroids influence astroglia in brain regions not involved in neuroendocrine regulation. Since cyclic fluctuations of synaptic numbers in the female have been described only for the endocrine hypothalamus, decrease of immunoreactive GFAP in the IPN during estrus may reflect a down-regulation of GFAP synthesis.

Geniculo-cortical afferents form synaptic contacts with vasoactive intestinal polypeptide (VIP) immunoreactive neurons of the rat visual cortex.

The lateral geniculate nucleus of the rat was injected with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) to see if geniculo-cortical axons terminate on vasoactive intestinal polypeptide immunoreactive (VIP-IR) neurons of the primary visual cortex. PHA-L-labelled boutons attached to VIP-IR perikarya and dendrites were identified as presynaptic parts of asymmetrical synapses. This geniculo-cortical projection to VIP-IR cells in the visual cortex and comparable findings in the somatosensory cortex suggest that sensory input from specific thalamic nuclei may influence local circuit inhibition and the metabolic state within the cortical domain via VIP-IR neurons.

On the nerve plexus of the chicken gizzard.

The Auerbach's plexus of the gizzard was stained in toto in adult chicken and in young and newly-hatched chicks. The plexus lies immediately beneath the serosa and extends over 55% of the surface of the organ, covering its cranial and caudal poles and the two curvatures. The areas into which the plexus does not extend (i.e., most of the ventral and the dorsal surface) are those where the muscle is covered by the laminar tendon of the gizzard. The ganglia are large, often with hundred of neurons, and have short and broad connecting strands. They are surrounded by a capsule of connective tissue. The ganglion neurons are discoidal and in the adult they measure up to 50 microns in diameter, each being surrounded by a set of glial cells. A few small neurons persist in the adult; in the newly-hatched chick these are predominant, but some large neurons up to 25 microns in diameter are already present. The ultra-structural features of the ganglia of the Auerbach's plexus include the abundance of axo-somatic synapses, as well as numerous axo-dendritic synapses, the presence of intra-ganglionic bundles of collagen fibrils and blood vessels, the abundance of glial cells. In addition to the plexus beneath the serosa, the gizzard has many small intramuscular ganglia located throughout the musculature (which is exclusively circular). These ganglia do not have a connective tissue capsule and are made of small and tightly packed neurons.

Consecutive diaphorase-acetylcholinesterase histochemistry in the myenteric plexus of frog stomach.

An investigation was made on the frog stomach myenteric plexus with 2 different histochemical techniques. Neuronal perikarya were stained with nicotinamide-adenine-dinucleotide-diaphorase (NADHd), while the acetyl-cholinesterase (AChE) staining showed rather the axoarchitectonic arrangement of the frog myenteric plexus. In double-labelled "whole mounts", NADHd-positive cell bodies and AChE-positive nerve processes were revealed. Some of the nerve cells and neuronal processes did not exhibit AChE activity at all. Since glyoxylic acid-induced fluorescence (GIF) was not detected in the myenteric plexus, the presence of catecholamines can be excluded. As a consequence of these observations, we suggest the presence of a non-cholinergic, non-adrenergic intrinsic neuronal system in the frog stomach myenteric plexus, containing purines or peptides as transmitters.

Acetylcholinesterase in the alimentary canal of fish: light and electron microscopic detection, quantitative distribution in different segments of the gut.

The acetylcholinesterase activity was measured and histochemically localized in the alimentary tract of 2 fish species, carp (Cyprinus carpio) and tench (Tinca tinca). A comparison was made of the activities in the different gut segments. Light and electron microscopic histochemistry revealed acetylcholinesterase-positive cell bodies along the entire length of the alimentary canal in both species, between the muscular layers. Acetylcholinesterase-positive, cholinergic motor endplates were frequent in the esophagus of both carp and tench, and they were also present in the striated muscular layers of the tench stomach and midgut. The enzyme activity detected by the method of Ellman et al. (1961) was highest (16.3 U/mg protein) in the tench foregut and midgut, while it was at the same lower level (9.5 U/mg protein) in each segment of the carp gut and in the tench hindgut. The morphological findings and the higher acetylcholinesterase activity in the tench foregut and midgut suggest that the enteric striated musculature is endowed with denser cholinergic innervation than the enteric smooth musculature.

Fine structure of the neuromuscular junctions in the alimentary tract of phylogenetically different animal species.

Authors studied the fine structural characteristics of the neuromuscular junctions in the alimentary tract of phylogenetically different animal species. Nearly in each studied species the so-called close contacts were observable, where the sarcolemma and axolemma establish a junction; the gap of this contact is 10-100 nm wide, and the neurotransmitters can affect the muscles through non-synaptic release (exocytosis). The junctional gap is widest in the gut wall of earthworm: 100-200 nm. Only close contacts are to be found in the alimentary canal of snail, and here, the junctional gap is as narrow as 10-15 nm. Beside close contacts synaptic neuromuscular junctions also occur in the locust gut. Their fine structural organization refer to that of chemical synapses. As in the tench intestine both striated and smooth muscular elements can be found, motor endplates as well as close contacts take part in their innervation. Only close contacts are in the smooth muscular layer of domestic fowl. Authors emphasize the role of close contacts in the regulation of gut peristalsis and only secondary importance is attached to other different junctions.

Sexual dimorphism of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus.

The intensity of immunostaining for the glial fibrillary acidic protein (GFAP) is outstandingly high in the interpeduncular nucleus. This nucleus was compared in males and females for its GFAP immunoreaction. Immunohistochemistry was carried out on free floating vibratome slices and evaluated by surface densitometry. While in males the reactions were similar, females showed individual variations. Since the interpeduncular nucleus is a hormonally inactive brain area where gonadal hormones do not induce plastic synaptic changes, it is concluded that concerning this astroglial marker a sexual dimorphism exists also outside the "endocrine brain".

[Injection sclerotherapy in acute peptic ulcer hemorrhage from the upper gastrointestinal system]. / A felsö gasztrointesztinális rendszer akut fekélyvérzéseinek injekciós szklerotizáló kezeléséröl.

In the last 7 years the author has used urgent endoscopy together with injection sclerotherapy with 1% Aethoxysklerol at 172 times on 153 acute bleeding peptic ulcers patients. He has successfully ceased the bleeding at 92.9% of the active bleeding patients. Only 11 patients had to be operated on in acute bleeding. Repetitive bleeding occurred at 9.15%, most of them could be overcome by resclerosing. The mortality among the patients treated by injection sclerotherapy was 4.92%, among the operated and partly repetitive bleeding patients 27.2%. The total mortality rate of the 153 patients was 6.5%. In his essay the author discusses the indications and contraindications of the injection sclerotherapy, the marking of correct time limit between the injection sclerotherapy and consideration of operation, the questions of acute bleeding peptic ulcer patients admission to hospital and hospitalizing.