Inhibitory effect of vasopressin receptor antagonist OPC-31260 on experimental brain oedema induced by global cerebral ischaemia.

The effects of the non-peptide vasopressin V(2) receptor antagonist 5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride (OPC-31260) on the cerebral oedema induced by general cerebral hypoxia were studied in rats. The general cerebral hypoxia was produced by bilateral common carotid ligation in Sprague-Dawley rats of the CFY strain. By 6 h after the ligation, half of the rats had died, but the survival rate was significantly higher following OPC-31260 administration. Electron microscopic examinations revealed typical ischaemic changes after the carotid ligation. The carotid ligation increased the brain contents of water and Na(+) and enhanced the plasma vasopressin level. The increased brain water and Na(+) accumulation was prevented by OPC-31260 administration, but the plasma vasopressin level was further enhanced by OPC-31260. These results demonstrate the important role of vasopressin in the development of the disturbances in brain water and electrolyte balance in response to general cerebral hypoxia. The carotid ligation-induced cerebral oedema was significantly reduced following oral OPC-31260 administration. The protective mechanism exerted by OPC-31260 stems from its influence on the renal vasopressin V(2) receptors. These observations might suggest an effective approach to the treatment of global hypoxia-induced cerebral oedema in humans.

Influence of the polyphenolic tannic acid on the toxicity of the insecticide deltametihrin to fish. A comparative study examining both biochemical and cytopathological parameters.

Humics and pesticides are present in aquatic environment and the toxicological consequences of their chemical interaction is well studied. However, data concerning the mechanism of the biochemical action of humic-pesticide combinations are scarce, especially in vertebrates. Thus we have chosen to study the in vivo effects of the plant polyphenolic tannic acid and the pyrethroid insecticide deltamethrin [Decis] alone or in combination on hepatic xenobiotic-metabolizing enzyme activities and the associated redox-parameters in carp, as the complex assessment of these systems are regarded to serve as a relevant biomarker of environmental pollution. Stress effects and tissue damage were followed by determination of the plasma glucose level, the activities of plasma transaminases, and by electron microscopy. Tannic acid alone exerted weak prooxidant effect due to its marked antioxidant enzyme inhibitory activity. Deltamethrin, applied in a very low dose, induced oxyradical production in fish via activation of cytochrome P450 isozymes. This effect was promoted by the antioxidant enzyme inhibitory action of tannic acid, when the two chemicals were combined; however, the ultrastructural damage of the hepatocytes was reduced by the common cytoprotective capacity of the phenolic. Numerous humics are known to alter the toxicity of pesticides and their influence depends on their type and concentration. Therefore, our work taken together with other comparative studies may contribute to the assessment of the impact of humics in nature, especially in case of environmental pollution.

Biochemical and morphological changes in carp (Cyprinus carpio L.) liver following exposure to copper sulfate and tannic acid.

As a consequence of human activity various toxicants reach the aquatic ecosystems; humics may interact with them and may change their toxicity. Many fish are exposed to a considerable concentration of humics and pollutants. Because of paucity of data on the biochemical action of tannins in the presence of the fungicide CuSO4 a comparative study was undertaken. The alterations of redox-parameters in carp liver were monitored and tissue necrosis was followed by measuring the plasma transaminase activities and by electron microscopy. Tannic acid, a representative phenolic/humic compound, exerted prooxidant effects in carp, which may be partially due to formation of prooxidant intermediates/end-products via its biotransformation. Alternatively, tannic acid may partially inhibit the antioxidant enzymes of fish. The response to CuSO4 was more severe. Although tannic acid alone acted as a prooxidant in fish, electron micrographs demonstrated that it reduced the necrotizing effect of copper, which may be due to the complexing activity of tannic acid with the biomolecules of the hepatocytes and to the H2O2-degrading activity of tannin-CuSO4 combination. Our results indicate that the heavy metal-detoxifying capacity of tannin may be significant; however, tannin-exposure alone or combined with metals may be toxic for fish due to enzyme inhibition and oxidative stress induction.

Comparative study of the neuronal plasticity along the neuraxis of the vibrissal sensory system of adult rat following unilateral infraorbital nerve damage and subsequent regeneration.

The aim of the present study was to examine the physiological consequences of a unilateral infraorbital nerve lesion and its regeneration at different levels of the somatosensory neuraxis. In animals whose right infraorbital nerve had been crushed, a large unresponsive area was found in the main brainstem trigeminal nucleus (Pr5). Responses evoked by ipsilateral vibrissal deflection in the middle of Pr5 reappeared only on days 22-35 after the nerve had been transected, whereas recovery from the nerve crush took only 7-9 days. However, no sign of short-term neuronal plasticity was observed in Pr5 after peripheral nerve injury. An enlargement of the receptive fields in two-thirds of the units and a lengthening in the delay of the evoked responses were observed as long-term plastic changes in Pr5 neurons after peripheral-nerve regeneration. In the ventral posteromedial nucleus of the thalamus (VPM) of partly denervated animals, however, only minutes or hours after the nerve crush, certain units were found to respond in some cases not only to the vibrissae, but also to mechanical stimulation of the face over the eye (two units), the nose (one unit), and the midline (one unit). Apart from the experiments involving incomplete denervation, the vibrissal representation areas of the VPM were unresponsive to stimulation of both the vibrissae and other parts of the face until nerve regeneration had occurred. In the somatosensory cortex, an infraorbital nerve crush immediately resulted in a large cortical area being unresponsive to vibrissal deflection. It was noteworthy, however, that shortly after the nerve crush, this large unresponsive whisker representation cortical area was invaded from the rostromedial direction by responses evoked by stimulation of the forepaw digits. In spite of the reappearance of vibrissa-evoked responses 7-10 days after the nerve crush, an expanded digital representation could still be observed 3 weeks after the nerve crush, resulting in an overlapping area of digital and vibrissal representations. The withdrawal of the expanded representation of forepaw digits was completed by 60 days after the nerve crush. The results obtained in Pr5, the VPM, and the cortex strongly suggest that the higher the station in the neuraxis, the greater the degree of plasticity after infraorbital nerve injury.

Comparative electrophysiological aspects of aluminum actions on central neurons and neuronal synapses of invertebrate and vertebrate animals.

Aluminum compounds (Al) increased the membrane potential (Em) and decreased the input resistance (Rin) of identified snail neurons. The neuronal excitability increased after Al withdrawal in the washing period. Cumulative Al applications caused dose-dependent modulation of Em and Rin in most of the studied neurons. Two phase actions were recorded on stimulus evoked excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) at pH 6.5-6.9. The Al induced facilitation followed by attenuation were statistically significant, time- and dose-dependent events. They could be recorded at each Em. Low affinity and slow binding kinetics could characterize the Al actions on the neurons. Al showed pH- dependent suppression of EPSPs or EPSCs in some neurons. Our findings are partially comparable with Al induced electrophysiological and pharmacological modifications reported on vertebrate neurons.

Neonatal monocular enucleation-induced cross-modal effects observed in the cortex of adult rat.

The cortices of neonatally enucleated rats were explored for somatosensory responses with special reference to an extension into the occipital cortex. Monocular enucleation was performed on rats at birth. The animals were raised and from the age of three months the activity evoked by either electric stimulation of the vibrissa pad or bending of the vibrissae was tested in the contralateral cortex by electric recording and autoradiography. It was found that early enucleation caused an expansion of the somatosensory responses, among others into the visual area. Neurons responsive to visual and somatosensory stimuli were demonstrated in the anterior part of the primary and secondary visual areas, contralateral to the enucleation. Electrophysiological and autoradiographic studies unambiguously proved that early enucleation exerted a significant cross-modal effect on the somatosensory responsive area.

Aggressive role of vasopressin in development of different gastric lesions in rats.

The effects of endogenous or exogenous vasopressin in models of gastric mucosal injury with a different pathophysiology (ethanol, indomethacin, reserpine, cold-restraint stress and haemorrhagic shock-induced lesions) were investigated in rats. [Mca1,TyrMe2,Arg8]vasopressin, a vasopressin pressor (V1) receptor antagonist, was found to reduce dose dependently the extent of the lesions in all models, and to protect the deeper layer of the mucosa (assessed by histology). Endogenous vasopressin deficiency, as in Brattleboro homozygous rats, had a similar effect. [Lys8]Vasopressin injected exogenously aggravated all types of lesions in normal rats. Circulating vasopressin levels were increased by ethanol, reserpine, cold-restraint stress and haemorrhagic shock, but not by indomethacin, whereas the intramucosal vasopressin content was found to be elevated in all models. Additionally, specific binding sites for vasopressin were shown on the blood vessels of the gastric mucosa (assessed by autoradiography). It is concluded that vasopressin plays a significant aggressive role in the generation of these types of lesions.

Rapid synthesis of a glycine-rich protein in the cerebral cortex of the rat during activity.

A glycine-rich protein was previously discovered (Rojik and Fehér, 1976) in the nervous system, which proved to be neuron-specific and in its appearance highly dependent on the actual intensity of neural activity. It was named by us neuragin. Time relations and regulation of neuragin synthesis were examined by the glycine labelling method in the cerebral cortex of the rat. During activation of the somato-sensory area, 2 min stimulation sufficed to provoke the appearance of neuragin in the neurons. The newly synthesised protein persisted in the nerve cells at least for 6 hours after stimulation. Phorbol-12-monoacetate strongly enhanced, sphingosin depressed the synthesis of neuragin. It is concluded, that neuragin is an immediate early gene product, similar to proteins encoded by genes c-fos and c-jun.

Modification in primary visual cortical activity of rat induced by neonatal monocular enucleation, an electrophysiological and autoradiographic study.

Mononuclear enucleation was performed in rats at birth. The animals were raised and from the age of 3 months the evoked activity was tested in the contralateral visual cortex both by mapping of evoked potentials and autoradiography. It was found that monocular enucleation changed the distribution of evoked activity characteristically. The focus of activity shifted laterally and restricted itself to the binocular part of the primary visual cortex (Oc1B), while, in the medial part of it (Oc1M) hardly any evoked activity or labelled neurons were found

Modification induced in visual cortical activity of rat by neonatal monocular enucleation.

Monocular enucleation was performed on rats at birth. The animals were raised and from the age of 3 months the evoked activity was tested in the contralateral visual cortex both by mapping of evoked potentials and by autoradiography. It was found that monocular enucleation changed the distribution of the evoked activity characteristically. The focus of activity shifted laterally and was restricted to the binocular part of the primary visual cortex, while hardly any evoked activity or labelled neurons were found in its medial part.

Application of the glycine labelling method to the cerebellum, hippocampus and spinal cord.

3H-glycine was applied to the cat cerebellar cortex under resting conditions and during inferior olive stimulation which activated the climbing fiber system on a restricted area. Electric recording was made. The autoradiograms showed, that under resting condition labelled glycine was incorporated mainly in granule, Golgi and basket cells and only a few Purkinje and stellate cells were active. Also cerebellar glomeruli remained without labelling. On climbing fiber stimulation Purkinje cells became activated singly and grouped, also Golgi and stellate cells increased in number. Granule cells were totally inhibited. 3H-glycine, when applied to the rat hippocampus, the dentate gyrus, CA1 and CA4 fields showed labelling at low frequency stimulation. When 400 Hz high frequency stimulation periods were interposed, long-term potentiation ensued. The overall labelling of each hippocampal region was intensified significantly, indicating that glycine incorporation may be a sign not only of excitation but also of long-term potentiation. 3H-glycine was applied to frog spinal cord during rest and dorsal root stimulation. Interneurons and motor neurons excited by the afferent fibers showed intensive glycine uptake. It is concluded that the glycine labelling method is suitable for detecting neural excitation in the structures dealt with in this paper.

A study on the tonotopic organization in the auditory cortex of the cat; an application of the glycine labelling method.

3H-glycine was locally applied to the auditory cortex of chloralose anaesthetized cats. Upon tonal stimulation the 3H-glycine was taken up and incorporated into the proteins of nerve cells. The selectively activated neurons were visualized by serial light microscopic autoradiography. Systematic application of this experimental setup revealed tonotopic organization in the primary auditory cortex. The distribution of nerve cells responding to spectrally pure, continuous tones of 0.34, 3.3, 8.0, 16 and 30 kHz was mapped. At these frequencies, distinct but overlapping representations were found, whose area increased in parallel with the elevation of frequencies. Tone pips and ramp stimuli resulted in generalized labelling, independently of pitch.

The functional projections of the anterior ventralis nucleus, as revealed by autoradiography.

The effect of stimulation of ventral anterior nuclei of the thalamus (VA) was studied in different areas of cat cortex (sensory, motor and association) with [3H]glycine autoradiography. One-hour VA stimulation resulted in the most intensive [3H]glycine incorporation in the second, third and fifth layers of the anterior suprasylvian gyrus and of the motor cortex. The labelling was less intensive in the sensory (auditory) and association (posterior middle suprasylvian association area) cortices, but the effect of VA stimulation reached these regions as well. The VA stimulation resulted in evoked potentials over the whole area examined, with highest amplitude and shortest latency in the anterior suprasylvian gyrus. It is suggested that the [3H]glycine autoradiography is suitable to study the functional projection of non-specific thalamocortical systems.

Afferent fibers to the anterior suprasylvian gyrus from the medial geniculate body of cat.

Afferents to the anterior suprasylvian gyrus (ASG) from the medial geniculate body of cat were demonstrated by means of autoradiography. [3H]Glycine was injected stereotactically into the medial division of the medial geniculate body (mMGB). After a 3-day survival period, the auditory cortices and the ASG were excised. Labelled terminals were found in the ASG, in the anterior auditory field (AAF) and in the acoustic cortex (AI). The density of labelling was highest in the ASG and lower in the AAF and AI. The afferents from the mMGB made synaptic contacts in the 3rd layer of the cortices examined.

Function-dependent glycine incorporation into neurons of the cat motor cortex.

Filter papers soaked with 3H-2-glycine solution were applied to the motor cortex of chloralosed and nembutalized cats for one hour. Then underlying cortical samples were excised, fixed in Bouin solution and processed for light-microscopic autoradiography. Sections stained with haematoxylin-eosin were made to determine total cell counts. The cortical samples were sectioned in series so that 10 micron sections at every 50 micron were preserved. From resting and stimulated cortices 10 sections were used for counting pyramidal cells and interneurons in different layers. The electric background activity and the potentials evoked by thalamic VL and antidromic PT stimulation were recorded and averaged. In chloralose anaesthesia, only 7%, while under nembutal 22% of the cells incorporated glycine, on the average. VL stimulation, which induced the appearance of typical evoked potentials, enhanced the glycine incorporation of pyramidal cells in layers II-III and Vb and some activation in the interneurons of layer II was also seen. Antidromic PT stimulation did not change the autoradiographic pattern in chloralosed preparations, while in nembutalized cats it depressed the labelling of pyramidal cells in layers II, III and Va drastically with some enhancement in interneurons of layer II. The changes of autoradiographic patterns are in harmony with theories about cortical effects of VL and antidromic PT stimulation.

Morphological and biochemical studies on liver, kidney and gill of fishes affected by pesticides.

Effect of a herbicide, paraquat (1,1'-dimethyl-4,4'-bipyridilium-dichloride), the fungicide copper sulphate, and zinc chloride was studied on the histological structure of liver, kidney and gill of three fish species with different feeding habits, viz.: a herbivorous, silver carp (Hypophthalmichthys molitrix); an omnivorous, common carp (Cyprinus carpio L.) and a carnivorous, sheatfish (Silurus glanis L.). The organs were studied electron microscopically after fixation according to Karnovsky. The toxic effect manifested itself characteristically on the respective species, regardless of the type of the chemical applied and the species specificity. Upon the effect of the treatments applied the cytoplasm of the respiratory cells of the gill became electron transparent and the cytoplasmic organelles disappeared almost totally. In the chloride cells showing focal necrosis, residuals of nuclear, mitochondrial and endoplasmic origin were seen. Pillar cells and the pericytes remained intact. In the nucleus of the liver cells, electron dense heterochromatin was not present. The degree of the damage in the liver cells was indicated by swollen mitochondria with electron transparent matrix and by dilatation and vacuolation of the endoplasmic reticulum system. Epithelial cells decreased in electron density, the endoplasmic reticulum was vesiculated, mitochondria were swollen. Leucocytes increased in number, and empty vacuoles and vacuoles filled with dense granules appeared in them during toxicosis. Copper sulphate or paraquat increased serum transaminase enzyme activities (glutamic acid-oxalacetic acid transaminase, glutamic acid-pyruvic acid transaminase) in all the three fish species. These damages can cause serious disturbances in energy uptake and secretion processes of fish.

Effect of epileptogenic agents on the incorporation of 3H-glycine into proteins in the cat's cerebral cortex.

Filter paper strips soaked in 3H-glycine solution were applied to acoustic cortex of cats, anaesthetized with Nembutal and pretreated with epileptogenic agents (Metrazol, G-penicillin, and 3-amino-pyridine) and cycloheximide. The untreated contralateral hemisphere served as control. After 1 h incubation, both cortical samples were excised simultaneously and fixed in Bouin solution for autoradiography. Incorporation was blocked by cycloheximide. There was no glycine incorporation on the penicillin-treated side, while pyramidal cells were intensively labelled in layers II-V of the mirror focus. 3-Aminopyridine produced the same result. Metrazol as convulsant proved to be far weaker than the previous two. The intensity of incorporation was significantly more intensive in the mirror focus than in the primary one. Penicillin and 3-aminopyridine, while provoking cortical seizures, seem to inhibit glycine incorporation into a neuron-specific, function-dependent protein contained by the labelled cells in the autoradiogram.

Correlations between glycine incorporation and cerebral cortical activity.

3H-glycine was applied on the acoustic cortex of cats at different levels of anaesthesia and its incorporation into the cortical cells was studied using light microscopic autoradiography. Glycine was incorporated only by nerve cells. The extent of incorporation depended on the level of activity: it was most intensive in waking cortices and diminished gradually with deepening of anaesthesia. In light barbiturate anaesthesia, when only spontaneous spindles were present in the electrocorticogram, labelling was restricted to layers I-III of the cortex. Acoustic click stimulation intensified and extended it to layer V. It seems that glycine incorporation reflects neuronal activity with the resolution of an extracellular microelectrode.

Incorporation of labelled amino acids into the acoustic cortex of the cat.

Labelled amino acids, 14C-leucine, 3H-glycine, 14C-GABA, 14C-glutamic acid and 14C-aspartic acid were applied to the surface of the cat's auditory cortex in dried filter paper strips for 40 min. The distribution of the amino acids taken up by the cortex was examined by autoradiography.The right acoustic area was stimulated by 2 cps acoustic clicks, through an ear-phone. Leucine showed cellular localization which was considerably dispersed by cortical excitation. Glycine was concentrated in nerve cells, mainly in layers I-II, but excitation intensified and extended its incorporation. GABA was accumulated rather diffusely in layers I-II with scattered cellular labelling which was depressed by stimulation. Glutamic and aspartic acids did not show any characteristic distribution pattern. The authors emphasize that the incorporation of glycine seems to be a good indicator of neurons being in excitation. The localizations of GABA uptake shows close correlation with the site of its physiological action.