Increase of the serotonin metabolite, 5-hydroxyindoleacetic acid, in cerebro-spinal fluid and spinal cord of bovines affected with the paraplegic syndrome.

The paraplegic syndrome of bovines is a condition characterized by impairment of locomotion, hypoalgesia and finally death within 72 h. The pathogenesis of the syndrome has not been established. In the present work we determined the levels of monoamines and their metabolites in cerebro-spinal fluid and spinal cord of affected animals in order to investigate the functional state of these neurotransmitters. The content of the main metabolite of serotonin, 5-hydroxyindoleacetic acid, was elevated in the cerebro-spinal fluid and in the gray matter of the spinal cord of paraplegic bovines. Serotonin content in the spinal cord did not differ with respect to control animals, but was decreased in the cerebro-spinal fluid of affected animals. Modifications in the noradrenergic system were also observed, but were less consistent, for which reason further studies are needed. These observations indicate an increase in the turnover rate of serotonin in the paraplegic syndrome. The meaning of the described alterations is unknown at the moment.

Taurine-induced regeneration of goldfish retina in culture may involve a calcium-mediated mechanism.

A possible mechanism of action of taurine as a trophic substance was studied in goldfish retina by investigating the effect of extracellular and intracellular calcium chelators on in vitro outgrowth, and the effect of taurine on calcium influx into postcrush retinal cells in culture. The amino acid stimulated the outgrowth from goldfish retinal explants, an effect that was blocked by EGTA and 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid methyl ester (BAPTA). The influx of calcium into cultured cells from postcrush retina was increased by taurine by day 5 in culture, but not by day 10, supporting previous results indicating a critical period in which taurine stimulates outgrowth. The present results suggest that taurine partially exerts its regenerative effect on postcrush retinal explants by increasing calcium influx.

[3H]serotonin binding sites in goldfish retinal membranes.

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [3H]5HT was sensitive to pre-treatment of the membranes at 40 degrees or 60 degrees C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [3H]5HT binding to retinal membranes. The 5HT2 agonist, 1-(-naphthyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodophenyl-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B(max) 2.45 pmol/mg of protein, kd 6.86 nM), and a low affinity component (B(max) 53.46 pmol/mg of protein, Kd 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (Kd 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existence of 5HT1 receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a subtype of receptor expressed in glial cells.

Possible neurotransmitter role of noradrenaline in the teleost retina.

The role of dopamine as a neurotransmitter in the retina of different species has been clearly established; however, there is still some controversy as to whether noradrenaline (NA) is present as a neurotransmitter in this tissue. In this study, we show that, under controlled conditions, NA is present in the retina of goldfish at a concentration of 0.15 +/- 0.03 ng/mg protein and its biosynthetic enzyme, dopamine beta-hydroxylase shows an activity of 2.5 +/- 0.2 pmol NA/hr/mg protein. The amount of NA increases to 1.88 +/- 0.24 ng/mg protein in light adapted animals and decreases to undetectable levels in dark adapted ones. By contrast, dopamine-beta-hydroxylase levels are not affected by changes in light conditions. This finding provides further evidence in favor of a neurotransmitter role for NA in vertebrate retina.

Differential taurine uptake in central and peripheral regions of goldfish retina.

The transport system of taurine was investigated in fragments of goldfish retina prepared from the total tissue and from concentric regions: center and periphery. A high-affinity, saturable, sodium-dependent system was demonstrated in the three types of fragments. The Km for one-site analysis was similar in the two regions and the total retina. The analysis for two sites revealed a significant higher Km for the high-affinity site in fragments from the central region. The maximal uptake rate was higher in the central zone than in the total retina or the periphery. The Hill slopes obtained from saturation experiments of fragments of total retina, center, and periphery were similar to one other and near to 1. The slope of the time course uptake was intermediate for total retina and higher in the center than in the periphery. Hypotaurine and beta-alanine were found to inhibit taurine uptake, but GABA was a weak inhibitor. The values of Ki for hypotaurine by one- and two-site analysis were lower in the central region. The disruption of photoreceptors by shaking did not modify significantly the uptake of the amino acid. Remotion of endogenous taurine by dialysis of central and peripheral fragments increased the uptake in the periphery, but not in the center. The differences observed among the three samples revealed less affinity and high capacity for taurine uptake in the center, plus a higher sensitivity of inhibition. In addition, the peripheral zone had a greater affinity for taurine, and the maximal velocity of the entrance seems to be inhibited by the higher concentration of the amino acid in this zone. These observations may reflect differences between proliferating and non-proliferating regions of the retina (i.e., periphery and center).

Cerebral serotonin in viral encephalitis.

In order to evaluate central serotonergic function during viral encephalitis biochemical, behavioural and immunohistofluorescence studies were carried out. Mice were inoculated with the moderate virulent strain of venezuelan equine encephalomyelitis virus, Pixuna. Signs of encephalitis were observed in 50-60% of infected animals. Levels of serotonin and 5-hydroxyindolacetic acid, and the ratio of the indolamine and its metabolite in raphe and cortex did not change with respect to sham-inoculated mice. A differential decrease in turnover rate by pharmacological methods, such as pargyline, p-chlorophenylalanine and probenecid administration, was observed in raphe and cortex. The ratio serotonin turnover rate/steady state concentration of serotonin was only decreased in the raphe of sick animals. The response to 5-methoxy-N,N-dimethyltryptamine was greater in infected animals. The duration of immobility in the swim test was shorter in the infected group. A greater number of viral antigen particles was localized in raphe and periraphe areas than in cortex, brain stem or striatum. The results suggest a serotonin presynaptic deficit, a postsynaptic hyperreactivity of serotonin system, and a region-selective distribution of the virus.

Retinal neurotransmitter interaction as reflected in horizontal cell spatial behaviour.

The effects of 5-hydroxytryptamine (5-HT) and its precursors 5-hydroxytryptophan (5-HTP) and L-tryptophan (L-Tryp) on the spatial properties of horizontal cells were studied in the isolated and perfused retina of the teleost Eugerres plumieri. All three compounds produce a contraction of the receptive field, evaluated by the ratio of responses evoked by local and distant light stimuli. This is the result of cell uncoupling, revealed by the hindrance to diffusion of intracellularly injected Lucifer yellow. Similar effects are produced by dopamine (DA) and the effectiveness is DA much greater than 5-HT greater than 5-HTP greater than L-Tryp. All these effects are blocked by Haloperidol. HPLC studies of endogenous DA release reveal that it occurs when isolated retinas are incubated with 50 mM potassium, 10 microM 5-HT or 5-HTP, but is not found with up to 1 mM L-Tryp. The results indicate that indolaminergic cells induce the release of DA from interplexiform cells, which in turn uncouples horizontal cells in the fish retina.

Cyclic adenosine monophosphate as a second messenger in horizontal cell uncoupling in the teleost retina.

The reduction in the receptive field of horizontal cells of the teleost Eugerres plumieri observed upon dopamine (DA) superfusion is thought to be due to cell uncoupling. The possible mechanisms by which activation of DA receptors modify the electric coupling between horizontal cells were studied in the present work. It was found that the effect of DA in different preparations is mediated by a modification of intracellular concentration of cAMP and H+. The effects of intracellular injection of cAMP and H+ were studied in retinal horizontal cells of the teleost E. plumieri. A triple microelectrode was used to inject the ion iontophoretically, to pass current pulses, and to record voltages from the same cell, while a fourth microelectrode was used to record voltages from a neighboring cell in the same retinal layer. Responses evoked by light spots and annuli were evaluated simultaneously. Coupling ratios between neighboring horizontal cells ranged from 0.22 to 0.45. The intercellular resistance (Rc), 0.5-3.5 x 10(6) ohms, and that of the remaining cell membrane resistance (Rm), 2.5-18 x 10(6) ohms, were calculated by means of a passive electrical model that has a hexagonal array. The microinjection of H+ with injection current from +5 to +30 nA for 40 to 100 sec led to temporary and reversible light response reduction. The coupling ratio between two impaled cells was reduced by about 30%, and intercellular resistance (Rc) increment was 320% while cell membrane resistance (Rm) did not change consistently. There was also a temporary and reversible Rm reduction (70-85%) and an Rc increment of 170-330% when cyclic adenosine monophosphate was iontophoretically injected with current from -30 to -40 nA for 50 to 170 sec. The coupling ratio between two impaled cells was reduced by about 40%, and light responses recorded from the injected cell showed a reduction in amplitude with the same time course as that of the resistive changes. The injection of Lucifer yellow into a horizontal cell under normal conditions always results in pronounced fluorescence for more distant cells; however, under constant injection of H+ or cAMP only the injected cell is fluorescent, which provides direct evidence of the reduction in the effectiveness of coupling between horizontal cells. The observed effects of intracellular H+ or cAMP injection correspond to the resistive changes in Rc and coupling ratio that occur in the horizontal cell network upon superfusion with a dopamine (DA) solution.

Spatial distribution of glutamate, taurine and GABA in teleosts and mammals retina: in vivo and in vitro study.

Teleost and amphibian retina grows throughout life at the circumferential margin distal to the center. Retinas of two teleosts, goldfish and Eugerres plumieri; and two mammals, rat and rabbit were dissected into concentric regions; center and periphery; or center, intermediate and periphery. The concentration of glutamic acid, taurine, and gamma-aminobutyric acid was determined in these areas. A non-significant difference was found between the center and periphery of retinas dissected into two regions. By dissecting the teleost retina in three concentric regions a progressive increase of gamma-aminobutyric acid and taurine for center to periphery was observed, which was statistically significant for taurine. Rat and rabbit retina did not present this difference. The concentration of these amino acids was also determined in two concentric regions of goldfish retina 24 hr after crush of the optic nerve. Taurine content significantly increased in the center of the regenerating retina, but not in the periphery. Outgrowth was measured in explants prepared from central and peripheral retina in the presence of exogenous taurine. The amino acid produced an increase of the nerve growth index of central explants, but inhibited the growth from peripheral explants. This report presents a correlation between taurine concentration in a region of the retina that could be considered as embryonic tissue, suggesting that this amino acid plays a role in the formation of new cells. Moreover, the results from post-crush and cultured retina, with respect to taurine concentration or regenerating effect, suggest a saturation of the taurine-enriched zone.

The interaction of substrate and taurine modulates the outgrowth from regenerating goldfish retinal explants.

Retinal growth from explants attached to different substrates was evaluated in the absence and the presence of various concentrations of taurine. Fiber thickness was higher in poly-L-lysine, collagen and laminin than in the other substrates tested. Explants on wheatgerm, soybean, and red kidney bean lectins did not show emission of neurites at 5 days in culture; growth on laminin was very poor at this period of time. By day 10 nerve growth index was possible to evaluate on soybean and laminin. Outgrowth from retinal explants on wheatgerm lectin was only observed in the presence of taurine. The percentage of growing explants on laminin was increased by the amino acid. Emission of neurites from explants on concanavalin A and lentil lectin was constant from 5 to 10 days in culture and was not accelerated or increased by taurine. Growth on poly-L-lysine and collagen was stimulated by the addition of taurine in a concentration-dependent manner. The amino acid stimulates outgrowth from explants attached to peanut lectin in a bell-shaped concentration dependency similar to that observed on poly-L-lysine. The effect of taurine on retinal explants is substrate-dependent, as an indication of the interaction of soluble molecular components plus charge and structure of the extracellular matrix in order to modulate the outgrowth from regenerating tissue.

Taurine effect on neuritic growth from goldfish retinal explants.

Explants from retinas from which the optic nerve was previously crushed exhibited neuritic outgrowth after 4-5 days in culture, and up to 10 days in the presence of the same nutrient medium. Intact retinas did not exhibit vigorous outgrowth. Taurine added in the medium to post-crush explants accelerated the emission of neurites in 1-2 days, and increased the nerve growth index in a concentration-dependent manner up to 4 mM. Greater concentration did not produce any effect on the nerve growth index. The effect of taurine was produced between 0 and 5 days in culture; from day 5 to day 10 explants did not exhibit further growth in the presence of the amino acid. However, post-crush control explants exhibited growth between 5 and 10 days. Taurine effect is blocked in the absence of fetal calf serum, and is reduced in the presence of dialysed serum. Providing new medium on days 1 and 2, but not later, elevated the nerve growth index with respect to explants regenerating in the same medium since day 0. In addition, taurine supplied at various days after plating produced the same pattern of growth as that observed by adding fresh medium, but the nerve growth index was statistically greater in the presence of the amino acid. The mechanisms for these observations remain to be elucidated.

Identification of horizontal cells generating different spectral responses in the retina of a teleost fish (Eugerres plumieri).

Six different types of spectral responses were recorded from horizontal cells under mesopic conditions in perfused retina, isolated from the dark-adapted mojarra (Eugerres plumieri). They were tentatively termed photopic Lr-, Lg1-, Lg2-, Lb-, and C-type, and scotopic L-type. The Lr-, Lg-, and Lb-type responses showed a maximum peak at 605, 550, and 516 nm respectively, while the C-type was composed of hyperpolarizing potentials in response to shorter wavelengths and depolarizing potentials in response to longer wavelengths (so-called R/G-type). The scotopic L-type has a peak at 516 nm in the spectral response and a slow decay phase in the waveform response. Following a brief period of diffuse illumination, it was found that the Lg1-type response is altered to the Lr-type, while both Lg2- and Lb-type responses change to the C-type. Intracellular marking with Lucifer or Procion yellow identified the cellular origins of different response types: external (He) and medial horizontal (Hm) cells for the Lr-type, internal horizontal (Hi) cells for the C-type, and rod-horizontal (Hr) cells for the scotopic L-type. Only He cells were found to possess an axon, while dye coupling was seen between axonless Hm, Hi, or Hr cells but not between He cells. The morphology of these fluorescent dye-marked cells was the same as that of the respective cells observed in Golgi-stained materials.

Behavioural effects produced in mice infected with venezuelan equine encephalomyelitis virus.

Pixuna, a strain of intermediate virulence of venezuelan equine encephalomyelitis virus, was inoculated intracranially to 24-day-old mice. Signs of encephalitis were present in 60% of the animals between 6 and 9 days with a maximum at 7 days postinoculation. The rest of the infected mice did not show clinical signs of encephalitis. In order to study the functional state of serotonergic systems a series of tests susceptible to modifications by serotonin activity were carried out. Locomotor activity was measured in an open field test. Virus-inoculated animals presented a variety of changes in their locomotor behavior at various days postinoculation with respect to the sham-inoculated group, however, they were not significant. Central serotonergic function was examined by the production of the serotonergic syndrome with the receptor agonist, 5-methoxy-N,N-dimethyltryptamine and the precursor, 5-hydroxytryptophan, both administered 4 days after the inoculation. The dose of the agonists was established by a prior drug-response analysis. Intensity of the syndrome was significantly higher in infected mice than in the sham-inoculated group only in 5-methoxy-N,N-dimethyltryptamine-treated animals. The behaviour in the swim test was also measured. Duration of immobility was much shorter in infected than in control mice. The decrease in central serotonin turnover previously reported might be responsible for the modification in locomotor behaviour and for the supersensitivity of serotonin receptors observed in infected mice.

Dopamine and noradrenaline content in fish retina: modulation by serotonin.

The presence of noradrenaline (NA) and the possible interaction of serotonin (5-HT) with dopaminergic and noradrenergic neuronal elements was studied in the retina of the teleost Eugerres plumieri. By means of a histofluorescent technique, paired amacrine cells can be visualized after intravitreal injection of NA or 5,6-dihydroxytriptamine, suggesting their probable catecholaminergic and indoleaminergic nature. With a high-performance liquid chromatographic (HPLC) method and after pargyline treatment of the animal, 6.86 ng/mg protein of dopamine (DA) was detected, while NA content was 0.50 ng/mg protein. The NA levels of the retina increased, whereas the DA levels decreased in a significant manner after in vivo treatment with 5-HT. 5-methoxy-N,N-dimethyltryptamine, a 5-HT agonist, was able to mimic this effect partially, while the agonists tryptamine and quipazine did not affect the levels of DA and NA. The antagonists methysergide, metergoline, and cyproheptadine significantly blocked the 5-HT-induced NA increase, whereas only the first two antagonists were effective in inhibiting the 5-HT-induced DA decrease. The 5-HT modulation of NA and DA is apparently receptor mediated and is not due to a hetero-exchange, since imipramine was not able to block the 5-HT effect. These findings support the suggested interaction between serotoninergic and dopaminergic cells [Negishi et al: J Neurosci Res 5:621-635, 1980; Neurosci Lett 25:1-5, 1981]. Furthermore, they demonstrate the possible modulation which 5-HT exerts on the endogenous levels of NA.

Serotonin turnover rate in raphe and cortex of mice infected with Venezuelan equine encephalomyelitis virus.

The turnover of serotonin (5HT) was determined in the raphe area and cortex of mice infected with Pixuna, a strain of intermediate virulence of Venezuelan equine encephalomyelitis virus (VEEV). NMRI-mice, 24 days old, were inoculated intracerebrally (ic) with 300 LD50 of the virus. The animals were sacrificed 4, 7, 15, 21, 30, and 60 days postinoculation. 5HT and 5-hydroxyindoleacetic acid (5HIAA) in raphe and cortex were determined by high performance liquid chromatography (HPLC) with electrochemical detection. Turnover rate of 5HT was determined by the administration of pargyline, p-chlorophenylalanine, and probenecid. The content of 5HT or 5HIAA and 5HT/5HIAA ratios were not significantly different in infected compared with control mice. However, a decrease of 5HT turnover rate, determined after pargyline treatment, was observed in the raphe and not in the cortex of infected mice at 4 and 7 days after the inoculation. The turnover rate/(5HT)0 in raphe is decreased in infected mice with signs of illness, suggesting a lower density of 5HT innervation in this brain area. The administration of p-chlorophenylalanine and probenecid showed that the cortex is also affected, but the synthesis is less modified than metabolism or elimination. Cell bodies of 5HT neurons seem to be more susceptible than projections to infection by Pixuna strain of VEEV.

Taurine- and penicillin-induced epileptic activity.

The present study has been performed to investigate the effect of i.v. administration of taurine on the electrical activity of the epileptogenic focus induced by penicillin applied to the right sensory motor cortex of adult rats. Taurine (100 mg/kg body weight) was administered 15, 30, 60, and 120 min before the application of penicillin. The EEG was unipolarly recorded by means of electrodes applied to the pia. Taurine caused a decrease of the frequency as well as the spike amplitude of epileptic discharge. The spread of epileptogenic foci to the opposite hemisphere was retarded when compared to that of control animals. The maximal antiepileptic effect of taurine was observed when the amino acid was administered 30-60 min previous to penicillin. It is suggested that high concentrations of taurine in the brain might be necessary to inhibit the epileptic activity.

Coagulant proteinase from Bothrops colombiensis venom.

The venom of Bothrops colombiensis, like other Crotalidae venoms, contains thrombin-like activity. We purified a mixture of isoenzymes by chromatography of the crude venom on DEAE-Sephacel where coagulant proteinase were separated from other proteolytic enzymes. By subsequent chromatography on Sephadex G-100 we obtained coagulant proteinase as a single band on acrylamide gel electrophoresis at pH 7.5 which showed 4 major protein bands when subjected to flat gel isoelectric focusing. This heterogeneity is presumably due to carbohydrates present in this glycoprotein. The native molecular weight of the coagulant proteinase was found to be over 90000 by gel filtration. SDS electrophoresis showed, however, that the monomer molecular weight is around 67000. The specific coagulant activity of the purified enzyme was increased 13 fold by purification and was 231 NIH units/mg. The optimal pH for coagulation of bovine fibrinogen was at pH 7.0. The enzyme shows maximal stability in the pH range 5-6 when incubated for 1 hr at 37 degrees C. The intraperitoneal LD50 for white mice was 4.0 mg/kg. The enzyme is similar to other known coagulant proteinases from snake venoms and thus potentially useful as a therapeutic agent.