Günther Sperk

Simultaneous Determination of Serotonin, 5‐Hydroxyindoleacetic Acid, 3,4‐Dihydroxyphenylacetic Acid and Homovanillic Acid by High Performance Liquid Chromatography with Electrochemical Detection

Abstract: A rapid and highly sensitive procedure for simultaneous determination of serotonin, 5‐hydroxyindoleacetic acid, 3,4‐dihydroxyphenylacetic acid and homovanillic acid is described. After precipitation of proteins with perchloric acid the samples are applied directly to a high performance liquid chromatograph, with electrochemical detection. As little as 20 pg of serotonin, 5‐hydroxyindoleacetic acid, and 3,4‐dihydroxyphenylacetic acid and 200 pg of homovanillic acid can be detected. One chromatographic run requires less than 10 min.

The role of brain edema in epileptic brain damage induced by systemic kainic acid injection.

Edema formation and blood-brain barrier permeability was studied in animals with epileptic seizures induced by subcutaneous injection of kainic acid. Brain edema was most pronounced between 3 and 24 h after kainic acid injection. It was reflected by massive swelling of perineuronal and perivascular astroglia. Three hours after kainic acid perivascular astroglia swelling resulted in disturbance of local microcirculation in the affected brain areas. In addition, compression of drainage veins by the edematous brain induced focal perivenous hemorrhages similar to herniation damage in human brain edema. Tracer studies with sodium fluorescein, Evans blue, albumin and horseradish peroxidase revealed only a mild increase in the permeability of cerebral vessels, topographically unrelated to areas of brain edema. This finding indicates the presence of cytotoxic brain edema in kainic acid-induced epileptic brain damage. Treatment of brain edema with dexamethasone did not influence the incidence and severity of kainic acid-induced epileptic brain damage. However, in 54% of animals injected with kainic acid, lesions were completely prevented by treatment of brain edema with mannitol. The present results indicate that brain edema plays an important role in the pathogenesis of epileptic brain damage following systemic kainic acid intoxication. It is suggested that in this model of limbic epilepsy the brain edema is due to the massive ionic imbalance elicited in the affected brain regions by the kainic acid-induced persistent neuronal excitation.

Kainic acid-induced changes of serotonin and dopamine metabolism in the striatum and substantia nigra of the rat.

Levels of the putative neurotransmitters serotonin (5-HT) and dopamine (DA) and their respective metabolites 5-hydroxyindoleacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC) was determined in the rat striatum after unilateral intrastriatal injection of the convulsive neurotoxin kainic acid. Two days after intrastriatal kainic acid injection, levels of the 5-HT metabolite 5-HIAA were increased by abut 200% in the injected striatum and by about 150% in the contralateral striatum. An elevated striatal 5-HIAA content was still detectable 10 days after the kainate lesion, but approached normal values 10 weeks after the injection of the neurotoxin. Two days after the lesion, but not at the other time intervals, a moderate increase of 5-HIAA also occurred bilaterally in other brain areas such as the substantia nigra, frontal cortex and hypothalamus. Levels of 5-HT were decreased significantly in the injected striatum 2 days after the intrastriatal application of kainic acid and increased by about 40% after 10 weeks. The 5-HT concentration in the contralateral striatum or in the three other brain areas examined was unchanged at all time intervals. Levels of the DA metabolite DOPAC and DA turnover were increased in the lesioned striatum 2 days after kainic acid injection; concomitantly the DOPAC level was increased in the substantia nigra of the contralateral side. DOPAC levels of the contralateral striatum were unchanged or slightly reduced 2 days after the injection. Ten days as well as 10 weeks after the lesion there was a slightly increased DOPAC concentration in both striata. The levels of DA were not altered at any time interval after the injection of kainic acid.

Effect of mannitol treatment on brain neurotransmitter markers in kainic acid-induced epilepsy.

The effect of mannitol treatment on the behavioural, morphological and neurochemical brain damage induced after subcutaneously applied kainic acid (10 mg/kg) was studied in the rat. Mannitol at a dose of 1.5 g/kg was injected intravenously 10 min, 1.5 h and 3 h respectively after kainic acid administration. A protective effect of mannitol was observed only when mannitol was given 1.5 h after kainic acid application, i.e. within the early phase of kainic acid-induced brain oedema development. At this time period, mannitol prevented the development of kainic acid-induced seizures as well as irreversible brain lesions and neurochemical changes, the latter being reduction of noradrenaline levels in amygdala/pyriform cortex measured 3 h, and reduction of glutamate decarboxylase and choline acetyltransferase activities measured 3 days after kainic acid treatment. Similarly loss of glutamate decarboxylase activity in dorsal hippocampus induced by kainic acid was prevented by mannitol treatment. It is concluded that by washing out brain oedema, mannitol treatment may prevent propagation of seizures and brain damage in the kainic acid model of epilepsy.

α2-adrenoceptors modulate kainic acid-induced limbic seizures

We have tested several compounds interfering with the brain monoamine (noradrenaline, dopamine, serotonin) and acetylcholine systems for their effects on limbic seizures produced by systemically (s.c.) injected kainic acid as well as on neurochemical changes in amygdala/pyriform cortex resulting from the kainic acid treatment. The characteristic neurochemical changes induced by s.c. kainic acid were a decrease in noradrenaline and an increase in 5-hydroxyindoleacetic acid in the acute (3 h after kainic acid injection) suggesting strongly increased neurotransmitter turnover in noradrenergic and serotonergic neurons. This was followed by a reduction of glutamic acid decarboxylase and choline acetyltransferase activities during the chronic phase (3 days) of the kainic acid action, indicating destruction of GABAergic and cholinergic neurons. The compounds tested in this model of limbic epilepsy included 1-propranolol, prazosin, clonidine, yohimbine, metergoline, atropine and haloperidol. Among these compounds the alpha 2-adrenergic agonist clonidine (0.1 mg/kg, i.p.) exhibited a powerful protective action on kainic acid-induced limbic seizures as well as on the neurochemical changes in the amygdala and pyriform cortex. In addition, the adrenoceptor antagonists prazosin (alpha 1) and propranolol (beta) as well as the dopamine receptor antagonist haloperidol had significant but less potent - protective actions upon kainic acid-induced seizures and subsequent neurochemical changes. On the other hand, yohimbine (alpha 2-antagonist) and metergoline (serotonin-antagonist) potentiated the limbic seizure syndrome and no effect was found with atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β‐hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A‐Sepharose (Con A‐Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A‐Sepharose. The adsorption of the DBH to Con A‐Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu2+ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu2+ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc‐ec). This procedure resulted in an approx. 10‐fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co‐factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.

In vivo synthesis of substance P in the corpus striatum of the rat and its transport to the substantia nigra

Incorporation of [35S]methionine into substance P in the striatum of the rat and the subsequent transport of the labelled peptide to the substantia nigra has been demonstrated in vivo. After a 4-h infusion of [35S]methionine into the corpus striatum and an additional interval of 4 h radiolabelled substance P was found in the striatum and the substantia nigra of the animals. The criteria for concluding that the labelled product was substance P were: (a) gel chromatography and subsequent ion exchange chromatography of an acetic acid extract of the infused striatum of the ipsilateral substantia nigra yielded a peak of radioactivity co-eluting with endogenous immunoreactive substance P or a sample of synthetic substance P; (b) the radioactive material from this peak also co-chromatographed with synthetic substance P on high-voltage paper electrophoresis or high-pressure liquid chromatography; and (c) bound specifically to the substance antibody. Intracisternal injection of colchicine (70 microgram, i.c.) completely suppressed the appearance of radiolabelled substance P immunoreactive material in the substantia nigra. The data indicate that synthesis of substance P occurs in nerve cell bodies located in the corpus striatum and that substance P is transported to the substantia nigra by a colchicine sensitive mechanism.

The Topographical Distribution of the Monoaminergic Innervation in the Basal Ganglia of the Human Brain

Publisher Summary Biochemical studies on post-mortem human brain material are fraught with many difficulties and limiting factors including age of the patient, disease state, immediate pre-mortem status and time elapsed between death and freezing of the brain. A largely neglected factor in human brain studies is the possible occurrence of a regionally uneven distribution of neurotransmitters within an individual brain nucleus. In the basal ganglia complex conspicuous changes in neurotransmitter levels have been observed in several neuropsychiatric diseases. Within this complex the striatum comprises a large subcortical mass and receives its main afferents from the cerebral cortex, thalamus, substantia nigra and dorsal raphe nucleus. There is increasing neurochemical evidence of inhomogeneities in the distribution of various neurotransmitter systems in the striatum of various species. The purpose of this chapter is to determine, in the normal human brain, the biochemical distribution pattern of the known monoaminergic aff erents (dopaminergic, noradrenergic, serotonergic) to the various nuclei of the basal ganglia complex.

Capsaicin does not change tissue levels of glutamic acid, its uptake, or release in the rat spinal cord.

Abstract: Capsaicin treatment (50 mglkg, subcutaneous) of newborn rats resulted in a 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high‐affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 μM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.

Changes in histamine in the rat striatum following local injection of kainic acid

Abstract The influence of local administration of kainic acid on the metabolism of histamine in the striatum of the rat has been investigated. Intrastriatal injection of kainic acid (1 μg) resulted in a more than 100% increase of the striatal content of histamine, which persisted from two days after the lesion up to ten weeks. Using two independent methods this accumulation of histamine has been found to be associated with a neuronal compartment of the striatum: (a) the concentration of histamine in synaptosomes prepared from kainic acid lesioned striatum was higher than in synaptosomes derived from the untreated (contralateral) striatum; (b) electrolytic lesions of the medial forebrain bundle, in animals whose striatum had been lesioned with kainic acid, abolished the increase in histamine levels induced by the neurotoxin. Since the rate of disappearance of histamine from the tissue after inhibition of its synthesis by α-fluoromethylhistidine (20 mg/kg, i.p.) was reduced in the lesioned striatum, the kainic acid induced increase in striatal histamine level may be due to a decrease of histamine turnover. Specific binding of [ 3 H]histamine and [ 3 H]cimetidine was measured in the lesioned striatum, revealing a 40% and 20% decrease in binding sites, respectively, when compared with the untreated striatum; the affinity for either ligand was not changed in the lesioned tissue. The data demonstrate considerable neurochemical changes involving the neuronal histamine system in the striatum after destroying the interneurons and output neurons of this region with the convulsive neurotoxin kainic acid.