Nikolai Klemm

Dopamine receptor binding in vivo: the feasibility of autoradiographic studies.

Abstract Several minutes after the injection of small quantities of 3 H-spiroperidol into the tail vein of the rat, radioactivity in the brain exhibited the characteristics of a major association with dopamine receptors. These characteristics included: a regional distribution paralleling that of dopamine receptors; saturability; an appropriate pharmacology in that only dopaminergic drugs blocked the accumulation; lack of metabolism of 3 H-spiroperidol. Autoradiographs revealed a great density of 3 H-spiroperidol binding sites in the neuropil of the striatum, nucleus accumbens and olfactory tubercle, all areas receiving a dopaminergic input. Other areas without such an input exhibited background levels of autoradiographic grains.

POST‐MORTEM CHANGES IN HIGH AFFINITY CHOLINE UPTAKE

Abstract— When animals were killed by decapitation and the heads kept refrigerated at 2–4°C. high affinity choline uptake was maintained up to 3 days post‐mortem. At 5 days post‐mortem, there was a significant reduction in uptake. In tissues kept at 2–4°C for 1 day, the ionic dependence, drug sensitivity and kinetic parameters of uptake were identical to that of control tissues. At 3 days post‐mortem, intact synaptosomal profiles, although with features characteristic of degenerating neuronal tissues, were observed in electronmicroscopic studies. In tissues maintained at room temperature, however, the uptake activity was nearly completely gone by 1 day. It is concluded that high affinity choline uptake is maintained for days, a surprisingly long time, in tissues kept in the cold immediately after death.

Neuroleptic and dopamine receptors: autoradiographic localization of [3H]spiperone in rat brain.

Rats were administered [3H]spiperone (SP: spiroperidol) by tail vein injection and 2 h later the brain was processed for light microscopic autoradiography. High densities of autoradiographic grains were found in all areas known to have a dopaminergic innervation, including the olfactory tubercles, nucleus accumbens, nucleus caudate-putamen, lateral septum, zona incerta, nucleus subthalamicus, arcuate nucleus, nucleus of the central amygdala, areas in the ventral tegmentum and the claustrum. There were also increased autoradiographic grain densities in other areas such as the midbrain and the frontal cortex indicating that binding occurred to other neurotransmitter receptors besides dopamine receptors. These studies delineate with a high resolution at an anatomical level the major binding sites for neuroleptic drugs in the forebrain. They suggest which areas of the brain are the most involved in neuroleptic drug action and they add further evidence that important regions are those receiving a dense dopaminergic innervation.

Detection of dopamine, noradrenaline and 5-hydroxytryptamine in the cerebral ganglion of the desert locust,Schistocerca gregaria forsk (insecta: orthoptera)

Abstract In this study, the locust cerebral ganglion was shown to contain intraneuronal dopamine and noradrenaline, with a predominance of dopamine. The neuropil structures contained mostly one of these catecholamines, but a possible mixed intraneuronal appearance could not be excluded. 5-Hydroxytryptamine-containing cell bodies were found in the optical lobe. After incubation with nialamide, a group of cell bodies in the pars intercerebralis exhibited a fluorescence with an excitation maximum typical for 5-hydroxytryptamine but with an atypical emission maximum. This fluorescence might have come from a mixture of two amines, from precursors or metabolites of 5-hydroxytryptamine, or from an additional presence of accumulated catecholamines. Chemical analysis confirmed the presence of dopamine, noradrenaline and 5-hydroxytryptamine.

Monoamine-containing neurons in the optic ganglia of crustaceans and insects

SummaryWith the fluorescence method of Falck and Hillarp, the presence and localization of monoaminergic neurons in the optic ganglia of several crustaceans and insects have been investigated. It was found that in both classes the monoaminergic terminals, when present, appeared (especially in the medullae externa and interna of the crustaceans and the medulla of the insects) in strata specific for each species. So far, the only monoamine (visualized by this technique) present in the crustacean optic ganglia is dopamine, whereas in the Insecta, the catecholamines dopamine and noradrenaline, and the indolamine, 5-hydroxytryptamine, are found in the optic lobe. But in the Insecta, different species show different content of these amines.

Phylogenetic distribution of [3H]kainic acid receptor binding sites in neuronal tissue

The phylogenetic distribution of specific binding sites for kainic acid was determined in 14 species including invertebrates and vertebrates. The highest level of binding was observed in brains of the frog (Xenopus laevis), followed by the spiny dogfish (Heterodontus francisci), the goldfish (Carasius auratus) and the chick (Gallus domesticus). Although significant specific binding was noted in some of the lowest forms tested (e.g. Hydra littoralis), this was not a consistent observation in the invertebrates. In most cases, specific binding to both high and low affinity sites was detected; notable exceptions were the cockroach brain (Periplaneta americana), which had negligible high affinity binding, and the crayfish brain (Procambarus) which had negligible low affinity binding. In the spiny dogfish, the smooth dogfish and the chick, the highest level of binding occurred in cerebellum with less in the forebrain and the least in the medulla; in the mammalian species, the highest level of binding occurred in the forebrain structures with less in the cerebellum and least in the medulla. Eadie plots of the saturation isotherms for [3H]kainic acid revealed similar kinetics of binding for frog whole brain, rat forebrain and human parietal cortex with two apparent populations of binding sites: KD1 = 25--50 nM and KD2 = 3--14 nM. While binding in the spiny dogfish forebrain and human caudate nucleus occurred exclusively at a high affinity component, the cerebella of chick, rat and man exhibited only a low affinity binding site. In the 3 species studied most extensively, frog, rat and man, unlabeled kainic acid was the most potent inhibitor of the specific binding of [3H]-kainic acid. L-Glutamic acid was 20--20-fold less potent than kainic acid, and D-glutamic acid was 4--2500-fold less potent than its L-isomer. Reduction of the isopropylene side chain of kainic acid to form dihydrokainic acid decreased the affinity of the derivative 115--30,000-fold. Hill coefficients derived from these displacement curves were 1.0 for unlabeled kainic acid but approximately 0.5 for L- and D-glutamic acids and dihydrokainic acid, which is compatible with negative cooperativity. In summary, these studies demonstrated a widespread distribution throughout the animal kingdom of specific binding sites for kainic acid in neural tissue; the characteristics of these receptor sites are remarkably similar from primitive vertebrates to man.

Origin, destination and mapping of tritocerebral neurons of locust

SummaryThe connectivities of the tritocerebrum of locust (Locusta migratoria L., Schistocerca gregaria (Forsk.)) were studied histologically and by means of cobalt chloride infusion. Its neuropil consists partly of fibers which traverse the tritocerebrum and areas consisting of neuropilar agglomerizations (“glomeruli”). The following direct connections between the tritocerebrum and other regions were observed: connections to 1) dorsal and lateral brain regions (mushroom body, optic lobe), 2) the ventral nerve cord, 3) the stomatogastric nervous system (here the protocerebrum and the subesophageal ganglion are also involved in these connections), 4) the retro-cerebral glands (corpora cardiaca, corpora allata), and 5) muscles of the foregut.

Monoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta) Teil II

SummaryThe distribution of monoamine-containing structures in the central nervous system of Trichoptera has been investigated with the histochemical fluorescence method of Falck and Hillarp (cf. also Klemm, 1968). In the protocerebrum, four groups of catecholamine containing perikarya can be distinguished. Another group of irregularly appearing cell bodies is situated in the optical lobe. Fluorescent varicosities occur scattered throughout the cerebral ganglion, being concentrated in the following neuropile areas: In the medulla, the lobula, the corpus centrale, the noduli, the corpora ventralia, and the α- and β-lobes, as well as in the lobus communis where the α- and β-lobes join the non-fluorescent pedunculus. In the lamina, the accessory medulla, the pons cerebralis, the tuberculum opticum, the tractus olfactorio-globularis, the pedunculus including its glomerular region, and the globuli-cells, no catecholamines are detectable. There is a sharp borderline between the monoamine-containing lobus communis and the non-fluorescent pedunculus. Fluorescent varicose fibres and single fluorescent perikarya occur in the Deuto- and Tritocerebrum. Two fluorescent tracts can be distinguished in the cerebral ganglion: 1. Stratum caudale. 2. Tractus ventralis. The pars anterior and pars posterior of the tractus ventralis begin in the dorsal fluorescent neuropile of the cerebral ganglion and join underneath the central body into their pars communis. From this, it can be traced as tractus ventralis into the tritocerebrum. The possible homologies of these tracts are discussed. Similar to the ganglion suboesophageale, the three thoracal and the five separate abdominal ganglia contain two paires of fluorescent cell bodies. Microspectrofluorometrically a content of dopamine in these cells could be established. One pair lies caudoventrally. The two other cell bodies are situated dorso-medially to dorso-caudally; their position varies notably, especially in the abdominal ganglia. The cell process and its arborisations are described. Occasionally a second dorsal cellpair could be observed. The last ganglion of the abdominal chain is composed of at least two gangliomeres with a reduced number of fluorescent perikarya. The neuropile of the thoracal- and abdominal ganglia is penetrated by monoamine-containing fibres, with a predominance in the dorsal half. In the ganglia, the fluorescent varicose fibres are mainly oriented dorso-ventrally in the lateral part and longitudinally in the medial part, where they branch and continue into the connectives. In this way, the catecholamine-containing neuropiles of adjacent ganglia are connected to each other.ZusammenfassungMit Hilfe der fluoreszenzmikroskopischen Methode nach Falck und Hillarp wurden die monoaminhaltigen Strukturen im Zentralnervensystem einiger Trichopteren untersucht (vgl. Klemm, 1968). Im Protocerebrum können vier Gruppen von katecholamin-haltigen Zellkörpern unterschieden werden. Eine weitere unregelmäßig darstellbare Gruppe von globulösen Perikarya liegt im Lobus opticus. Fluoreszierende Varikositäten durchsetzen locker das Cerebralganglion und sind in folgenden Neuropilstrukturen konzentriert: Medulla, Lobula, Corpus centrale, Nodulus, Corpus ventrale, α- und β-Lobus und Lobus communis. Letzterer verbindet den α- und den β-Lobus mit dem nicht fluoreszierenden Pedunculus. In der Lamina, in der accessorischen Medulla, in der Pons cerebralis, im Tuberculum opticum, im Tractus olfactorio-globularis, im Pedunculus, im Stielglomerulus und in den Globulizellen ließen sich keine Katecholamine nachweisen. Zwischen dem monoaminhaltigen Lobus communis und dem nicht fluoreszierenden Pedunculus besteht eine scharfe Grenze. Fluoreszierende variköse Fasern und einzelne fluoreszierende Perikarya befinden sich im Deuto- und Tritocerebrum. Zwei fluoreszierende Bahnen können im Cerebralganglion unterschieden werden: 1. Stratum caudale, 2. Tractus ventralis. Letzterer beginnt mit seiner Pars anterior und posterior im dorsalen fluoreszierenden Neuropil des Protocerebrum. Die beiden Teile laufen frontal und caudal um den Zentralkörper herum und vereinigen sich unterhalb des Zentralkörpers in der Pars communis. Von hier aus zieht der paarige Tractus ventralis bis in das Tritocerebrum. Die Frage einer Homologisierung dieser Bahnen wird diskutiert.Ähnlich wie im Ganglion suboesophageale sind in den drei thorakalen und fünf separaten abdominalen Ganglien zwei Paar fluoreszierender Zellkörper vorhanden, in denen Dopamin mikrospektrofluorimetrisch festgestellt werden konnte. Ein Paar dieser Zellen liegt caudoventral, das andere dorso-median bis dorso-caudal. Ihre Zellfortsätze und Abzweigungen werden beschrieben. In einzelnen Fällen konnte noch ein zweites dorsales Paar fluoreszierender Perikarya sichtbar gemacht werden. Das letzte Ganglion der Bauchkette setzt sich aus zwei bis drei Gangliomeren zusammen. Die Anzahl ihrer fluoreszierenden Perikarya ist reduziert. Das Neuropil der Ganglien im Bauchmark ist von monoaminhaltigen Fasern durchsetzt, wobei sich in der dorsalen Hälfte mehr fluoreszierendes Neuropil befindet als in der ventralen. Lateral in den Ganglien sind die monoaminhaltigen varikösen Fasern vorwiegend dorsoventrad angeordnet. Im medianen Teil laufen sie in Längsrichtung, verzweigen sich und setzen sich in die Konnektive fort und verbinden die katecholaminhaltigen Neuropilbereiche der einzelnen Ganglien miteinander.Mit Hilfe der fluoreszenzmikroskopischen Methode nach Falck und Hillarp wurden die monoaminhaltigen Strukturen im Zentralnervensystem einiger Trichopteren untersucht (vgl. Klemm, 1968). Im Protocerebrum konnen vier Gruppen von katecholamin-haltigen Zellkorpern unterschieden werden. Eine weitere unregelmasig darstellbare Gruppe von globulosen Perikarya liegt im Lobus opticus. Fluoreszierende Varikositaten durchsetzen locker das Cerebralganglion und sind in folgenden Neuropilstrukturen konzentriert: Medulla, Lobula, Corpus centrale, Nodulus, Corpus ventrale, α- und β-Lobus und Lobus communis. Letzterer verbindet den α- und den β-Lobus mit dem nicht fluoreszierenden Pedunculus. In der Lamina, in der accessorischen Medulla, in der Pons cerebralis, im Tuberculum opticum, im Tractus olfactorio-globularis, im Pedunculus, im Stielglomerulus und in den Globulizellen liesen sich keine Katecholamine nachweisen. Zwischen dem monoaminhaltigen Lobus communis und dem nicht fluoreszierenden Pedunculus besteht eine scharfe Grenze. Fluoreszierende varikose Fasern und einzelne fluoreszierende Perikarya befinden sich im Deuto- und Tritocerebrum. Zwei fluoreszierende Bahnen konnen im Cerebralganglion unterschieden werden: 1. Stratum caudale, 2. Tractus ventralis. Letzterer beginnt mit seiner Pars anterior und posterior im dorsalen fluoreszierenden Neuropil des Protocerebrum. Die beiden Teile laufen frontal und caudal um den Zentralkorper herum und vereinigen sich unterhalb des Zentralkorpers in der Pars communis. Von hier aus zieht der paarige Tractus ventralis bis in das Tritocerebrum. Die Frage einer Homologisierung dieser Bahnen wird diskutiert.

Monoamine-containing neurons and their projections in the brain (supraoesophageal ganglion) of cockroaches

SummaryFluorogenic monoamines were studied in the brain of three cockroach species by use of aldehyde-fluorescence techniques. All three optic ganglia contain fluorogenic monoamines. The lamina contains fibres with an indolylalkylamine-fluorophore. The medulla is innervated by local CA neurons which contribute to four fluorescent strata. The lobula receives both CA- and 5-HT-fibres, predominantly of central origin. CA occur in almost all areas of the brain. The areas are interconnected by a CA-fibre system. All parts of the mushroom body are innervated by CA-fibres from the surrounding neuropil. The CA innervation in the mushroom body divides it into a fronto-ventral part (α-lobe, β-lobe, anterio-ventral peduncle) and a dorso-caudal part (caudo-dorsal peduncle, calices) leaving a fluorescence-free central part of the peduncle in between. CA-fibres run between the mushroom bodies of both hemispheres and also between the mushroom body and the lobula. The central body complex contains CA. The pons aggregates indolylalkylamine-containing fibres. The olfactory glomeruli are surrounded by CA-fibres originating from deutocerebral cell bodies. CA-fibres are further linked to the protocerebral neuropil. CA-fibre tracts pass from the brain to the suboesophageal ganglion and the stomatogastric nervous system. The cell bodies of the frontal ganglion are of indolylalkylamine type. Non-fluorescent neuropils (n. ocellaris, tractus olfactorio-globularis, lobus glomerulatus) are innervated by the CA-fibre system.

Histochemical Demonstration of Biogenic Monoamines (Falck-Hillarp Method) in the Insect Nervous System

The biogenic monoamines (β-arylethy lamines and β-arylethanolamines) dopamine (DA),1 noradrenaline (NA), adrenaline (A), and 5-hydroxytryp-tamine (5-HT) are considered to be neuronal transmitter substances in vertebrates (Carlsson, 1974; Axelrod, 1975) and invertebrates (Sak-harova, 1970; Murdock, 1971; Welsh, 1972; Gerschenfeld, 1973; Kerkut, 1973; Klemm, 1976). They were found to be stored mainly in vesicles (Aden et al., 1969; Smith, 1972). Chemical and histochemical analyses for biogenic monoamine content have been performed on insect central nervous systems (CNS) (Frontali, 1968; Klemm, 1968a, 1971a, 1974, 1976; Bjorklund et al., 1970; Elofsson and Klemm, 1972; Klemm and Bjorklund, 1971; Klemm and Axelsson, 1973; Ramade and L’Hermite, 1971; Musko et al., 1973; Robertson, 1976) and stomatogastric nervous systems (Klemm, 1968b, 1971b, 1978; Chanussot et al., 1969; Chanussot and Pentreath, 1973; Gersch et al., 1974), on nerve fibers innervating visceral muscles (Klemm, 1972, 1978) and salivary gland (Klemm, 1972; Bland et al., 1973; Fry et al., 1974; Robertson, 1975), and in the corpora cardiaca (Klemm, 1971b, 1972, 1976; Klemm and Falck, 1978; Lafon-Cazal and Aurluison, 1976; Gersch et al. 1974).