Development of dopamine-containing neurons and dopamine uptake in embryos of Hirudo medicinalis

The embryonic development of neurons which contain or take up dopamine was studied with glyoxylic acid histofluorescence in Hirudo medicinalis. Beginning at the time of the formation of the tail ganglion, one pair of dopamine-containing neurons was stained per segmental ganglion. The normal outgrowth of the cell bodies into the anterior roots was prevented in isolated and cultured chains of embryonic ganglia. Preincubation of intact embryos in dopamine led to the staining of additional neurons at certain developmental stages. These neurons presumably are the precursors of serotonin-containing cells, which have a temporary capability of taking up and storing dopamine.     

GAS CHROMATOGRAPHIC–MASS SPECTROMETRIC ANALYSIS OF BIOGENIC AMINES IN IDENTIFIED NEURONS AND TISSUES OF HIRUDO MEDICINALIS

—Previous workers have reported that the colossal cells of Retzius in the segmental ganglia of the medicinal leech contain about 2.3 pmol of 5-hydroxytryptamme (5-HT)/cell body. We verify the identify of 5-HT in the Retzius cells by gas chromatography-mass spectrometry and derive concentrations of 1.3–4.1 pmol/neuron by analyses of eight individually dissected Retzius cell bodies. The Retzius cell bodies contain about 30% of the 5-HT in each ganglion. An average of 25 pmol 5-HT/mg tissue, a concentration about 500 times lower than that in the Retzius cell, was found in the fibrous, pigmented tissue surrounding the leech nervous system. We could not detect γ-aminobutyric acid, octopamine, dopamine or norepinephrine in the Retzius cells, in the pigmented tissue, or, with the possible exception of dopamine (±0.4 pmol/ganglion), in whole ganglia. Furthermore, we could not detect 5-HT in pooled samples of 100 non-chromaffin control neurons.     

大鼠脊神经节内交感神经支配的荧光组织化学与HRP示踪观察

本研究应用乙醛酸诱发儿茶酚胺（ＣＡ）荧光技术观察大鼠肾上腺素（ＮＡ）能神经在脊神经节内的分布;并应用ＨＲＰ顺、逆行追踪技术对脊神经节内ＮＡ能神经纤维的起源及其与脊神经节神经元的关系进行了探讨。荧光组织化学观察发现、有些神经节神经元胞体周围分布有带膨体的ＮＡ能神经末梢;有的紧密围绕脊神经节细胞——卫星细胞复合体。颈上交感神经节内注射霍乱毒素Ｂ亚单位结合ＨＲＰ（ＣＢ┐ＨＲＰ）,在同侧Ｃ３～６节段脊神经节内可见标记的点状纤维末梢紧邻于节细胞旁。Ｔ１１～Ｌ２节段脊神经节内注射ＨＲＰ后,在同侧椎旁交感链（Ｔ９～Ｌ１）内可见标记的交感节后神经元胞体。上述实验结果表明,交感节后神经元发出节后纤维可直接到达脊神经节内,与节细胞发生接触。本研究提示、交感神经在脊神经节水平可能参与躯体初级传入信息的调制     

Fluorescent properties of monoamine neurons following glyoxylic acid treatment of intact leech ganglia

Summary The SPG modification (de la Torre and Surgeon 1976) of the glyoxylic acid method for amine condensation is a straightforward procedure which can be used upon intact ganglia from the leech. Intense fluorescence of the neurosomata of identified neurons which contain either indoleamine (serotonin, 5-HT) or catecholamine (CA) is obtained in less than 30 min. The fluorescence of the 5-HT containing neurons is yellow (518–526 nm) and decays more rapidly than the dominant blue emission (478–480 nm) of the CA neurons. Most importantly, the SPG technique greatly enhances the visibility of the axonal processes of neurons which contain both classes of amines over the fluorescence produced by formaldehyde condensation techniques. Both blue and yellow fluorescent varicosities are easily distinguished in the longitudinal connectives and lateral roots of the leech C.N.S. Because of its simplicity and high fluorescence yields, the SPG method for histochemical fluorescence should contribute to investigations of amine functions in invertebrate nervous systems.Supported by NIH research grant No. NS14481-03 and NSF research grant BNS-7915/08     

Involvement of auxin and nitric oxide in plant Cd-stress responses

Cadmium (Cd) toxicity inhibited the seedling growth while inducing the occurrences of lateral roots (LR) and adventitious roots (AR). Further study indicated that auxin and nitric oxide (NO) are involved in the processes. In this study, we chose model plant Arabidopsis thaliana and Cd-hyperaccumulator Solanum nigrum as material to examine the involvement of Cd-induced auxin redistribution in NO accumulation in plants and the effect of NO on Cd accumulation. For this aim, the histochemical staining, NO fluorescence probe (DAF-2DA) detections combined with the pharmacological study were used in this study. By using DR5:GUS staining analysis combined with NO fluorescence probe (DAF-2DA) detection, we found that Cd-induced NO accumulation is at least partly due to auxin redistribution in plants exposure to Cd. Supplementation with SNP donor S-nitrosoglutathione (GSNO) increased the number of LR and AR. In contrast, NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (cPTIO) reversed the effects of NO on modulating root system architecture and Cd accumulation. These results suggest that manipulation of the NO level is an effective approach to improve Cd tolerance in plants by modulating the development of LR and AR, and provide insights into novel strategies for phytoremediation.     

Adrenergic nerves and 5-hydroxytryptamine-containing cells in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus

Summary The adrenergic innervation of the pulmonary vasculature of the file snake Acrochordus granulatus was examined by use of glyoxylic acid-induced fluorescence. Perivascular plexuses of blue-green fluorescent nerves are observed around the common pulmonary artery, the anterior and posterior pulmonary arteries, the arterioles leading to the gas exchange capillaries of the lung, the venules draining the lung, and the anterior and posterior pulmonary veins. Adrenergic nerves are also associated with the visceral smooth muscle of the lung septa and other tissues. Thus, adrenergic control of pulmonary blood flow may occur either at the common pulmonary artery or more regionally within the lung. Regional control of blood flow in the elongate lung of this snake may be important in matching pulmonary perfusion with the distribution of respiratory gas. Glyoxylic acid-histochemistry and immunohistochemistry revealed that populations of cells located in the common pulmonary artery contain the indoleamine 5-hydroxy-tryptamine. Many of the cells are intimately associated with varicose blue-green fluorescent nerves. It is proposed that the 5-hydroxytryptamine-containing cells may be involved in intravascular chemoreception.     

Biogenic monoamines in Hirudo medicinalis

Summary The distribution of certain catecholamines and indoleamines in the ventral nerve cord and the body segments of the medical leech, Hirudo medicinalis, was studied with the fluorescence microscope technique of Falck and Hillarp, with microspectrofluorometry, and with chemical determinations of the amines. The six cells of the segmental ganglia previously shown to be chromaffin were found to contain an amine, most probably 5-hydroxytryptamine. In the two giant cells, the amine was found on the surface of coarse intracellular granules, lying mainly at the cell membrane, and at the nucleus. The two giant cells send their axons to the body muscles, which thus seem to have a 5-hydroxytryptaminergic innervation. The four smaller amine-containing cells of the segmental ganglia send their axons to the neuropil of the ganglion.The only cell type found to contain a catecholamine (probably noradrenaline) was situated in the anterior segmental nerve in the cell cluster anterior of the nephridial duct, one cell in each nerve. The axon of this cell terminates in two or more segmental ganglia; thus these neurons seem to be afferent.This work was supported by grants from the Swedish Natural Science Research Council (project no. 99-35) and the Swedish Medical Research Council (projects no. B 68-12 X-712-03 B and B 68-14 X-56-04 B).     

太湖蓝藻滤液的遗传毒性研究

蓝藻爆发是环境污染引发的重要事件之一,随之产生的蓝藻毒素又直接危及区域水安全.该论文采用蚕豆和大蒜根尖微核试验研究了太湖蓝藻暴发期间蓝藻滤液的遗传毒性.结果表明,同阴性对照相比,所有试验处理对蚕豆根尖细胞微核发生率的影响显著增加;对大蒜根尖细胞微核发生率而言除蓝藻滤液8倍稀释液的影响不显著外,其它水平效应显著高于阴性对照,而且表现出一定的剂量效应.暴发期蓝藻滤液原液对蚕豆根尖细胞微核发生率影响显著高于阳性对照(0.8mg·mL^-1环磷酰胺)的效应,从而说明蓝藻暴发时期蓝藻滤液具有较强的遗传毒性.通过微核试验效果分析,蚕豆作为植物监测系统的敏感性和稳定性都优于大蒜材料.     

Localization and identification of catechol compounds in the ctenophore mnemiopsis leidyi

1. The ctenophore Mnemiopsis leidyi, was examined for monoaminergic nerves by glyoxylic acid-induced fluorescence.2. The meridional canals contained strands of fluorescent cells.3. High pressure liquid chromatography (HPLC), showed the presence of 2,3-dihydroxyphenylalanine (DOPA), 0.25–0.35 pmol/specimen, and 5-S-cysteinylDOPA, 0.3–0.85 pmol/specimen.4. Electronmicroscoplcal examination of tissues showing histofluorescence, revealed large cells (> 20 μm) containing irregular osmium-reducing vesicles, usually 100–500 nm in size. A second cell type, situated by the outside of the canal, contained dense core vesicles, 175–200 nm in diameter.5. It is suggested that the histofluorescence is caused by DOPA and 5-S-cysteinylDOPA. The presence of catecholamines is discussed.     

Adrenergic innervation of the human gall bladder.

Adrenergic innervation of the human gall bladder was studied using two specific fluorescence histochemical methods. Blue-green fluorescing varicose nerves were scarce and mostly followed the course of blood vessels as typical perivascular plexuses. However, some adrenergic nerves not associated with the vessels were occasionally seen, as well as structures suggestive of a pericellular arrangement of varicose adrenergic nerve terminals on non-fluorescing ganglion cells. A few enterochromaffin cells were seen in the epithelial lining, also in the deep invaginations obviously representing the Aschoff-Rokitansky sinuses. Occasionally, small rounded cells with a rounded, relatively large nucleus, and exhibiting a weak yellow-green to blue-green granular cytoplasmic fluorescence, were observed in the wall of the gall bladder. The possible functional and evolutionary significance of these neural and endocrine elements was discussed against the data on physiological and pharmacological studies obtained from the literature. It was concluded that their significance is, in all probability, secondary to the influence of the intestinal polypeptide hormones, vagal innervation and circulating catecholamines upon the normal function of the gall bladder. The glyoxylic acid-induced fluorescence histochemical method was found to be superior to the conventional formaldehyde technique in studies on human tissue.     

Adrenergic innervation of the human gall bladder

Summary Adrenergic innervation of the human gall bladder was studied using two specific fluorescence histochemical methods. Blue-green fluorescing varicose nerves were scarce and mostly followed the course of blood vessels as typical perivascular plexuses. However, some adrenergic nerves not associated with the vessels were occasionally seen, as well as structures suggestive of a pericellular arrangement of varicose adrenergic nerve terminals on non-fluorescing ganglion cells. A few enterochromaffin cells were seen in the epithelial lining, also in the deep invaginations obviously representing the Aschoff-Rokitansky sinuses. Occasionally, small rounded cells with a rounded, relatively large nucleus, and exhibiting a weak yellow-green to blue-green granular cytoplasmic fluorescence, were observed in the wall of the gall bladder. The possible functional and evolutionary significance of these neural and endocrine elements was discussed against the data on physiological and pharmacological studies obtained from the literature. It was concluded that their significance is, in all probability, secondary to the influence of the intestinal polypeptide hormones, vagal innervation and circulating catecholamines upon the normal function of the gall bladder. The glyoxylic acid-induced fluorescence histochemical method was found to be superior to the conventional formaldehyde technique in studies on human tissue.     

Fine structure of the doliolaria larva of the feather star Florometra serratissima (Echinodermata: Crinoidea), with special emphasis on the nervous system

The epidermis of the doliolaria larva of the Florometra serratissima is differentiated into distinct structures including an apical organ, adhesive pit, ganglion, ciliary bands, nerve plexus, and vestibular invagination. All these structures possess unique cell-types, suggesting that they are functionally specialized in the larva, except the vestibular invagination that becomes the postmetamorphic stomodeum. The epidermis also contains yellow cells, amoeboid-like cells, and secretory cells. The enteric sac, hydrocoel, axocoel, and somatocoels have differentiated but are probably not functional in the doliolaria stage. Mesenchymal cells, around the enteric sac and coeloms, appear to be actively secreting the endoskeleton and connective tissue fibers. The nervous system is composed of a nerve plexus, ganglion, and sensory receptor cells in the apical organ. The apical organ is a larval specialization of the anterior end; the ganglion is located in the base of the epidermis at the anterior dorsal end of the larva. The nerve plexus underlies most of the epidermis, although it is more prominent in the anterior region. Here, processes from sensory receptor cells of the apical organ, as well as those from nerve cells, contribute to the plexus. These processes contain one or a combination of organelles including vesicles, vacuoles, microtubules, and mitochondria. The configuration of glyoxylic acid-induced fluorescence, revealing catecholamine activity, correlates to the apical organ, nerve cells, and nerve plexus. Morphological evidence suggests that the nervous system may function in initiation and control of settlement, attachment, and metamorphosis. The crinoid larval nervous system is discussed and compared to that found in other larval echinoderms.     

IDENTIFICATION OF SEROTONIN WITHIN VITAL-STAINED NEURONS FROM LEECH GANGLIA

Abstract— We have measured serotonin (5-HT) within large and small neurosomata which are vitally stained by Neutral Red dye. A micro-radioenzymatic technique which is sensitive to 50fmol of 5-HT was employed on intact ganglia, 75 μm Retzius Cells (RZ) and a 10 μm ventro-lateral cell (VL) taken from the leech Macrobdella decora. The stain does not affect the levels of 5-HT in either ganglia or RZ. The VL cell body contains 5-HT at concentrations of at least 100 m m . Microspectrofluorometry of all the ganglionic neurosomata which fluoresce following the Falck-Hillarp formaldehyde condensation reaction detected rapidly-fading emission peaks of 509–523 nanometers. We conclude that all seven fluorescent neurons in the leech ganglion very probably contain serotonin.     

Endodermally-derived and neural crest-derived differentiation antigens expressed by a human lung tumor.

The plasma membrane antigens of an undifferentiated small cell (oat cell) carcinoma of the lung were studied by the indirect immunofluorescence method on frozen section substrates with a rabbit antiserum prepared to the tumor plasma membrane fraction. After appropriate absorption of the antiserum, at least two differentiation antigens present on the tumor cells but undetectable on normal lung surface or glandular epithelial cells were identified. One antigen(s) was characteristic of certain normal, endodermally-derived epithelial cells of the digestive system, including those of colonic mucosa, hepatic ducts, pancreatic ducts and acini, and islets of Langerhans. The other antigen(s) was characteristic of certain normal, neural crest-derived cells in the peripheral nervous system, including cells in peripheral nerve, dorsal root ganglion, and anterior roots of the spinal cord; parasympathetic ganglion cells in the colon; and small nerves and nerve processes in the lung, colon, and skin. It was concluded that the presence of these differentiation antigens on the tumor cells resulted from the expression of gene products repressed in the normal cell of origin of the tumor.     

Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis

Summary Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis was investigated using indirect immunofluorescence with antibodies against dopamine, GABA, and serotonin, and glyoxylic acid-induced fluorescence of catecholamines. Serotonergic cells first appear in full gastrulae; dopaminergic and GABAergic cells are present in early four-arm plutei. The number of neurons and the complexity of the nervous system increases through development of the pluteus. In the pluteus the dopaminergic component of the nervous system includes a ganglion in the lower lip of the mouth and a pair of ganglia at the base of the post-oral arms which extend axons along the base of the circumoral ciliary band. The distribution of cells visualized by glyoxylic acid-induced fluorescence is similar to that of dopaminergic cells. GABAergic neurons occur in the upper lip and in the wall of the esophagus. Serotonergic neurons are present in the lower lip; the pre-oral hood contains an apical ganglion which extends axons along the base of the epidermis overlying the blastocoel. The dopaminergic and GABAergic components of the nervous system are associated with effectors involved in feeding and swimming. The serotonergic component is not associated with any apparent effectors but may have a role in metamorphosis.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

Thoracic output of crayfish giant fibres

Summary The thoracic homologue of the abdominal segmental giant neurone of crayfish Pacifastacus leniusculus is identified and described. It has a small cell body located in the anterior ventro-lateral quadrant of the ganglion and a large neuropil arborization, with dendrites aligned along the tracts of the giant fibres. The SG axon exits the ganglion within the major root which innervates the leg, usually in the anterior region of this root. Within 1–2 mm of the ganglion the axon terminates in a mass of fine branches, apparently randomly located within the base of the root.The SG receives suprathreshold input from the ipsilateral MG and LG fibres through rectifying electrical synapses. It makes output to FF motor neurones, also through electrical synapses. The SG also makes output to at least one corollary discharge interneurone. The SG receives depolarizing inhibitory synaptic potentials which can prevent its activation by the GFs. Some but not all of these synaptic potentials are common to similar potentials occurring in a large leg promotor motor neurone.Abbreviations AC anterior connective - GF giant fibre - IPSP inhibitory post-synaptic potential - LG lateral giant fibre - MG medial giant fibre - MoG motor giant neurone - PC posterior connective - PMM promotor motor neurone - r1 first root - r3 third root - rAD anterior distal root - rPD posterior distal root - rPM promotor muscle root - SG segmental giant neurone     

ACETYL-CoA SYNTHESIZING ENZYME ACTIVITIES IN SINGLE NERVE CELL BODIES OF RABBIT

Activities of five enzymes (pyruvate dehydrogenase complex; citrate synthase, EC 4.1.3.7; carnitine acetyltransferase, EC 2.3.1.7; acetyl-CoA synthetase, EC 6.2.1.1; and ATP citrate lyase, EC 4.1.3.8) were determined in cell bodies of anterior horn cells and dorsal root ganglion cells from the rabbit. For comparison, molecular layer, granular layer and white matter from rabbit and mouse cerebella and cerebral cortex and striatum from the mouse were analyzed. Samples (3–85 ng dry weight) were assayed in 180 to 370 ml of assay reagents containing CoASH and other substrates in excess. By using ‘CoA cycling’, the assay systems were devised to amplify and measure small amounts of acetyl-CoA formed during the enzyme reactions. Carnitine acetyltransferase was the most active enzyme in single nerve cell bodies and all layer samples, except for rabbit and mouse cerebellar white matter. Citrate synthetase was the lowest in single cell bodies. The activities of carnitine acetyltransferase and acetyl-CoA synthetase (656 and 89.8 mmoles of acetyl-CoA formed/kg of dry weight/h at 38°C) from dorsal root ganglion cells were about 2-fold higher than those from anterior horn cells. The activity of ATP citrate lyase (134mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) from anterior horn cells was approximately twice that from dorsal root ganglion cells. The activity of this enzyme was distributed in a wider range in anterior horn cells than dorsal root ganglion cells. The second highest activity (80.0 mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) of ATP citrate lyase was found in striatum where cholinergic interneurones are abundant. Relatively higher activities of this enzyme were found in cerebellar granular layer and white matter which are known to contain the cholinergic mossy fibers. These results suggested that cholinergic neurones contain higher activity of ATP citrate lyase which is thought to supply acetyl-CoA to choline acetyltransferase (EC 2.3.1.6) as a substrate to form acetylcholine.     

Dopamine and 5-hydroxytryptamine actions on the cardiac ganglion of the lobster Homarus americanus

The cardioexcitor monoamines dopamine (DA) and 5-hydroxytryptamine (5HT) accelerate bursting by isolated cardiac ganglia of the lobster Homarusamericanus most effectively when they act on a region of the ganglionic trunk anterior to the small cells which have been considered the pacemakers of the system. 5HT may exert its acceleratory action by depolarizing cell processes. Neither the somata nor the spike-initiating zones of the small cells have to be directly exposed to 5HT or DA in order for acceleration to occur. When 5HT is applied selectively to the small cells bursts are prolonged, probably as a result of increases in the duration of the endogenous burst-organizing potentials (driver potentials) generated by these neurons. This action on the small cells can lead to prolonged and intensified bursts of the full ganglion during the onset of 5HT action when the whole ganglion is exposed to the monoamine. Neither DA nor 5HT has a direct effect on the characteristics of large cell (motorneuron) driver potentials. Accepted: 3 September 1997     

Time-related changes in the labeling pattern of motor and sensory neurons innervating the gastrocnemius muscle,as revealed by the retrograde transport of the cholera toxin B subunit

Summary Morphological changes in the motor and sensory neurons in the lumbar spinal cord and the dorsal root ganglia were investigated at different survival times following the injection of the B subunit of cholera toxin (CTB) into the medial gastrocnemius muscle. Unconjugated CTB, visualized immunohistochemically, was found to be retrogradely transported through ventral and dorsal roots to motor neurons in the anterior horn, each lamina in the posterior horn, and ganglion cells in the dorsal root ganglia at L₃–L₆. The largest numbers of labeled motor neurons and ganglion cells were observed 72 h after the injection of CTB. Thereafter, labeled ganglion cells were significantly decreased in number, whereas the amount of labeled motor neurons showed a slight reduction. Motor neurons had extensive dendritic trees filled with CTB, reaching lamina VII and even the pia mater of the lateral funiculus. Labeling was also seen in the posterior horn, but the central and medial parts of laminae II and III had the most extensively labeled varicose fibers, the origin of which was the dorsal root ganglion cells. The results indicate that CTB is taken up by nerve terminals and can serve as a sensitive retrogradely transported marker for identifying neurons that innervate a specific muscle.