Fekete, Éva

English

Fekete Éva

University of Szeged

Acta Biol. Szeged. 32. pp. 61-120 (1986) E F F E C T O F 6 - H Y D R O X Y - D O P A M I N E O N T H E F L U O R O G E N I C M O N O A M I N E C O N T A I N I N G N E R V E E L E M E N T S I N T H E F O R E - G U T M U S C U L A T U R E O F HELIX POM ATI A É . F E K E T E (Received June 18,1985) Abstract Using glyoxylic acid induced fluorescence method an intensive green fluorescence characteristic to catecholamines was observed on nerve fibres with varicosities, nerve bundles and perykaryons on wholemounts stretch preparates o f snail's gut. On the effect of 6-hydroxy-dopamine ( 6 - O H D A ) administered intracardially (2.5 mg/animal) the amount of fluorescent nerve elements were significantly reduced. 48 hours after the injection o f 6 - O H D A practically no fluorescent fibres were seen. However several cell bodies showing intensive green fluorescence were detectable during the 72 hours of experimental periode. Key words-, fluorescence, catecholamines. 6-hydroxy-dopamine. snail Introduction Glyoxylic acid (GA) has been found to be suitable for the histochemical demonst ra t ions of biogenic monoamines (AXELSSON et al. 1 9 7 3 ) . It was shown that G A induced fluorescence ( G I F ) is specially very sensitive for the demons t ra t ion of catecholamines in peripheral adrenergic nerves ( F U R N E S S and C O S T A , 1 9 7 4 ) . The use of a different combina t ion of G I F techniques the wholemounts preparates of intact ganglia ( M A R S D E N and K E R K U T , 1 9 7 0 ) and a number of peripheral tissues such as the heart , prosta te gland, salivary gland (BARBER, 1983) provided valuable inform-ation on the localization of nerves, cell bodies and axons having associated biogenic monoamines . By the use of sucrose-phosphate-glyoxylic acid (SPG) technique (DE LA T O R R E and S U R G E O N , 1 9 7 6 ) on who lemoun t stretch prepara tes of snail 's gut, the distr ibution of fluorogenic monoamines containing nerve elements were followed in the whole length of snail a l imentary tract ( F E K E T E , 1 9 8 4 ) , and three segments were revealed concerning the a m o u n t o f fluorescent nerve elements and also the fluorescence intensity. It is likely f rom pharmacological studies (TRIMBLE et al., 1984) that a m o n g catecholamines in Mollusc only dopamine acts as neurotransmit ter . However preliminary biochemical (HALASY et al. 1985) and some physiological ( K A Z A C H E N K O et al. 1 9 7 8 ) investigations demonst ra ted that both adrenal ine and noradrenal ine a re present in snai l ' s gut and may act as neurot ransmit ters o r neuromodula tors . However noradrenergic axons have of ten been identified by their ability to take up noradrenal ine analogues, such as 5-, o r 6 -hydroxydopamine (COSTA et al. 1 9 7 6 , D R E Y F U S et al. 1 9 7 7 , F U R N E S S and C O S T A , 1 9 7 4 ) , the l i terature concerning 6 2 Ê . F E K E T Ï the selectivity and the mode of action of 6 - O H D A is very contraversary. ft is well documented that 6 - O H D A causes degeneration in the mammal ian adrenergically innervated organs ( P O R T E R et al. 1 9 6 3 , T H O E N E N and T R A N Z E R . 1 9 6 8 ) , due to degeneration of their noradrenal ine terminals. The typical degenerative changes seen in mammalian adrenergic neurons after 6 - O H D A treatment could not be detected in Octopus peripheral nerves ( M A R T I N and B A R L O W , 1 9 7 5 ) . An investigation on different Mussel neurons (ELEKES et al. 1977) showed that different part of mussel 's neurons have different sensitivity to 6 - O H D A . In the central nervous system of Planorbis corneus a giant dopaminergic cell ( G D C ) was identified, in which 6 - O H D A caused an increased catecholamine-specific fluorescence ( B E R R Y et al. 1 9 7 4 ) . To clarify the effect of 6 - O H D A in the enteric nerves of Helix pomalia we followed the nerve elements surrounding blood vessels in the fore-gut of the snail and evaluated the differences in the fluorescence intensity and the number of fluorescent nerve elements before and after intracardially administered 6 - O H D A . Materials and Methods Helix pomalia used in these studies were collected over the period of April to October. Seasonal changes in the intensity of histofluorescence were not observed during this period For the histochemical detection of biogenic monoamines the SPG method (DE LA TORRE and SURGEON. 1976) was applied to wholemount stretch preparates of snail's gut. The digestive tract were quickly dissected and incubated in a reaction mixture containing 6.8 g sucrose. 3.2 g K H 2 P O * and I g glyoxylic acid ( G A ) in 100 ml o f distilled water, at 4 C for IS minutes. The muscular layer o f the gut wall was then stretched on microscope slides, blotted with blotting paper and dried under coo l air for about 1/2 hour. Finally the specimens were placed in an oven at 95 °C for 4 min. and mounted with liquid paraffin. The preparations were viewed through a Leitz Ortoplan microscope equipped with indirect illumination and an HBO SOW super pressure mercury lamp. A n E - 3 filter block was used to observe induced fluorescence. Black and white photographs were taken on F O R T E - P A N 400 film. T o fo l low the efTect o f 6 - O H D A . 6 - O H D A HC1 (Sigma) was administered intracardially. The 6 - O H D A was dissolved in 0.1 ml of saline (NaCI 0.65%; KCI 0 .015%) and was administered as one dose o f 2.S mg animal The animals were killed and processed for histochemical detection o f fluorogenic m o n o a m i n e s after 2. 24. 48 and 72 h o f injection. Results In the untreated controls (Fig. 1-3., Fig.8.) the fluorescence was bright and well localized in the fore-gut. Fluorescent fibres showed a characteristic arrangement around blood vessels and these part of the specimens were the most comparable in the whole length of the fore-gut. Fluorescent fibres occurred singly or in bundles and were concentrated in several areas (Fig.I . , 8.). Most of the single fibres were varicous (Fig. 1-2., arrowheads) . Fibres running along the vessels divided a t some places and made connections with neighboring nerve trunks. Single cells with bright fluorescence were also seen. Most of them were in close association with nerve t runks (Fig.I . , 2. arrows). Processes of these cells could be followed on the surface of the EFFECT OF 6-HYDROXY-DOPAMINE ON THE FLUOROGENIC MONOAMINE 6 3 Fig. I. Glyoxyl ic acid-induced fluorescence in whole -mount stretch prepárales in the snail's fore-gut musculature. Arrows indicates cell bodies, arrow-heads varicosities. Scale bar: 50 (im Fig. 2. Fluorescent cell bodie in the fore-gut (arrow) being in close connect ion to nerve trunks. Scale bar: 50 urn Fig. 3. Single cells o f different sizes and shapes in the fore-gut with no visible connect ion to the bundles. In the ne ighborhood of these cells randomly oriented varicous fibres (arrows-head) are running. Scale bar: 50 jim 6 4 É F F K E T E 1 0 / -u * v • o • a 4 + y 6 — 7 Fig. 4. Fluorescent nerve cell bodie after 24 hours o f 6 - O H D A treatment in snail's fore-gut. The cell processes show varicosities (arrow-head). Scale bar: 50 urn Fig. 5. Varicouse fibres are concentrated at several areas after 48 hours o f 6 - O H D A treatment. Intensively fluorescent cell bodie (arrow) running parallel to the fibres. Scale bar: 50 urn Fig. 6. After 72 hours of 6 - O H D A treatment varicous fibres (arrow-heads) and intensively fluorescent cell bodie (arrow) still can be seen at several areas in the fore-gut. Scale bar: 50 pm Fig. 7. After 72 hours o f 6 - O H D A treatment some o f the remaining cells (arrow) s h o w much reduced fluorescence Scale bar: 50 jim E F F E C T OF 6 - H Y D R O X Y - D O P A M I N E O N THE F L U O R O G E N I C MONOAMINE 6 5 N N BV I ig. 8. Whole mount of fore-gut showing the characteristic pattern of cathecolaminc containing nerve fibres a long the blood vessels (BV) before 6 - O H D A treatment. Scale bar 50 Mm 66 Ê . F E K E T Ï vessels. Single cells showing a less intense fluorescence appeared a little farther with no visible connection to the nerve bundles (Fig.3.). The G I F in the perykarya of these cells was usually restricted to the cytoplasm. Most of the speciemens prepared from 6 - O H D A treated animals could be separated f rom the controls on a single blind bases (Fig. 8.. 9.). However 2 hours after 6 - O H D A treatment there was no visible changes neither in the intensity nor in the amount of fluorescent nerve elements. 24 hours after the d rug treatment most of the fluorescence disappeared (Fig. 4-7. , 9.). The rest of the fibres showed an irregular distribution and the fluorescence intensity was reduced comparing to the untreated samples. All of the remaining fibres were short and varicous in appearance (Fig. 4-6. , arrowheads). After additional 24 hours practically there was no change in the G I F (Fig. 6.). While 72 hours af ter the 6 - O H D A treatment a further decrease in the fluorescent nerve elements and fluorescence intensity was observable (Fig. 7., 9.). In these specimens a very few faintly fluorescent fibres were only seen on the surface of the vessels (Fig. 9.). Some of the cells with a very intense G I F having close connection to the vessels „survived" the 6 - O H D A treatment and revealed a very intense catecholamine fluorescence within the whole experimental periode (Fig. 4 6.). In some cells the fluorescence intensity was dropped by 72 hours of t reatment (Fig. 7.). No sign of regeneration in the fluorescent profiles was seen dur ing the experimental periode. Discussion The use of G I F technique on wholemount stretch prepárales of gut musculature in Helix pomaiia allowed us to map the distribution of aminergic nerve fibres and related cells in different segments of alimentary tract (FF.KETE, 1 9 8 4 ) . Since the fluorescent network was most pronounced in the fore-gut (FEKETE. 1984) and was most comparable around the blood vessels this segment of the gut was used to follow the effect of 6 - O H D A . The main finding of the present work was that 6 - O H D A treatment resulted a significant decrease in the fluorescent intensity and the number of fluorescent nerve elements. Al though a characteristic time-pattern appeared in the degeneration processe of CA nerves within the experimental period. Some of the cells being in close connection to vessels were not sensitive to drug treatment at all. The processes of these cells could be followed along the blood vessels and also several thin, varicous fibres were seen neighboring the cells. From these findings we assumed that different cells and connected fibres have different sensitivity to the neurotoxin 6 -OHDA which is known to be specific for noradrenergic nerve elements ( T H O E N E N and T R A N Z E R , 1 9 8 3 ) . We have several alternative explanations for this phenomenom ( H Ó K F E L T and U N G E R S T E D T , 1 9 7 3 ; F U R N E S S and C O S T A , 1 9 8 0 ) . These cells may not contain noradrenalin but some other kind of monoamines which are not sensitive to the drug. This alternative is supported by data (BERRY et al. 1974) which showed up increased catecholamine-specific fluorescence in identified dopa-mine-containing cells after 6 - O H D A treatment. We consider a possible higher EFFECT OF 6-HYDROXY-DOPAMINE ON THE FLUOROGENIC MONOAMINE 6 7 catecholamine level of these cells as an other explanat ion. This can cause a delayed exchange rate between the toxin and the fluorogen amine. This al ternative is partly supported by da t a f rom the literature ( L L E W E L L Y N et al. 1 9 8 1 ) , and also favored by our finding a f te r 72 hours of 6 - O H D A accumulat ion. Some of the cells showed very intense fluorescence a f te r 48 hours o f t reatment but lost fluorescence intensity by 72 hours of t reatment . The third possibility is that the selective amine pumpe present on adrenergic nerves and having an active role in monoamine and their an t imetab-olites uptake is missing f rom these cells. Biochemical and ul t rastructural investigations are in progress to decide the validity of these possible explanat ions . References AXELSSON. S . . BJORKLUND. A . . FALCK, B . , LINDVALL, O . and SVFNSSON. L A . (1973): Glyoxylic acid condensation: a new fluorescence method for the histochemical demonstration o f biogenic monoamines. — Acta Physiol Scand 87. 57 -62 . BARBER. A . (1983): A monoamine-detecting histofluorescencc technique for use on wholemounts of molluscan tissues. — Neurosci Methods 8. 171-175. BERRY. M . . PENTREATN. V . . TURNER. J. and COTTRELI. G . (1974): Effects of 6-hydroxydopamine on an identified dopamine containing neuron in the central nervous system of Planorhis corneus. — Brain Res 76. 309-324. COSTA. M . . RUSH, R . , FURNESS. J. and GEFFEN, L. 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MARTIN. R . and BARIX>W. J (1975): Muscle and gland cell degeneration in the Ottopus posterior salivary gland after 6 - O H D A administration. — J.UItrastruc.Res. 52. 167-178. 6 8 E F E K I T> PORTER. C . . TOTARO. J and STONE. C . (1963): F.ITccl o f 6 - h y d r o x y - d o p a m i n e and s o m e other c o m p o u n d s o n the concentrat ion o f norepinephrin in the heart o f mice J .Pharmacol exp THCR 140. 308 316. THOENEN. H and TRAN7.FR. J (1968) Chemical s y m p a t c c t o m y by selective destruct ion o f adrenergic nerve endings with 6 - h y d r o x y d o p a m i n e Ann Rev .Pharmaco l . 13. 169 DT l a T o r r e . J . and S u r g e o n . J ( 1 9 7 6 ) A methodo log ica l approach to rapid and sensitive m o n o a m i n e histof luoresccncc using a modif ied g iyoxyl ic acid technique: the S P G method His tochemistry 49. 8 1 - 9 3 . TRANZER. J and THOENEN. H (1967): Electron-microscopic local izat ion o f S - h y d r o x y d o p a m i n e a new-false sympatet ic transmitter. — Expericntia 23. 7 4 3 - 7 4 5 TRIMBLE. D BARKER. D and B I LI.ARD. B (1984): D o p a m i n e in mol luscan nervous system. Synthes is and fluorescence histochemistry . — J Ncurobyol . IS. 2 7 - 3 6 Address of the author: E . F e k e t e Department o f Z o o l o g y Attila JoTsef Univers i ty H - 6 7 2 2 Szeged. P.O. Box 659 Hungary 

Effect of 6-hydroxy-dopamine on the fluorogenic monoamine containing nerve elements in the fore-gut musculature of Helix pomatia

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Effect of 6-hydroxy-dopamine on the fluorogenic monoamine containing nerve elements in the fore-gut musculature of Helix pomatia

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