The occurrence of monoamines in Planorbis corneus: A fluorescence microscopic and microspectrometric study

Comp. gen. Pharmac., I97o, x, i o I - I I 6

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THE O C C U R R E N C E OF M O N O A M I N E S IN PLANORBIS

CORNEUS: A F L U O R E S C E N C E

MICROSCOPIC

MICROSPECTROMETRIC

AND

STUDY

C. M A R S D E N * AND G. A. K E R K U T Department of Physiology and Biochemistry, University of Southampton

(Received io Oct., i969) ABSTRACT i. The distribution and nature of the monoamines in the brain and oesophagus of Planorbis corneus were investigated using fluorescence microscopy and microspectrometry and spectrophotofluorimetric estimations. 2. Certain neurones in the brain contain dopamine and others 5-HT. There is one giant neurone in the right pedal ganglion that contains dopamine. This is in contrast to gastropod molluscs previously studied in which dopamine was confined to very small neurones. 3- The central neuropil of the ganglia contains nerve-fibres and varicosities that show a green or yellow fluorescence. 4. In the oesophageal wall there are small green fluorescing ceils with smooth processes. In the oesophageal musculature there are yellow fluorescent dots. 5. Reserpine depletes the specific fluorescence. Nialamide increases the number of yellow fluorescent varicosities but has no apparent effect on the green fluorescence. Tranylcypromine has no effect on the specific fluorescence.

SINCE the development of a histochemical method for the cellular localization of monoamines (Falk, Hillarp, Thieme, and Torp, I962 ) the technique has been employed in the study of monoaminergic systems in the invertebrates, in particular the annelids (Clark, I966; Rude, I966 , I969; Kerkut, Sedden, and Walker, 1967 a ; Myhrberg, 1967; Ehinger, Falk, and Myhrberg, 1968; Marsden and Kerkut, i969b ) and the molluscs (Dahl, Falk, von Mecklenberg, Myhrberg, and Rosengren, i966 ; Kerkut and others, I967b; Sakharov and Zs-Nagy, I967; Zs-Nagy; I967; Sedden, Kerkut, and Walker, i968; Sweeney, i968 ). There is now some evidence that dopamine (3-hydroxytyramine) and 5-HT (5-hydroxytryptamine) function as chemical transmitters in the central nervous system of certain molluscs. For instance, dopamine and

* Present address: Department of Pharmacology, University of Bergen, Norway.

5-HT are present in the brain of Helix aspersa (Kerkut and Cottrell, 1963 ; Kerkut, Sedden, and Walker, i966 ) where they are localized within neurones (Kerkut and others, I967b ). When applied to the isolated snail brain preparation, dopamine inhibits the spontaneous firing of certain nettrones whilst 5-HT excites certain neurones (Kerkut and Walker, I96I, I962 ). The similarity of the ionic mechanisms and the dopamine response led Kerkut, Horn, and Walker (I969) to postulate that dopamine was the transmitter involved in the inhibition of long duration response of the ' big D cell ' of Helix aspersa. Gerschenfeld and Stefani (1966) working on the related species Cryptomphallus aspersa concluded that 5-HT was the transmitter involved in the slow excitatory response of neurones showing inhibition of long duration. Certain problems have been encountered in the fluorescence microscopic studies on

MARSDEN AND KERKUT

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m o l l u s c a n g a n g l i a . T h e r e is s o m e c o n f l i c t as to whether yellow fluorescent varicosities, representing the terminal synaptic area of t h e n e u r o n e , a r e f o u n d . Also all t h e g r e e n ( c a t e c h o l a m i n e ) n e u r o n e s so f a r f o u n d i n t h e molluscan brain have been very small and u n s u i t a b l e for e l e c t r 0 p h y ~ i o l o g i c a l s t u d i e s o n a n e u r o n e k n o w n to c, ~, a particular a m i n e . I t w a s w i t h t h e s e facts i n m i n d t h a t Planorbis corneus ( w a t e r s n a i l ) w a s s e l e c t e d for t h e p r e s e n t i n v e s t i g a t i o n , as t h i s s n a i l h a s well defined ganglia containing several giant n e u r o n e s i n c o n s t a n t p o s i t i o n s (de N a b i a s , 19oo).

METHODS

Planorbis corneus were o b t a i n e d from a dealer a n d stored in a n a q u a r i u m at I o ° C. until required. T h e m e t h o d used for the cellular localization of the m o n o a m i n e s followed t h a t described by Falk a n d O w m a n (I965) with modifications to incorp o r a t e the Edwards-Pearse Tissue Freeze Dryer ( M a r s d e n a n d Kerkut, I969a ) a n d the use of whole m o u n t s (Rude, I966; M a r s d e n a n d Kerkut, I969b). T h e buccal a n d circumoesophageal ganglia a n d the oesophagus were rapidly r e m o v e d from the a n i m a l a n d q u e n c h e d in isopentane cooled in liquid nitrogen. T h e tissues were then placed on the precooled t h e r m o m o d u l e of the freeze-dryer a n d left for 6 - 2 4 hours at --3 °° C. Tissues used in whole m o u n t studies (the ganglia of P. corneus) were r e m o v e d from the a n i m a l a n d cleared of s u r r o u n d i n g connective tissue. W h e n the connective tissue was particularly tough it was digested away by soaking the tissue in a pronase solution (Io rag. per ml.) for 15 minutes. A t this c o n c e n t r a t i o n the pronase h a d no effect on the fluorescence of the cells after exposure to formaldehyde gas. T h e whole m o u n t s were then orientated o n pieces of coverslip glass, q u e n c h e d in isop e n t a n e , a n d freeze dried whilst still a t t a c h e d to the pieces of glass. After the period of freeze drying the tissues a n d whole m o u n t s were exposed to f o r m a l d e h y d e gas stored at a relative h u m i d i t y of 5o-7 o per cent for i - 3 hours at 8o ° C. in a closed c h a m b e r . I n certain cases, in a n a t t e m p t to increase the yield of the 5 - H T fluorophore, the tissues were first exposed to f o r m a l d e h y d e gas stored at a relative h u m i d i t y of 7o per cent for 2 hours at 8o ° C. followed by exposure to f o r m a l d e h y d e gas stored at 9 ° p e r cent relative h u m i d i t y for a further I h o u r at 8o ° C. (Fuxe a n d J o n s s o n , I967). T h e tissues to be p r e p a r e d as sections were t h e n e m b e d d e d in degassed wax, sectioned at 8-15 P, a n d m o u n t e d in E n t e l l a n (Merck) or F l u o r m o u n t (Gurrs). T h e whole m o u n t s were cleared in light

Comp. gen. Pharmac.

liquid paraffin u n d e r vacuum. Both the sections a n d whole mounts were viewed u n d e r the Zeiss Large Fluorescence Microscope using the excitation filters BG I2 a n d 38 a n d the barrier filters 47 a n d 5 ° . W h o l e mounts were studied u n d e r transmitted and epi-illumination. Photog r a p h y was carried out using T r i - X P a n a n d E.H. Ektachrome. After t r e a t m e n t with formaldehyde vapour in the presence of dried protein the catecholamines are converted to a green fluorophore a n d 5 - H T to a yellow one. T h e specificity of the fluorescence was established using the sodium borohydride reduction test (Corrodi, Hillarp, a n d Jonsson, I964) or i n c u b a t i n g the tissues at 8o ° C. for i - 3 hours in the absence of formaldehyde vapour. T h e following drugs were used: reserpine (Serpasil phosphate, CIBA), n i a l a m i n d e (Pfizer), a n d tranylcypromine (Smith, Kline, a n d French). T h e y were all dissolved in the required volume of distilled water (pH 6 6.5) a n d injected t h r o u g h the foot of the snail. T h e emission characteristics of the fluorophores p r o d u c e d after the exposure of the monoamines to formaldehyde v a p o u r were observed using a fluorescence microspectrometer modified from designs described by T h i e m e (I966) a n d V a n O r d e n , V u g m a n , a n d G i a r m a n (I965). T h e light source was a high pressure mercury l a m p ( H B O 2oo/W). A xenon l a m p is more suitable for fluorescence microspectrometry as it has a fairly continuous light distribution. T h e mercury lamp, however, with its stable o u t p u t a n d brightness, is suitable for measuring emission properties providing the absorption of the f u o r o p h o r e is high at one of the mercury lines. I n the case of the catechola m i n e fluorophores, which have absorption peaks at 220, 33 o, a n d 4 I o mla, the 4o4-4o7 m ~ mercury line can be utilized. T h e o u t p u t from the l a m p was stabilized with a Zeiss light modulator. T h e light source was then filtered t h r o u g h a Zeiss 4o4 mla interference filter so incorporating the 4o4-4o7 m g mercury line as the excitation wavelength. T h e emitted light was passed t h r o u g h barrier filters t h a t h a d the edge at 44 o, 47o, 5 °o mla. F r o m the barrier filters the light was passed into a p h o t o m e t e r h e a d containing a b e a m splitter t h a t permitted areas as small as I I~ to b e measured from. T h e light passed from the p h o t o m e t e r h e a d t h r o u g h a m o n o c h r o m a t o r (Hilger-Watts 2ooxooomla spectral b a n d ) a n d into a n E M I 6256 B photomultiplier a n d from there into a display unit containing a high-voltage power supply. Recordings from the display unit were m a d e graphically on a pen recorder. T h e power supply was stabilized with a G T F / v o l t a g e stabilizer. T h e i n s t r u m e n t was calibrated for the spectral response of the photomultiplier by means of a tungsten l a m p of k n o w n spectral characteristics.

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OCCURRENCE OF MONOAMINES

T h e emission characteristics of model 5-HT, dopamine, a n d n o r a d r e n a l i n e smears were obtained by dissolving the amines in 2 p e r cent aqueous bovine serum a l b u m i n at p H 6"5-7"5 to form o" I-5"o m. per ml. solutions. T w e n t y lalitres the a m i n e solution were smeared onto a piece of glass coverslip, air dried at room temperature, preh e a t e d at 5 °o C. for 15 minutes, a n d then incub a t e d with p a r a f o r m a l d e h y d e vapour, stored at

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microscopy when the emission characteristics of the fluorophores were studied on the fluorescence microspectrometer b u t with rigorous standardization of the reaction conditions. T h e emission spectra in graphic form are all expressed as relative q u a n t a per 5 m/l w a v e l e n g t h . T h e rate of U.V. photodecomposition of the m o n o a m i n e fluorophores in model a n d tissue systems was studied by making direct recordings

Fro. r . - - W h o l e m o u n t of the cerebral ganglia of P. corneus showing the position of the large yellow

fluorescent cells on the dorsal surface. Note the other non-fluorescent cells.

FIG. 3 . - - V e n t r a l view of a whole m o u n t of the pedal ganglia of P. corneus. T h e r e are several yellow fluorescent cells (5-HT) a n d a giant green (dopamine) fluorescing cell (G). T h e p e d a l nerves contain n u m e r o u s green a n d yellow fibres (arrowed). relative humidities of 50-70 p e r cent for 1- 3 hours at 8o ° C. (Johnsson, i967a , b). T h e emission peaks were d e t e r m i n e d on the microspectrometer using the m e r c u r y lines 404-407 m g as the excitation wavelength. Controls were p r e p a r e d by omitting the a m i n e from the a l b u m i n or i n c u b a t i n g the a m i n e / a l b u m i n solution in the absence of form a l d e h y d e v a p o u r for i - 3 hours at 8o ° C. Tissue sections were p r e p a r e d as for n o r m a l fluorescence

FIG. 2 . - - P h o t o m i c r o g r a p h of a cerebral ganglion of P. corneus showing the g r o u p of small green fluorescent cells near the large yellow fluorescent cell. Note the smooth, weakly fluorescent fibres of the catecholamine (green) cells (arrowed). of the fluorescence intensity of the fluorophores on a pen recorder a t t a c h e d to the microspectrom e t e r over a 2-minute period of time. T h e occurrence a n d c o n c e n t r a t i o n of the amines present in the ganglia were o b t a i n e d from extracts m a d e of 3 ° pooled circumoesophageal ganglia using the m e t h o d of Brownlee a n d Spriggs (I965). T h e 5 - H T was estimated in the extracts using the m e t h o d of Bogdanski, Pletscher, Brodie, a n d U d e n f r i e n d (1956), the d o p a m i n e a c c o r d i n g to Carlsson a n d W a l d e c k (x958), a n d the nora d r e n a l i n e by the m e t h o d of Shore a n d Olin (,958).

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Comp. gen. Pharmac.

MARDSEN AND KERKUT

RESULTS A. Fluorescence Microscopy

BUCCAL GANGLIA T h e p a i r e d b u c c a l g a n g l i a c o n t a i n several small green fluorescent cells t h a t lie n e a r the edge o f the green fluorescent neuropil. T h e r e are green fibres in the nerves t h a t arise from

fluorescent cells (IO--I 5 ~.1) a n d a few yellow cells of similar size (Fig. 2). T h e cells in these ganglia a n d all the ones t h a t show specific fluorescence have non-fluorescent nuclei. T h e axons o f b o t h the green a n d yellow fluorescent cells enter the central neuropil which contains a mass of green fibres a n d

FIG. 4.--Photomicrograph of the right pedal ganglion of P. corneus showing the large green fluorescent cell (G) and two yellow fluorescent cells (Y). There is some autofluorescence in the other non-fluorescent cells. The neuropil shows a very strong specific fluorescence (N).

FIG. 5.--Photomicrograph of the right pedal ganglion of P. corneus showing the giant green fluorescent neurone, with its non-fluorescent nucleus, large axon hillock with the fluorescent axon running towards the neuropil.

A

B

FIG. 6.--Serial sections through the right pedal ganglion of P. corneus showing the course of the thick fluorescent axon of the giant dopamine-containing neurone. There are several fine branches entering the neuropil (arrowed). There is a 5-HT-containing neurone next to the green cell (Y). the g a n g l i a connective.

including

the

cerebrobuccal

T H E CIRCUMOESOPHAGEAL GANGLIA

Cerebral Ganglia. I n an a n t e r o d o r s a l position a n d on the surface of each ganglion there is a large ( i o o - I 5 O g) yellow fluorescent cell (Fig. i) n e a r b y w h i c h there is a g r o u p of small green

varicosities a n d r a t h e r fewer yellow fibres a n d varicosities. T h e yellow fluorescence in the n e u r o p i l c a n be increased b y using a c o m b i n a t i o n of p a r a f o r m a l d e h y d e stored at 7 ° a n d 9 ° per cent relative h u m i d i t y r a t h e r t h a n just p a r a f o r m a l d e h y d e stored at a relative h u m i dity of 7o p e r cent as was r o u t i n e l y used. After the c o m b i n a t i o n t r e a t m e n t there a p p e a r e d to be several large yellow fluorescent fibres

I97o, x

O C C U R R E N C E OF M O N O A M I N E S

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running through the neuropil and numerous yellow varicosities. There are no fluorescent cells in the cerebropleural or pedal connectives but in both these and the other cerebral nerves there are thick smooth green fluorescent fibres.

right pedal ganglion a n d leave the ganglion by the pedal nerves (Fig. 6B). Near the exit of the pedal nerves there are small groups of green fluorescent cells (15 25 g) and several cells showing a yellow fluorescence that are somewhat larger in size (2o-3o g). T h e neuropil is intensely fluores-

Fie. 7.--Photomicrograph of the pedal ganglia of P. corneus showing some of the medium-sized yellow fluorescent cells after treatment with paraformaldehyde stored at a relative humidity of 7° and 9° per cent. The axon of one cell is clearly visible and there are yellow fluorescent fibres in the neuropil (arrowed).

FIG. 8.--Photomicrograph of the yellow fluorescent cells in the visceral and the right parietal .ganglia of P. corneus. The neuropil (N) shows Intense fluorescence.

Pedal Ganglia O n the ventral and to a lesser degree the dorsal surface of the ganglia there is a group of m e d i u m to large yellow fluorescent neurones (5o-2oo p) (Fig. 3). T h e intensity of the fluorescence in these cells is variable. T h e preterminals are weakly fluorescent as they enter the neuropil. T h e pedal ganglia show a curious asymmetry in that in the right pedal ganglion but not in the left there is a giant green fluorescent cell (28o-3oo la) (Fig. 4). This cell is found amongst the yellow fluorescent cells in a ventroposterior position. T h e cell has large characteristic non-fluorescent nucleus, a large axon hillock, and a thick smooth axon that runs towards the neuropil where it divides (Fig. 5). O n e b r a n c h of the axon passes along the edge of the neuropil and into the pedal-pleural connective where it continues through the right pleural ganglion and terminates, as far as it is possible to trace, in the right parietal ganglion. O t h e r branches o f the axon pass t h r o u g h the neuropil o f the

Fro. 9.--Whole mount of the dorsal surface of the visceral (V) and the right parietal (RP) ganglia of P. corneus. A montage made from two photographs. The fluorescence in certain cells is yellow and fluorescent fibres are visible in the visceral left parietal connective (C). cent and contains m a n y varicosities, but as pedal nerves emerge the n u m b e r of the varicosities decline a n d smooth green and yellow fibres are found in the pedal nerves. T h e use of a combination of paraformaldehyde stored at relative humidities of 7 ° and 9 ° per cent increases the n u m b e r of yellow

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Comp. gen. Pharmac.

MARSDEN AND KERKUT

fibres and varicosities found in the neuropil (Fig. 7). The yellow fluorescence is very sensitive to U.V. light, suggesting it is due to the presence of 5-HT.

The Left Parietal and the Pleural Ganglia There are no fluorescent cells in these ganglia. The neuropil of them all does, however, contain fluorescence that is specific.

A

C

The Right Parietal and Visceral Ganglia These ganglia contain many fluorescent cells mainly grouped on their adjacent dorsal surfaces. The cell~ vary in size (3o-I5 ° It) and they all show the golden yellow fluorescence characteristic of 5-HT (Fig. 8). It is difficult to persistently locate individual fluorescent cells though there are always fewer in the right parietal than in the visceral ganglion (Fig. 9). All these cells have thick smooth preterminals that fluoresce yellow and enter the neuropil and, in some cases, leave the ganglia by the nerves. In several cases the axons of the yellow fluorescent cells were seen to merge before reaching the neuropil.

One of the yellow fluorescent neurones ill the visceral ganglion is bipolar (Fig. io). Apart from the occasional very small green fluorescent bipolar cell in the cerebral ganglia this was the only fluorescent bipolar cell found in P. corneus. On several occasions it was possible to observe green fluorescent dots in close contact

B

FIo. Io.--Serial sections of the bipolar neurone (B) in the visceral ganglion of P. corneusthat has a yellow fluorescence. In B and C the small green fluorescent neurone found in the visceral ganglion is visible (G) near the strongly fluorescent neuropil. with the yellow fluorescent fibres in the right parietal and visceral ganglia. These dots may represent the varicosities of the terminals of the green fibres. There are many green fluorescent varicosities visible in the neuropil of the ganglia. There are only two small green fluorescent cells in each of the ganglia and these are found in a ventroposterior position close to the neuropil (Fig. IoB, C). The position of the fluorescent cells in the circumoesophageal ganglia of Planorbis corneus are summarized in Figs. I I - i 3. AUTOFLUORESCENCE Only about 5-IO per cent of the total cell population of the ganglia contain specific fluorescence after treatment with formaldehyde vapour. Many of the other cells contain yellow or red autofluorescence. The nonspecificity of this autofluorescence is readily established by the sodium borohydride reduction test (Corrodi and others, 1964) or by incubating the tissues for I - 3 hours at

I97o , I 80'-' C. in vapour.

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OCCURRENCE OF MONOAMINES

the

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smooth processes in the wall of the oesophagus. I n the muscle layer of the oesop h a g u s there are some yellow dots t h a t fade r a p i d l y u n d e r U . V . light. This m a y indicate the presence o f 5 - H T in the muscle layer.

of formaldehyde

T H E OESOPHAGUS

T h e r e are n u m e r o u s small green fluorescent cells w i t h non-fluorescent nuclei a n d

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Fro. xi . - - D i a g r a m showing the position of the cells on the dorsal surface of the cerebral ganglia of P . corneus that exhibit a specific fluorescence after paraformaldehyde treatment. I. Main cerebral nerves. 2. Cerebropleural connective, O, Yellow fluorescent neurones. 0 , Green fluorescent neurones. = , Fluorescent fibres.

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FIG. 12.--Diagram of the dorsal surface of the circumoesophageal ganglia of P . corneus showing the position of the cells exhibiting a specific fluorescence. The cerebral ganglia are viewed from their ventral surface as they have been separated to display the pedal ganglia. Drawing made from whole mount preparations and photographs. R.C/L.C, right and left cerebral ganglia; R.Pe/L.Pe, right and left pedal ganglia; R.P1/L.P1, right and left pleural ganglia; R.P/L.P, right and left parietal ganglia; V.P, visceral ganglion. Other symbols as in F i g . I I.

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Comp. gen. Pharmac.

MARSDEN AND KERKUT

TI~E EFFECT OF DRUGS ON THE SPECIFIC FLUORESCENCE

Reserpine A dose o f 2 "5 mg. p e r a n i m a l for 2 4 a n d 4 8 hours caused a m a r k e d decrease in the intensity o f the specific fluorescence. T h e green fluorescence was m o r e resistant to reserpine t r e a t m e n t b u t this m a y simply reflect the g r e a t e r

fluid balance. N i a l a m i d e (2. 5 mg. per a n i m a l for 4 8 hours) also h a d no effect on the green a n d yellow fluorescence in the cells a n d the green fluorescence in the neuropil. I n certain areas of the neuropil of the cerebral, visceral, a n d right p a r i e t a l ganglia there a p p e a r e d to be an increase in the n u m b e r of yellow fluorescent varicosities after n i a l a m i d e

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F1o. 13.--Diagram of the ventral surface of the circumoesophageal ganglia of P. corneus showing the position of the cells that exhibit specific fluorescence after paraformaldehyde treatment. Lettering and symbols as in Figs. ix, 12. fluorescence yield o f the catecholamines. T h e autofluorescence was unaffected b y reserpine (Fig. 14).

Monoamme Oxidase Inhibitors T r a n y l c y p r o m i n e , a fast-acting M A O inhibitor, h a d no effect on the specific fluorescence at a dose o f 0"2 mg. p e r a n i m a l for 2-8 hours. H i g h e r levels o f the d r u g caused the r a p i d d e a t h o f the snail w i t h a p p a r e n t loss of

t r e a t m e n t . T h e varicosities were small a n d f a d e d r a p i d l y u n d e r U . V . light.

B. Fluorescence Microspectrometry M o d e l systems of i rag. p e r ml. solutions of 5 - H T , d o p a m i n e , a n d n o r a d r e n a l i n e dissolved in 2 per cent aqueous bovine s e r u m a l b u m i n gave characteristic emission peaks after exposure to f o r m a l d e h y d e gas at 8o ° C.

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OGGURRENGE OF MONOAMINES

for i - 3 hours. 5-HT had an emission peak at 525 mix using the 4o 4 mix interference filter as the excitation wavelength, whilst dopamine and noradrenaline both had peaks of emission at 480 mix.

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and the group of yellow cells in the right parietal a n d visceral ganglia all h a d emission m a x i m a of 53o-54 ° mg (Fig. 15). Model systems of dopamine p r e p a r e d at a concentration of 5 rag. per ml. exhibited a

A

B

FIo. I4.--A , Photomicrograph of a pedal ganglion from P. corneus after paraformaldehyde treatment showing the occurrence of yellow and green fluorescent neurones and the fluorescence in the neuropil. B, Photomicrograph of a pedal ganglion after reserpine. Note the absence of any specific fluorescence.

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Fio. 15.--The emission characteristics of some fluorescent cells in P. corneus measured on the fluorescence mxcrospectrometer, i. The small green fluorescent cells in the cerebral ganglia have a peak at 47o-480 mla. 2. The giant green fluorescent neurone in the right pedal ganglion has an emission peak. 3. One of the yellow fluorescent neurones in the visceral ganglion has an emission peak at 53o-54 ° mla. The large green fluorescent neurone found in the right pedal ganglion of P. corneus a n d the small green neurones found adjacent to the large yellow fluorescent neurone in each of the cerebral ganglia had emission peaks of 47o-48o mix. On the other hand, the large yellow fluorescent cells in the cerebral ganglia

shift in the emission peak of the fluorophore from the normal 480 mix to a broad peak of 49o-55o mla. This concentration shift was not observed in any of the a p p a r e n t green fluorescent structures in the tissue sections. A m i n e / a l b u m i n smears incubated in the absence of formaldehyde gas or albumin

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MARSDEN A N D K E R K U T

incubated in the presence of formaldehyde gas but in the absence of an amine showed a very weak and broad emission peak between 420 and 45 ° m g when activated at 404 m~. Smears containing 5 - H T and exposed to U.V. light during which time direct recordings of the fluorescence intensity were made

Comp. gen. Pharmac.

these neurones confirmed that the highest level of specific fluorescence was found in the axon hillock region with relatively low levels of fluorescence around the nucleus. Specific fluorescence was absent from the nucleus (Fig. i7). S P E G T R O I ~ H O T O F L U O R I M E T R I C ESTIMATIONS OF THE MONOAMINES

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Dopamine was found at a concentration of 9"53 gg. per g. wet weight in the circumoesophageal ganglia ofP. corneus and 5 - H T at 3"34 gg. per g. wet weight. No noradrenaline was found in the ganglia. Reserpine reduced both the dopamine and 5 - H T levels in the ganglia ( Table I).

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80

TIME (sees) FIG. I6.--A direct recording of the effect of U.V. light on the intensity of the fluorescence, after paraformaldehyde treatment, in the giant green neurone (G) in the right pedal ganglion and a yellow fluorescent neurone (Y) in the visceral ganglion. Note the rapid fading of the yellow fluorescence in contrast to the green. Recorded on a pen recorder from readings made with the fluorescence microspectrometer. showed a decrease in the intensity of 3 ° 35 per cent within 9 ° seconds. The dopamine fluorophore only showed a 3-5 per cent decrease in intensity during the same time period. In P. corneus the neurones that had emission characteristics similar to 5 - H T models also faded at a similar rate to the models (Fig. 16), whilst the neurones with emission m a x i m a similar to the dopamine and noradrenaline models had similar fading properties to those of the dopamine models. Subjective observations of tissue sections suggested that there was a particular distribution of the fluorescence in the neurones containing a specific yellow fluorescence. Quantitative readings made with the fluorescence microspectrometer in different areas of

DISCUSSION The distribution of monoaminergic structures has been studied in the nervous systems of several gastropod molluscs. Dahl and others (1966) studied Helix pomatia, Sakharov and Zs-Nagy (I967) Lymnaea stagnalis, and Sedden and others (i968) Helix aspersa. The distribution of the fluorescent structures in Planorbis corneus show several marked similarities with that found in other gastropods. For example the occurrence of the large yellow fluorescent cell in the cerebral ganglia, the group of fuorescent cells in the right parietal and visceral ganglia, the general small size of the green fluorescent neurones and the presence of green fluorescent varicosities in the central neuropil. The most outstanding difference between the distribution of the fluorescent structures in Planorbis corneus and other gastropods is the presence of the giant green neurone in the right pedal ganglion. The emission peaks of the monoamine fluorophores in model systems correspond to those obtained by other workers (Van Orden and others, 1965; Caspersson, Hillarp, and Ritzen, 1966). The peaks obtained from the fluorophores on neurones of the ganglia imply that the yellow fuorescence is due to the presence of 5 - H T and the green to the presence o f a catecholamine. There is further evidence for this supposition from the rate of photodecomposition of the fluorophores under U.V. light. It has been demonstrated, with a

IIl

O C C U R R E N C E OF MONOAMINES

I97 o, I

workers have shown that the green fluorophore in the neuropil of Anodonta piscinalis is due to the presence of dopamine. Dopamine is the only catecholamine found in several lamellibranch and gastropod molluscs (Sweeney, i963; Dalai and others, I966; Kerkut and others, 1966 ). More recently

fluorescence microspectrometer, that 5-HT fades at a much greater rate than the catecholamine fluorophore (Caspersson and others, I966 ; Ritzen, i967). In the present case the cells showing an emission maxima of 53o-54o m~t faded by 3° per cent within 9 ° seconds while the cells with an emission

A B FIG. I7.--A, Photomicrograph of a yellow fluorescent neurone in the pedal ganglia of P. corneus showing the high intensity of the fluorescence in the axon hillock region (AH). B, Diagram of the same cell showing the relative fluorescence intensity in different parts of the cell as measured with the fluorescence microspectrometer. Note the high reading obtained in the axon hillock region (AH) and the absence of fluorescence in the nucleus (N). T a b l e / . - - T H E EFFECT OF RESERPINE ON THE MONOAMINES FOUND IN THE BRAIN OF Planorbis corneus TREATMENT

TIME

EXPERIMENT

(rag. per animal)

(hours)

No.*

none

Reserpine (2.5mg.) Reserpine (2'5 rag.)

I6 48

5-HT ( gg. per g.)

( lag. per g.)

3 "34 i .32 o.6o

9"53 3 "4° o'5 °

DOPAMINE

* Each extraction consisted of 3° pooled eircumoesophageal ganglia. maxima of 480 m~t showed only a 3-5 per cent decline in fuorescence intensity during the same period of time. In the spectrophotofluorimetric estimations of the monoamines in the ganglia of P. corneus dopamine was the only catecholamine isolated, so it would appear that the green specific fluorescence is due to the preserLce of dopamine. By measuring changes in the excitation wavelengths of the dopamine and noradrenaline fluorophores after exposure to hydrochloride gas it is possible to distinguish between dopamine and noradrenaline in tissue sections (Bjorklund, Ehinger, and Falk, 1968 ). These

Cottrell (i967) found appreciable amounts of noradrenaline in the lamellibranch Spisula solida and the cephalopod Eledone cirrosa; dopamine, however, was the most abundant catecholamine. Sweeney (I968) has also found dopamine and noradrenaline in the lamellibranch Sphaerium sulcatum. 5-HT is known to have a wide distribution in the molluscan ganglia (Welsh, i968 ) and Welsh and Moorhead (I96O) demonstrated the presence of 5-HT in Planorbis corneus in whole animal extracts. There has been a certain amount of discussion as to whether the fibres, derived from

112

MARSDEN A N D K E R K U T

molluscan cell bodies containing the yellow 5 - H T fluorophore, terminate in fine varicosities showing the yellow fluorescence. It is the varicosities that are assumed to be the presynaptic endings of the nerve-fibres (Elfin, I963). Dahl and others (I966) found yellow varicosities in the neuropil of the lamellibranch Anodonta piscinalis and the gastropod Helix pomatia that were particularly apparent after nialamide treatment. Kerkut and others (i967b) demonstrated that the neuropil of the pedal ganglia of Helix aspersa appeared yellow after the pretreatment of the animal with 5-HTP. O n the other hand, Zs-Nagy (i967) concluded that any yellow fluorescence in the neuropil of Anodonta cygnea was not due to the presence of nerve terminals but to the presence of gliosomes in glial cells. Sakharov and Zs-Nagy (I967) were also unable to find yellow varicosities in the neuropil of the gastropod Lymnaea stagnalis and on the basis of these results questioned the role of 5 - H T as a chemical transmitter in the molluscan brain as it was not found in the area of the neurone where the synapse occurs. One of the major problems with the fluorescence histochemical method for the cellular localization of monoamines has been the low fluorescence yield of 5 - H T in cornparison to that of catecholamines (Jonsson, i967a , b). This problem has in part been recently overcome by the use of extreme paraformaldehyde treatment in a manner that avoids loss of the amines by diffusion (Fuxe and Jonsson, i967). Under these conditions yellow varicosities are found in the neuropil of the pedal, cerebral, visceral, and right parietal ganglia of P. corneus. The difficulty in locating yellow fluorescent terminal varicosities is probably due to the low reactivity of 5 - H T with paraformaldehyde and the rapid fading of the 5 - H T fluorophore. Numerous green varicosities are found in the neuropil of P. corneus as they are in all the other molluscs that have been studied (Dahl and Falk, i962; Dahl and others, I966; ZsNagy, x967; Sakharov and Zs-Nagy, I967; Kerkut and others, 1967 a, b ; Sweeney, 1968). T h e occasional occurrence of bright green varicosities on the yellow fluorescent axons of

Comp. gen. Pharmac.

certain neurones in the visceral ganglion of P. corneus is of interest as these may represent synaptic junctions. Dahl and Falk (t96~) found fluorescent varicosities encircling nonfluorescent cell bodies in Anodonta piscinalis, though it is believed that the molluscan neurone has no synapses above the axon hillock (Gerschenfeld, i963). The neurones of the gastropod molluscs are in general unipolar (Gerschenfeld, x963). In the present work a single yellow fluorescent bipolar neurone was found in the visceral ganglion of P. corneus. Kerkut, French, and Walker (I97o) have recently found a bipolar neurone in a similar position in Helix aspersa after tracing the axons with procion yellow dye. In the related species Cryptomphallus aspersa a few bipolar neurones have also been isolated (Sanchis and Zambrano, 1969). Bertaccini (196I) recorded a rise in the dopamine content of the optic ganglia of the cephalopod Eledone moschata after treatment with monoamine oxidase inhibitor. Kerkut and Cottrell (1963) were unable, however, to find monoantine oxidase activity in the brain of Helix aspersa. Kerkut and others (I966) also found that the monoamine oxidase inhibitor iproniazid had no effect on the dopamine level of the brain of H. aspersa. Several workers, however, have found that nialamide increases the intensity of the fluorescent fibres, in particular those showing a yellow fluorescence, in certain molluscs (Dahl and others, x966; Zs-Nagy, I967; Sakharov and Zs-Nagy, I967). In the present study there was a noticeable increase in the number of yellow fluorescent varicosities in the cerebral, visceral, and right parietal ganglia after treatment with nialamide, but tranylcypromine had no effect. There was no similar effect on the number of green fluorescent varicosities; this may, however, reflect that the dopamine is present in the terminals at a level above the threshold value determined by Ritzen (I 966) and that any increase in the dopamine concemration would not result in a linear increase in the fluorescence intensity. It would appear that if monoamine oxidase is present in the snail brain it has a low activity. This need not prevent the

197o, t

OCCURRENCE

OF MONOAMINES

II 3

monoamines from being considered candi- those found in mammalian systems in comdates as chemical transmitters. Other in- mon with other molluscs so far studied activation processes may be considered such (Dalai and others, 1966; Sedden and as re-uptake by the presynaptic membrane as others, i968; Sweeney, I968) and several found in mammalian systems (Fuxe and other invertebrates (Clarke, I966; Rude, Ungerstedt, I968 ). Gerschenfeld and Stefani I966 , i969; Elofsson, Kauri, Nielson, and (i966, i968) proposed that 5-HT was the Strombcrg, 1966; Myhrberg, 1967; Ehinger transmitter involved in the slow EPSP and others, x968; Klemm, x968a, b; Marsden (excitatory post-syrmptic potential) of cells and Kerkut, I969b ). The major morphoshowing inhibition of long duration in logical features are the occurrence of partiC9~Otomphallus aspersa and that the inactiva- cular neurones containing a monoamine and tion process was by diffusion away from the that the amine is concentrated in terminal varicosities that are believed to be the areas receptor. In common with other molluscs reserpine, of synaptic contact. The study of the electroat relatively high doses, depletes the mono- physiological properties of a neurone known amines (Piccinelli, I958; Mirolli and Welsh, to contain dopamine should be aided by the i964; Dahl and others, I966; Kerkut and size and constant appearance of the giant others, I966; Cottrell, I967; Sakharov and neurone in the right pedal ganglion of Zs-Nagy, i967; Zs-Nagy, i967). Curtis and Planorbis corneus. Kerkut (I969) have recently shown that SUMMARY reserpine reduces the number of dense I. The distribution and nature of the granules in the cerebral ganglia of Helix monoamlnes in the central nervous system ad~Oefsa. The maximum intensity of specific fluores- and the oesophagus of Planorbis corm,us were cence in the cell body of the neurones con- studied using fluorescence microscopy and taining 5-HT in the visceral ganglion of P. microspectrometry and spectrophotofluoric o r n ~ is found in the axon hillock. This co- metric estimations. 2. In the cerebral, pedal, visceral, and incidcs with a high level of metabolic activity in this area (Kerkut, I967). As the tissues right parietal ganglia there are neurones that were freeze dried followed by formaldehyde contain a monoamine. The neurones showgas fixation the apparent distribution is ing a yellow fluorescence are generally unlikely to be due to a diffusion artifact. medium to large in size whilst the green Semory cells containing a catceholamine fluorescent neurones are small. There is found in the peripheral systems have been one giant green neurone in the fight isolated in the earthworm (Rude, I966; pedal ganglion the axon of which sends Myhrberg, I967) and in certain molluscs branches out along the pedal nerves and (S.-Rozsa and Perdnyi, i966; Sweeney, another branch to the right parietal gan1968). Planorbis corneus has many small green glion. 3. The neuropil of the ganglia contain fluorescent cells with smooth processes in the oesophageal wall that may function as pri- numerous green fibres and varicosities and mary sensory cells. The rapidly fading yellow after extreme formaldehyde gas treatment fluorescent dots in the oesophageal wall yellow fibres and varicosities. musculature may be the terminals of 5-HT 4. The buccal ganglia have no yellow fluorescent ceils but several small green motor nerves. The morphological criteria for a trans- fluorescent cells. The neuropil shows a green mitter function of dopamine and 5 - H T in the specific fluorescence. mammalian central nervous system have been 5. Fluorescence microspectrometry and fulfilled (Fuxe, Dahlstrom, and Hillarp, spectrophotofluorimetric estimations indicate 1965; Fuxe, Hokfelt, and Ungerstedt, 1968). that the green fluorescence is due to the Morphologically the monoaminergic neur- presence of dopamine and the yellow to the onal systems of P. c0meus closely resemble presence of 5-HT.

MARSDEN AND KERKUT

1,4

6. T h e greatest concentration o f 5 - H T within the cell bodies of the yellow fluorescent cells is found in the axon hillock. 7. Reserpine depletes the specific fluorescence. Nialamide increases the n u m b e r of yellow fluorescent varicosities in the neuropil b u t has no effect on the green fluorescence. T r a n y l c y p r o m i n e has no effect on the specific fluorescence. 8. T h e r e are several small green fluorescent cells with smooth processes in the wall o f the oesophagus. Yellow fluorescent dots that m a y be 5 - H T terminals are found in the muscles o f the oesophagus. 9. T h e morphological evidence for the possible role o f 5 - H T a n d d o p a m i n e as chemical transmitters is discussed. ACKNOWLEDGEMENT We are indebted to the Royal Society for a grant for the fluorescent microscope.

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COTTR~LL, G. A. (1967) , 'Occurrence of dopamine and noradrenaline in the nervous tissue of some invertebrate species ', Br. 3. Pharmaz. Chfm0t/wr., 29, 63-69. CuaTm, D.J., and K~RKUT, G. A. (i969) , ' T h e effect of reserpine on the vesicle content of Helix asl~rsa cerebral ganglia ', Comp. Biochem. Physiol., 30, 835-84o. DA~m, E., and F~K, B. (1962), ' Monoamines in mollusc neurones ', K. fysiogr. 8dllsk. Lund F#rh., 32, 89--92. VON MECKLENBERO, C . MYHRBERO~

H., and ROSENOm~N, E. (I966), 'Neuronal localization of dopamine and 5-hydroxytryptamine in some mollusca ', Z. Zellforsch. mikrosk. Anat., 7 t, 489-498. EHmOER, B., FALK,B., and MYHREERO,H. (1968), 'Biogenic monoamines in Hirudo medidnalis ', Histochemie, 15, 140-I49. ELFIN, L. (I963), ' T h e uhrastructure of the superior sympathetic cervical ganglion of the cat, II. The structure of the pre-ganglionic end fibres and synapses as studied by serial sections ', 07. Ultr~trua. Res., 8, 441-476. ELOrSSON, R., KAUm, T., NmLSON, S.-O., and STRO~mERO, J.-O. (1966), 'Localization of monoaminergic neurons in the central nervous system of Astacus ~tacus Linne (Crustacea) ', Z. Zdlforsch. mikrosk. Anat., 74, 464-473. FALK, B., HILLARP, N.- A., T H m ~ , G., and ToaP, A. (196o), ' Observations on the possibilities of the cellular localization of monoamines by a fluorescence method ', Acta physiol, scand., 56, Suppl. 197, 1-25. ----and OWMAN, C. (1965), ' A detailed methodological description of the fluorescence methods for the cellular demonstration of biogenie amines ', Acta Univ. lund., Sect. II, No. 7, 1-2 3 .

FuxE, K., DAHLSTaOM,A., and HmLARP, N.-A. (i965) , 'Central monoamine neurons and monoamine neurotransmission ', Proc. int. Union physiol. Sd., 4, 419-434 . - - - - HOV~LT, T., and UNOEaSTEDT, U. (1968), 'Localization of indolealkylamines in the central nervous system ', Adv. Pharmac., 6A, 035-251. and JoNssON, G. (1967), ' Modification of the histochemical fluorescence method for the improved localization of 5-hydroxytryptamine ', Histochemie, xl, 161-166. a n d UNOERSTEDT, U . (I 968), ' Histochemical studies on the effect of ( + ) amphetamine, drugs of the imipramine group and tryptamine on central catecholamine and 5hydroxytryptamine neurons after intraventricular injection of catecholamines and 5-hydroxyo tryptamine ', Eur. 07. Pharmac., 4, I35-144. GERSCHEm~LD, H. (1963) , ' Observations on the ultrastructure of synapses in some pulmonate molluscs ', Z. Z d l f °rsch. mikrosk. Anat., 60, 258276.

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GERSCHENI~LD, H. and STm,Am, E. (1966), ' A n electrophyslological study of 5-h.ydroxytryptamine receptors of neurones m the molluscan nervous system ', .7. Physiol., Lond., x S ~ n 684-700. (t968), 'Evidence for an excitatory transmitter role of serotonin in molluscan central synapses ', Adv. Pharmac., 6A~ 369-39a. JONSSON, G. (1967a), ' F u r t h e r studies on the specificity of the histochemical fluorescence method for the demonstration of catecholamines ', Acta histochem., a6~ I - I I. - - - - ( I 9 6 7 b ) , 'Fluorescence methods for the histochemical demonstration of monoamines, V I I . Fluorescence studies on biogenic monoamines and related compounds condensed with formaldehyde ', Hist0chem/e, 8, 288-296. KERKUT, G. A. (1967) , 'Biochemical aspects of invertebrate nerve cells ', in The Invertebrate .Nervous System (ed. WmRSMA, C. A. G.), pp. 5 37" Chicago: University Press. ----and COTTSZLL, G. A. (I963), 'Acetylcholine and 5-hydroxytryptamine in the snail brain ', Comp. Biochem. Physiol., 8, 53-63. --FRZNCH, M. C., and WALteR, R . J . (I97o), ' T h e location of axonal pathways of identifiable neurones of Helix aspersa using the dye Procion Yellow. M-4R ', Comp. Biochem. Physiol., 3 a , 681-69 o. - - - - H O R N , N., and WAt.~R, R. J. (I969) , ' Long lasting synaptic inhibition and its transmitter in the snail Helix aspersa ', Ibid., $o, xo6txo 7 4 . - - - - S~VDEN, C. D., and WALg~R, R . J . (x966), ' T h e effect of DOPA, a-methyl DOPA and reserpine on the dopamine content of the brain of the snail ', Ibid., xs, 92x-93o. . . . . . . . (I967a), ' Cellular localization of monoamlnes by fluorescence microscopy in

Hirudo medidnalis and Lumbricus terrestris ', Ibid., ax, 687-69o. (t967b), ' U p t a k e of DOPA and 5-hydroxytryptophan by monoamine formhag neurones in the brain of Helix aspersa ', Ibid., aa, 159--I62. - - - - and WALKER, R. J. (x96t), ' The effect of drugs on the neurones of Helix ~persa ', Ibid., 3~ I43-16o. (I96~), ' T h e specific chemical semitivity of Helix nerve ceils ', Ibid., 7~ 277 -288. K ~ m ~ , N. (I968a), ' M o n o a m i n e containing neurons in the central nervous system in Tricoptera (Insecta), part I ', Z. ZelOr°rsch. mikrosk. Anat., 92, 487-5o2. - - - - (I968b), ' Monoamine cell elements in the stomatogastric nervous system of Tricoptera (Insecta) ', Z- .Naturf. B, ~3, I283--Xo84MAmDEN, C. A., and K~gmrr, G. A. (1969a), ' T h e cellular localization of monoamines in invertebrates using the Edwarcls-Pearse Tissue Freeze Dryer ', in Laboratory Experiments in Physiology and Biochemistry (ed. Km~xuT, G. A.),

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MAmDSN, C. A., and K m ~ t r r , G. A. (1969b), 'The cellular localization of monoamines in

Himdo medidnalis ', Comp. Bioshem. Physiol., 3x, 85 ~-862. MIROLLI, M., and WELSH, J. H. (I964), ' T h e effects of reserpine and LSD on molluscs ', in

Comparative .Neurochemistry : 5th International .Neurochemistry Symposium (ed. RICHTER, D.), pp. 433444. Oxford: Pergamon Press. MYrn~sRO, H. (x967), ' Monoaminergic mechanisms in the nervous system of Lumbricus terrestris L.', Z. Zellforsch. mikrosk. Anat., 81, 31 x-343. DE N.~mIAS,B. (I9OO), ' Nouvelles recherches sur le syst~me ndrveux des gastropodes pulmones aquatiques. Cerveaux des planorbes (Plan-

orbis corneus)', C. r. Ass. fr. Avanc. Sci., a9, 726-730.

PICGINELLI,D. (t958), ' Azione della reserpina su alcune localizzazione di indolalchilamine e fenilalchilamine in vertebrati inferiori e mollmchi ', Archs int. Pharmacodyn. 77dr., xxT, 452459. RXTZEN, M. (t966), 'Quantitative fluorescence microspectrometry of catecholarnine-formaldehyde products. Model experiments ', Exp. Cell Res., 44, 505-520. - - - - ( x 9 6 7 ) , 'Quantitative fluorescence microspectrometry of 5-hydroxytryptamine-formaldehyde products in models and in mast ceils ', Ibid., 45, I78--I94. RuDz, S. (I966), ' Monoamine-containing neurones in the nerve cord and body wall of Lumbrkus terrestris ', .7. comp. .Neurol., xas, 397-412. - - - - (1969) , ' Monoamine-containing neurom in the central nervous system and peripheral nerves of the leech, Hirudo medidnalis ', Ibid., x 3 6 , 349-37 I. S.-RozsA, K., and PER~NYX, L. (1966), ' Chemical identification of the excitatory substance released in Helix heart during stimulation of the extracardial nerve ', Comp. Biochem. Physiol., x9, IO5-I 13 . SAm~AROV, D. A., and Zs-NAoY, I. (x967) , ' Localization of biogenic amines in the cerebral ganglia of Lymnaea stagnalis L.', Acta biol. hung., x9, I45-I57. SANCtuS, C. A., and ZAUmRANO,D. (I969) , ' The occurrence of bipolar neurons in the abdominal mass ganglia of a pulmonate mollusc (Cryptomphallus aspersa) ', Experientia, aS, 385-386. SEDn~.N, C. B., KeRKUT, G. A., and W ~ R , R.J. (I968), ' T h e localization of dopamine and 5-hydroxytryptamine in neurones of Helix

aspersa ', Syrup. zool. Soc. Lond., ~2~ I9-32. SHORE, P., and OLIN, S. (I958), 'Identification and chemical assay of norepinephrine in brain and other tissues ', .7. Pharmac. exp. Ther., x22~ 295-300. SwE~r~Y, D. (x963), ' Dopamine, its occurrence in molluscan ganglia ', Ssieme, ~.N.T., x39, io5 I.

I 16

MARSDEN AND KERKUT

Swx~m~Y, D. (I968), ' The anatomical distribution ofmonoamines in the freshwater bivalve mollusc Sphaerium ~lcatum (L.) ', Comp. Biochem. Physiol., 25~ 6oi-6t3. THmME, G. (t966), A versatile device for microscopic spectrofluorimetry ', Acta physiol, stand., 67, 5x4-52o. VAN ORD~-N, L., VUOMA~, I., and GIAm~.~, N. (1965) , '5-Hydroxytryptamine in single neoplastic mast cells. A microscopic spectrofluorimetric study ', Sdeme, 3i'.~'., x48, 64~--644. WELSH, J. H. (x968), ' Distribution ofserotonin in the nervous system of various animal species ', Adv. Pharma¢., 6A, x7I-x88.

Wzt.m, J. H., and Moom-m~, M. (x96o), ' The quantitative distribution of 5-hydraxytryptamine in the invertebrates especially in their nervous systems ', 07. dVeuroctwm.,6j 146--I69. Zs-NAGY, I. (I967) , 'Histochemical demonstration of biogenic amines in the central nervous system of the lamellibranch mollusc Anodonta cygnea L.', dcta biol. hung., x8, t-8.

Key Word Index: Planorbis corneus, dopamine, 5-hydroxytryptamlne, monoamines, fluorescence microscope, catecholamines, reserpine, nialamide, tranylcypromine, chemical transmission.