Brain Research, 578 (1992) 221-234 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
221
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Ultrastructural, biochemical and electrophysiological changes induced by 5,6-dihydroxytryptamine in the CNS of the snail Helix pomatia L. L~iszl6 Hern~idi, L~iszl6 Hiripi, /~gnes Vehovszky, GyOrgy Kemenes* and Katalin S.-R6zsa Balaton Limnological Research Institute of the Hungarian Academy of Sciences, Tihany (Hungary) (Accepted 10 December 1991)
Key words: Gastropod; Central nervous system; Neurotoxin; 5.6-Dihydroxytryptamine; Serotonin
The serotonin neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT), was injected into the body cavity of snails. Changes induced in the central nervous system (CNS) by the neurotoxin were studied by morphological, electrophysiological and biochemical techniques for up to 90 days following injection. The neurotoxin induced a variety of ultrastructural alterations during the early phase (1st to 6th days) following treatment. On day 6 after treatment, membranous structures first appeared in the synaptic-like areas and apparently migrated to cell bodies where they were detected by day 14. Their number increased with time. Neurotoxin-induced structural alterations were found in neuronal processes and cell bodies of the serotonergic metacerebral giant cells injected intracellularly with horseradish peroxidase and in serotonin immunoreactive axons. These findings suggest that the toxin-induced alterations are rather selective for the serotonin-containing neuronal elements. The neurotoxin decreased the concentration of 5-HT in and [3H]5-HT uptake into cerebral and pedal ganglia, with a maximum effect between the 3rd and 5th day following drug administration. 5-HT levels and 5-HT uptake returned to normal by 19-21 days after treatment. The concentration of dopamine and of [3H]DA uptake capacity were reduced between 3-5 days after injection of 5,6-DHT by 6-7 days following treatment. The transmission from identified serotonergic synapses to targets was reduced beyond day 5 after 5,6-DHT administration. By 15 days after treatment, synaptic transmission between the metacerebral giant cell (MGC) and buccal followers was blocked. Transmission recovered by day 21 after 5,6-DHT. Comparison of the time-course of functional and structural recovery indicates that while functional recovery takes place within 21 days after treatment, certain structural alterations, e.g. the membranous structures and dense particles, remain in the nerve fibres and cell bodies. These may serve as specific intracellular markers of the serotonin-containing neuronal elements long after functional recovery from the effect of 5,6-DHT. INTRODUCTION N e u r o p h a r m a c o l o g i c a l studies have d e m o n s t r a t e d that serotonin (5-HT) can act b o t h as a neurotransmitter and a n e u r o m o d u l a t o r in the central nervous system (CNS) of invertebrates, including molluscs 14'15'2x'22'46'52'54. Using biochemical techniques, the presence of e n d o g e n o u s serotonin was also proved in the gastropod C N S 29'47"49" 59. In gastropods, 5-HT can affect and m o d u l a t e a variety of behavioral and physiological processes, such as feeding 39'4°'44'56'5s, withdrawal and escape reaction 43, circulation 45'52, and some forms of learning 2'19'24"25'34'35' 36 Studies on the role of serotonergic neurons and pathways in various behaviors have benefited from the discovery that 5,6- and 5,7-dihydroxytryptamine ( 5 , 6 - D H T and 5,7-DHT), two dissimilar serotonin-related substituted tryptamines, are capable of causing selective degeneration and ablation of serotonergic neurons and pathways in the vertebrate CNS 4"5'7'9'11'12'33'42. In the CNS of invertebrates, including molluscs, the short term
(lasting from a few hours to about 3 weeks) effects of 5 , 6 - D H T include electrophysiological, biochemical and structural changes ts'3°'39"4°'41'51. A long t e r m effect, developing in 14-60 days after the injection of 5 , 6 - D H T or 5 , 7 - D H T into the b o d y cavity of different gastropod species, is that brown or orange pigmentation appears in particular groups of neurons in the CNS of the animals
(Helix pomatia 55, Helix lucorurn t, L ymnaea stagnalis 38, Aplysia californica31). C o m p a r a t i v e immunocytochemical studies in the CNS of Helix 26"27 and Lymnaea 3s have indicated that such pigmentation only appears in serotonergic neurons affected by the neurotoxic drug, and that this labelling could be used as a durable indicator of such neurons in the snail ganglia. Since the pigment-containing neurons have normal electrophysiological characteristics4°'55's7, the neurons apparently have recovered from the neurotoxic effects of 5,6- and 5 , 7 - D H T by the time the pigmentation develops. This is s u p p o r t e d by behavioral, electrophysiological and biochemical d a t a from Lymrnaea and Helix treated with 5 , 6 - D H T 4°'41"5v. However, no information is available on the causes of the
* Present address: Department of Biology, University of York, Heslington, York, Y01 5DD, UK. Correspondence: L. Hern~di, Balaton Limnological Research Institute of the Hungarian Academy of Sciences. Tihany, Hungary H-8237.
222 pigment formation and on the ultrastructural changes underlying the slowly developing and reversible effect of these neurotoxins in the molluscan CNS. The aim of the present study is to give a detailed and comparative description of the ultrastructural, biochemical and electrophysiological changes induced by 5,6DHT in the CNS of the snail Helix pomatia. These changes occur over a period of 3 months following the injection of the snails with 5,6-DHT. Using serotonin immunocytochemistry and intracellular horseradish peroxidase (HRP) labelling of serotonergic neurons in the CNS of 5,6-DHT-treated snails we provide further data which support the idea that the ultrastructural consequences of 5,6-DHT are specific for this neurotoxin as well as for neurons containing serotonin. Furthermore, we compare the temporal pattern of the changes with those described in the vertebrate central nervous system. MATERIALS AND METHODS
Treatment of the experimental animals The experiments were performed on adult specimens of Helix pomatia L., collected locally on the Tihany peninsula. The animals
ation the samples were dehydrated in graded alcohols and embedded in Spur's medium. During dehydration the samples were blockcontrasted in 70% ethanol saturated with uranyl acetate. Sections were cut from each individual ganglion, the thin sections were contrasted with lead citrate and investigated under electron microscope. To see if the neurotoxin 5,6-DHT affected the ultrastructure of serotonergic neuronal fibres and somata, two different techniques were used: 1. Fourteen days after the toxin injection, when pigment granules started to appear in the neurons, one of the serotonergic MGC's was intracellularly injected with HRP in two control and two 5,6-DHT injected preparations. The toxin-induced alterations were studied in the HRP-labelled neuronal fibres and cell bodies of the MGC's using the EM methods described above. 2. To see if serotonin-containing neuronal fibers in the neuropil were affected by 5,6-DHT, the CNS was dissected from 3 snails 12 days after the injection with 5,6-DHT and the CNS was fixed for electron microscopical immunocytochemistry in 4% paraformaldehyde buffered with 0.1 M cacodylate (pH 7.2). The ganglia were cut with vibrotome, the 100/~m thick slices were collected in phosphate-buffered saline (PBS) and used for electronmicroscopical immunocytochemistry according to Priestley5°. After osmification in 1% OsO4 buffered with 0.1 M cacodylate, the slices were dehydrated in graded ethanol and embedded in Spur epoxy resine. Block staining was performed in 70% ethanol saturated with uranyl acetate. The thin sections were contrasted with lead citrate and studied with the electron microscope.
were kept in laboratory for two weeks on wet tissue paper. 5,6DHT was injected in a single dose of 10 mg/kg b.w. into the body cavity of 250 snails in the form of 5,6-dihydroxytryptamine creatinine sulphate dissolved in 25/tl ascorbic acid solution 0.5 mg/ml). Control animals (n = 250) were only injected with 25/A of the cartier. The 5,6-DHT induces dark brown pigmentation and labels individual serotonergic cell bodies that can be seen with a stereomicroscope with normal illumination55. A detailed description of the injection protocol has been given by S.-Rrzsa et al. 55. It was found earlier that the pigmentation, serotonin content and immunoreactivity of the CNS and the serotonergic synaptic transmission of the metacerebral giant cell (MGC) showed seasonal variations 16"26"27'29. Therefore, the analysis of the effects of 5,6-DHT was carried out in autumn.
Electrophysiological experiments to monitor changes in the serotonergic synaptic transmission after injection of snails with 5,6-DHT
Neuroanatomical methods to follow ultrastructural changes after injection of snails with 5,6-DHT
Biochemical measurements to follow changes of serotonin (5-HT) and dopamine (DA) levels after injection with 5,6-DHT
Brains from 3 animals were dissected after 30 min, 1 h, 4 h, 24 h, as well as 2, 6, 12, 14, 21, 30 and 90 days following injection with the neurotoxin. The development of the pigmentation was tested on the serotonergic MGC in fresh wholemount preparations. For electron microscopy, the CNS was put into cold fixative containing 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) and kept in the fixative for 4 h at 4°C. The thick connective tissue sheath of the CNS was removed in the buffer and the individual ganglia of the CNS were separated and postfixed in 1.5% OsO4 buffered with 0.1 M s-collidine (pH 7.2) for 2 h at 4°C. After fix-
The monoamines were assayed by a Waters high-performance liquid chromatograph equipped with an electrochemical detector. Samples were collected from the pedal ganglia which contains most of the serotonergic neurons of the CNS, from the cerebral ganglia which contains a smaller number of the serotonergic neurons, and from the buccal ganglia which contains no serotonergic cell bodies, only a large number of serotonergic fibres26. These ganglia were dissected from 5 5,6-DHT-treated and control snails, respectively, 3 h, 24 h, 2, 3, 6, 9, 14, 17, 20 and 24 days after injection with vehicle or 5,6-DHT. In addition, from each animal 100 ml hae-
Electrophysiological experiments were carried out on isolated CNS preparations consisting of the ring ganglia and the buccal ganglia connected by the cerebro-buccal connectives. The serotonergic synaptic transmission was tested on the excitatory connection between the MGC and ipsilateral follower neurons A and M in the buccal ganglion, described by Cottrell and Macon ~6. The synaptic efficacy was tested daily in two preparations of both 5,6-DHT injected and vehicle injected animals during the first month after injection with 5,6-DHT. For another 2 months the synaptic efficacy was tested weekly in 3 preparations. For intracellular recording and stimulation conventional microelectrophysiological methods were used. All the experiments were carried out in normal Helix saline tr.
Figs. 1-7. Early structural alterations in the central nervous system (CNS) of Helix pomatia following injection of 5,6-dihydroxytryptamine (5,6-DHT) into the snails. Fig. 1. Thirty minutes after injection with the toxin the extracellular spaces are dilatated, predominantly in the fine neuropil areas (o). The glial elements are swollen (A). Fig. 2. Higher magnification picture of a swollen glial element ( A ) in the extraceUular space. Fig. 3. Thirty minutes after injection small dense deposits (small arrows) can be observed on the neuronal membranes. Fig. 4. Two days after injection with 5,6-DHT dense synaptic areas appear in the fine neuropil areas (o) of the ganglia. Fig. 5. Higher magnification picture of the fine neuropil (e) shows dense deposits (arrows) in the mitochondria. Fig. 6. Structural appearance of the neuropil area 6 days after the treatment. In the fibers and synaptic areas membranous structures appear (thick arrows). Fig. 7. At higher magnification, dense deposits (thin arrows) can still be detected in the mitochondria 6 days after injection with 5,6-DHT. The glial elements in the extracellular spaces are swollen (A). Original magnifications: Fig. 1, 5,000x; Fig. 2, 28,000x; Fig. 3, 48,000x; Fig. 4, 6,000×; Fig. 5, 16,000x; Fig. 6, 16,000x; Fig. 7, 28,000×. Bars = 1/~m,
223 molymph was used for assaying 5,6-DHT. The samples were homogenized in 0.1 M perchloric acid and centrifuged at 30,000×g
for 20 rain at 4°C. Aliquots of the clear supernatant were injected onto reverse phase C18 Nucleosil column (Whatman, 15 cm x 4.6
224
Figs. 8-12. Late structural alterations in the CNS of Helix pomatia following injection of 5,6-DHT into the snails. Fig. 8. Twelve days after injection, numerous fibres contain myelin-bodies which are frequently rolled up (arrows) and surrounded by gathered dense particles. Fig. 9. Similar rolled up myelin-bodies (arrows) can also be found in the synaptic areas of the CNS. Fig. 10. In some areas of the fine neuropil extremely dense synaptic areas with numerous empty vesicles can be observed, sometimes showing glomerular arrangement (A). Fig. 11. The structural appearance of the buccal neuropil 12 days after injection. The highly dense synaptic profiles with numerous small dense particles and empty vesicles are the most characteristic structural alterations (arrowheads). These profiles usually terminate on thick neuronal processes (ax). Fig. 12. Higher magnification picture from the same preparation showing dense particles (arrowheads) near the neuronal processes (ax). Original magnifications: Fig. 8, 10,000x; Fig. 9, 16,000x; Fig. 10, 17,500x; Fig. 11, 7,000x; Fig. 12, 17,500x. Bars = 1 ~m.
225 ram, 5/~m). The mobile phase contained 0.05 M sodium acetate, 0.2 M EDTA, 1 mM octanesulfonic acid, 10% methanol and the final pH was adjusted to 4.0 with citric acid. The flow rate was 1.0 ml/min and the column temperature was 4°C. The potential of the electrochemical detector was set at 0.7V. Biochemical measurements to follow changes of the uptake of serotonin (5-HT) and dopamine (DA) into neural tissue after injection with 5,6-DHT To investigate the direct pharmacological effect of 5,6-DHT on the in vitro uptake of [3H]5-HT and [3H]DA, the CNS were dissected from 30 uninjected animals and incubated for 30 min in
[3H]5-HT (n = 15) or [3H]DA n = 15) according to Osborne et al. 48. Radioactivity measurements were carried out on homogenized samples from 6 control and 24 experimental brains. The latter were incubated with the tritiated amines in the presence of 5,6DHT in the incubation medium at concentrations from 10 -6 t o 10 -3 M. The 50% inhibitory concentrations (IC50) of 5,6-DHT for the tritiated amines were determined graphically from log-probit plots. A detailed description of the measurement and evaluation procedure has been given by Kemenes et al. 41. To investigate the in vivo toxic effect of 5,6-DHT on the serotonin uptake mechanism, the [3H]5-HT uptake was also measured in ganglia dissected from vehicle (n = 5) and 5,6-DHT-injected (n = 5) animals on the 5th and 30th days following the injection. RESULTS
Time course of ultrastructural changes induced by 5,6DHT in the CNS of Helix In samples taken during the first day after the treatm e n t extreme dilatation of the extracellular spaces was the most conspicuous ultrastructural alteration in the CNS. This was most p r o m i n e n t in the d e e p fine neuropil areas of the ganglia. This effect was already seen within 30 min of the neurotoxin injection (Fig. 1). The glial elements in the CNS were also swollen (Fig. 2). Parallel with this p h e n o m e n o n , small dense deposits were seen scattered r a n d o m l y on the surface of the neuronal membrane (Fig. 3). Two days after injection neuronal fibres with increased diffuse density were observed in each ganglion. These fibers had a large n u m b e r of clear and a small n u m b e r of dense core vesicles (Fig. 4). In some terminals or
synaptic structures the mitochondria had small dense particles (Fig. 5). A t this time ultrastructural alterations in the perikarya of the serotonergic M G C were not yet observed. By the 6th day after injection with 5,6-DHT, m e m b r a nous structures a p p e a r e d in synaptic-like areas, and these contained small clear and dense-core vesicles (Figs. 6 and 7). O n the neuronal fibres swellings and irregularities were frequently observed (Figs. 6 and 7). O n the 12th day following the injection, m e m b r a n o u s structures such as myelin-bodies were already seen both in the synaptic-like areas and the neuronal fibres. These structures often had a rolled-up a p p e a r a n c e (Figs. 8 and 9). The affected synaptic-like areas did not possess true synaptic specializations such as thickening of the pre- or postsynaptic m e m b r a n e (Figs. 8, 9, 10 and 12). A r o u n d the myelin-bodies, electronlucent hollow and small dense particles were usually observed (Figs. 8 and 9). In some parts of the neuropil of the ganglia, the numerous nerve terminals showed increased osmiofilia and sometimes app e a r e d in a glomerular a r r a n g e m e n t (Fig. 10). In the buccal ganglia where there are no serotonergic cell bodies only axons 26, the nerve terminals with increased osmiofilia r e p r e s e n t e d the only toxin-induced structural alterations (Figs. 11 and 12). Following an increase in the n u m b e r of structures in the synaptic-like areas and axons, similar structures app e a r e d in the p e r i k a r y o n of the M G C by 14 days after the injection. These were first seen in the area of the axon hillock (Fig. 19). This was also the time when the first dark pigment granules were seen in the soma of the M G C (Fig. 18) in wholemount preparations. By the 21st day after the injection cytosome- and lipofuscin-like granules together with numerous myelinbodies started to a p p e a r in the p e f i k a r y o n of the serotonergic M G C . The n u m b e r of these a b n o r m a l structures then increased throughout the rest of the postinjection p e r i o d studied (Figs. 18 and 19). By the 30th day after
Figs. 13-15. Structural appearance of the neuropil of the CNS of Helix pomatia in the recovery phase following injection of 5,6-DHT into the snails. Fig. 13. By the 30th day after injection the dilatation of the extracellular spaces has disappeared, but the myelin-bodies and the gathered dense particles can be still detected in the synaptic areas (arrows). Fig. 14. Myelin-bodies and gathered dense particles are present even 90 days after treatment. Fig. 15. In the buccal ganglia the toxin-affected dense synaptic profiles show an inner clear area with scattered dense particles (curved arrows). Original magnifications: Fig. 13, 8,000x; Fig. 14, 20,000×; Fig. 15, 20,000×. Bars = 1/~m. Figs. 16-21. The appearance of different granular forms in the somata of metacerebral giant cell (MGC) at different times after the injection of the neurotoxin. Fig. 16. In wholemount preparations, in the control MGC dark pigment granules can not be observed, only natural pigments (arrowhead) are present. Fig. 17. In thin sections made from a control preparation only lipofuscine-like dense granules can be detected (arrows). Fig. 18. In wholemount preparations by 14 days after the injection dark pigment granules (arrowhead) appear in the MGC. Fig. 19. In thin sections made from a preparation 14 days after injection numerous rolled-up myelin-bodies (arrows) are present in the cytoplasm. Fig. 20. Thirty days after the injection the MGC is full of dark pigment granules (arrow) in the wholemount preparation. Fig. 21. In thin sections made from preparation 30 days after treatment different transient forms of the pigment granules can be seen. Besides the lipofuscine-like dense granules (arrows), various forms of membranous bodies (arrowheads) can be also be detected. Original magnifications: Fig. 16, 200x: Fig, 17, 15,000x: Fig. 18, 200x; Fig. 19, 16,000x; Fig. 20, 200x; Fig. 21, 12,000x. Bars = 1 pm on Figs. 17, 19 and 21; 100 /~m on Figs. 16, 18 and 20.
226
Figs. 13-21. For legend see p. 225.
227
Figs. 22-29. For legend see p. 228.
228 Figs, 22-29. Neurotoxin-induced alterations in horseradish peroxidase (HRP)-labelled and 5-HT immunostained neural elements of the Helix CNS. Fig. 22. Light microscopical appearance of a HRP-injected MGC from a control preparation. The unipolar MGC gives off two main branches, an ipsilateral (il) and a contralateral one (cL). Fig. 23. At higher magnification numerous side branches can be observed at the proximal segment of the contralateral branch of the HRP-filled MGC. The side branches run close to cell bodies and terminate near or on cell bodies (arrows). Fig. 24. Neurotoxin-induced structural alterations in a HRP-labelled serotonergic MGC 14 days after the injection of 5,6-DHT into the snail. In the perikaryon groups of rolled-up myelin-bodies can be seen (arrows) around the nucleus (N), Fig. 25. In the labelled neuronal processes myelin-bodies surrounded by dense particles can be detected (small arrows). The inset in Fig. 25 shows the area in the rectangle at higher magnification. Fig. 26. Neurotoxin-induced structural alterations in serotonin immunoreactive neuronal fibers 12 days after the injection. In the immunoreactive fibers large empty vesicles (A) and myelin-bodies (arrows) are present. Fig. 27. Higher magnification picture from the same preparation showing myelin-bodies also characteristic of 5,6-DHT-treated but not immunostained preparations 12 days after treatment (see Figs. 8 and 9). Figs. 28 and 29. In other sections from the same preparation gathered dense particles (o) can be identified in immunostained fibers. Original magnifications. Fig. 22, 120×; Fig. 23, 200x. Bars = 100 mm; Fig. 24, 8,000x; Fig. 25, 6,000x; insert, 15,000x; Fig. 26, 15,000x; Fig. 27, 42,000x; Fig. 28. 30,000x: Fig. 29, 20,000x. Bars = 1 pro.
the injection they became d o m i n a n t in the perikaryon and appeared as pigment granules in the wholemount preparations (Figs. 20 and 21). These structures were observed only in a low n u m b e r in the perikaryon of the control M G C (Figs. 16 and 17). By 30 days following the treatment, the n u m b e r of myelin-bodies increased considerably and they invaded the whole perikaryon (Figs. 20 and 21). By this time,
when the serotonergic cell bodies were already fully pigmented in w h o l e m o u n t preparations, the dilatation of the extracellular spaces had already decreased, The myelin-bodies were frequently surrounded by highly dense particles in the fibres and synaptic areas at the end of the first m o n t h after injection with 5 , 6 - D H T (Fig. 13). They were detected even 90 days after the injection, but less often than in the earlier periods following the treatment (Fig. 14). By the 90th day after the injection, in the neuropil of
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03 h 1 3 5 7 9 11 13 15 17 19 21 23 OAYS Fig. 30. Mean 5-hydroxytryptamine (5-HT), dopamine (DA) and 5,6-DHT levels in the cerebral (A), pedal (B) and buccal (C) ganglia at different times following injection of 5,6-DHT into snails. 5-HT and DA levels are expressed as a percentage of concentrations measured in vehicle-injected control snails. 5,6-DHT concentration is expressed in pmol/ganglion. The S.E.M.s were less than 14% of the mean values which are the means of 5 preparations at each point,
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Fig. 31. Excitatory synaptic connection between the metacerebral giant cell (MGC) and its follower neurons. A: discharge of the MGC evoked by current injection evokes increased activity in the buccal follower neuron A. B: after hyperpolarizing the membrane of the buccal follower neuron M summated EPSPs can be evoked by stimulating the presynaptic MGC.
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ls Fig. 32. Injection of snails with 5,6-DHT transiently impairs the synaptic transmission between the MGC and the buccal followers A and M. In the first period (1-3rd day) after the injection no change in the excitatory connection can be recorded. On the 5th day following the treatment the synaptic transmission between the MGC and its followers is weaker and ceases by the 15th day following the injection. On the 21st day the excitatory connections are restored between the MGC and its follower neurons. bodies and the dense particles became the most dominant alterations.
Ultrastructural analysis of the 5,6-DHT-pigmented and HRP-labelled MGC In the perikaryon of the HRP-labelled MGC numerous myelin-bodies were seen on the 14th day following the injection of snails with 5,6-DHT (Fig. 24). In the processes of the cell, typical toxin-induced alterations, such as various myelin-bodies and gathered glycogen-like particles were observed. These were most prominent in the contralateral projection of the main axon and in its side branches (Fig. 25) which run close to the neuronal cell layer and terminate near or on neuronal cell bodies in the metacerebrum (Figs. 22 and 23). Distribution of serotonin immunoreactivity in neuronal elements of the CNS of 5,6-DHT-treated snails Serotonin immunoreactive fibers were observed in small groups in the neuropil areas. The immunoreactivity was found in neuronal processes and synaptic-like areas showing 5,6-DHT-induced morphological alteration affected appearance (Fig. 26). These labelled neuronal elements contained myelin-bodies surrounded by large electronlucent areas (Figs. 26 and 27), and dense particles (Figs. 28 and 29) that were typical in samples from 5,6-DHT treated snails. Frequently, the myelin-bodies showed immunoreactivity (Figs. 26 and 27). The effect of 5,6-DHT on serotonin (5-HT) and dopamine (DA) levels of the CNS In the cerebral and pedal ganglia 5,6-DHT increased
both 5-HT and D A levels within 3 h following injection of the snails with the toxin (Fig. 30A,B). The increase was more prominent in the pedal ganglia (Fig, 30B). In the buccal ganglia, an increase of the concentration of endogenous D A took place between 3 and 24 h after treatment but 5-HT levels started to drop. (Fig. 30C). In all 3 ganglia the concentration of both 5-HT and D A was lower during the first week after the injection in 5,6-DHT-treated animals than in controls. The concentrations reached a minimum at 50-70% of the control level by the 2nd or 3rd day after the application of the drug (Fig. 30). The decrease was smallest in the cerebral ganglia and largest in the buccal ganglia which has no serotonergic cell bodies, only serotonergic axons (Fig. 30). The DA concentration returned to the control level by the 7-8th days after injection and then had a slight overshoot. The 5-HT concentration returned to the control level only by the 19th-21st day following the injection and showed a slight overshoot later. On the day of the injection, 5,6-DHT was detected in high concentration in each ganglion of the CNS but it decreased to a low constant level by the 3rd-5th day. Some free toxin was still present even 30 days after the treatment (Fig. 30). In the haemolymph (not shown in Fig, 30) 5,6-DHT was detected in a very variable concentration during the first 3 days following the injection, but could not be assayed from the 5th to the 7th day following the treatment.
The in vitro pharmacological and the in vivo toxic effects into the CNS 5,6-DHT, when applied in vitro at concentrations from 10 -6 t o 10 -3 M significantly reduced the in vitro uptake of both [3H]5-HT and [3H]DA. The concentration of 5,6-DHT sufficient to reduce uptake by 50% (IC50) was 8.5)
221
BRES 17643
Ultrastructural, biochemical and electrophysiological changes induced by 5,6-dihydroxytryptamine in the CNS of the snail Helix pomatia L. L~iszl6 Hern~idi, L~iszl6 Hiripi, /~gnes Vehovszky, GyOrgy Kemenes* and Katalin S.-R6zsa Balaton Limnological Research Institute of the Hungarian Academy of Sciences, Tihany (Hungary) (Accepted 10 December 1991)
Key words: Gastropod; Central nervous system; Neurotoxin; 5.6-Dihydroxytryptamine; Serotonin
The serotonin neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT), was injected into the body cavity of snails. Changes induced in the central nervous system (CNS) by the neurotoxin were studied by morphological, electrophysiological and biochemical techniques for up to 90 days following injection. The neurotoxin induced a variety of ultrastructural alterations during the early phase (1st to 6th days) following treatment. On day 6 after treatment, membranous structures first appeared in the synaptic-like areas and apparently migrated to cell bodies where they were detected by day 14. Their number increased with time. Neurotoxin-induced structural alterations were found in neuronal processes and cell bodies of the serotonergic metacerebral giant cells injected intracellularly with horseradish peroxidase and in serotonin immunoreactive axons. These findings suggest that the toxin-induced alterations are rather selective for the serotonin-containing neuronal elements. The neurotoxin decreased the concentration of 5-HT in and [3H]5-HT uptake into cerebral and pedal ganglia, with a maximum effect between the 3rd and 5th day following drug administration. 5-HT levels and 5-HT uptake returned to normal by 19-21 days after treatment. The concentration of dopamine and of [3H]DA uptake capacity were reduced between 3-5 days after injection of 5,6-DHT by 6-7 days following treatment. The transmission from identified serotonergic synapses to targets was reduced beyond day 5 after 5,6-DHT administration. By 15 days after treatment, synaptic transmission between the metacerebral giant cell (MGC) and buccal followers was blocked. Transmission recovered by day 21 after 5,6-DHT. Comparison of the time-course of functional and structural recovery indicates that while functional recovery takes place within 21 days after treatment, certain structural alterations, e.g. the membranous structures and dense particles, remain in the nerve fibres and cell bodies. These may serve as specific intracellular markers of the serotonin-containing neuronal elements long after functional recovery from the effect of 5,6-DHT. INTRODUCTION N e u r o p h a r m a c o l o g i c a l studies have d e m o n s t r a t e d that serotonin (5-HT) can act b o t h as a neurotransmitter and a n e u r o m o d u l a t o r in the central nervous system (CNS) of invertebrates, including molluscs 14'15'2x'22'46'52'54. Using biochemical techniques, the presence of e n d o g e n o u s serotonin was also proved in the gastropod C N S 29'47"49" 59. In gastropods, 5-HT can affect and m o d u l a t e a variety of behavioral and physiological processes, such as feeding 39'4°'44'56'5s, withdrawal and escape reaction 43, circulation 45'52, and some forms of learning 2'19'24"25'34'35' 36 Studies on the role of serotonergic neurons and pathways in various behaviors have benefited from the discovery that 5,6- and 5,7-dihydroxytryptamine ( 5 , 6 - D H T and 5,7-DHT), two dissimilar serotonin-related substituted tryptamines, are capable of causing selective degeneration and ablation of serotonergic neurons and pathways in the vertebrate CNS 4"5'7'9'11'12'33'42. In the CNS of invertebrates, including molluscs, the short term
(lasting from a few hours to about 3 weeks) effects of 5 , 6 - D H T include electrophysiological, biochemical and structural changes ts'3°'39"4°'41'51. A long t e r m effect, developing in 14-60 days after the injection of 5 , 6 - D H T or 5 , 7 - D H T into the b o d y cavity of different gastropod species, is that brown or orange pigmentation appears in particular groups of neurons in the CNS of the animals
(Helix pomatia 55, Helix lucorurn t, L ymnaea stagnalis 38, Aplysia californica31). C o m p a r a t i v e immunocytochemical studies in the CNS of Helix 26"27 and Lymnaea 3s have indicated that such pigmentation only appears in serotonergic neurons affected by the neurotoxic drug, and that this labelling could be used as a durable indicator of such neurons in the snail ganglia. Since the pigment-containing neurons have normal electrophysiological characteristics4°'55's7, the neurons apparently have recovered from the neurotoxic effects of 5,6- and 5 , 7 - D H T by the time the pigmentation develops. This is s u p p o r t e d by behavioral, electrophysiological and biochemical d a t a from Lymrnaea and Helix treated with 5 , 6 - D H T 4°'41"5v. However, no information is available on the causes of the
* Present address: Department of Biology, University of York, Heslington, York, Y01 5DD, UK. Correspondence: L. Hern~di, Balaton Limnological Research Institute of the Hungarian Academy of Sciences. Tihany, Hungary H-8237.
222 pigment formation and on the ultrastructural changes underlying the slowly developing and reversible effect of these neurotoxins in the molluscan CNS. The aim of the present study is to give a detailed and comparative description of the ultrastructural, biochemical and electrophysiological changes induced by 5,6DHT in the CNS of the snail Helix pomatia. These changes occur over a period of 3 months following the injection of the snails with 5,6-DHT. Using serotonin immunocytochemistry and intracellular horseradish peroxidase (HRP) labelling of serotonergic neurons in the CNS of 5,6-DHT-treated snails we provide further data which support the idea that the ultrastructural consequences of 5,6-DHT are specific for this neurotoxin as well as for neurons containing serotonin. Furthermore, we compare the temporal pattern of the changes with those described in the vertebrate central nervous system. MATERIALS AND METHODS
Treatment of the experimental animals The experiments were performed on adult specimens of Helix pomatia L., collected locally on the Tihany peninsula. The animals
ation the samples were dehydrated in graded alcohols and embedded in Spur's medium. During dehydration the samples were blockcontrasted in 70% ethanol saturated with uranyl acetate. Sections were cut from each individual ganglion, the thin sections were contrasted with lead citrate and investigated under electron microscope. To see if the neurotoxin 5,6-DHT affected the ultrastructure of serotonergic neuronal fibres and somata, two different techniques were used: 1. Fourteen days after the toxin injection, when pigment granules started to appear in the neurons, one of the serotonergic MGC's was intracellularly injected with HRP in two control and two 5,6-DHT injected preparations. The toxin-induced alterations were studied in the HRP-labelled neuronal fibres and cell bodies of the MGC's using the EM methods described above. 2. To see if serotonin-containing neuronal fibers in the neuropil were affected by 5,6-DHT, the CNS was dissected from 3 snails 12 days after the injection with 5,6-DHT and the CNS was fixed for electron microscopical immunocytochemistry in 4% paraformaldehyde buffered with 0.1 M cacodylate (pH 7.2). The ganglia were cut with vibrotome, the 100/~m thick slices were collected in phosphate-buffered saline (PBS) and used for electronmicroscopical immunocytochemistry according to Priestley5°. After osmification in 1% OsO4 buffered with 0.1 M cacodylate, the slices were dehydrated in graded ethanol and embedded in Spur epoxy resine. Block staining was performed in 70% ethanol saturated with uranyl acetate. The thin sections were contrasted with lead citrate and studied with the electron microscope.
were kept in laboratory for two weeks on wet tissue paper. 5,6DHT was injected in a single dose of 10 mg/kg b.w. into the body cavity of 250 snails in the form of 5,6-dihydroxytryptamine creatinine sulphate dissolved in 25/tl ascorbic acid solution 0.5 mg/ml). Control animals (n = 250) were only injected with 25/A of the cartier. The 5,6-DHT induces dark brown pigmentation and labels individual serotonergic cell bodies that can be seen with a stereomicroscope with normal illumination55. A detailed description of the injection protocol has been given by S.-Rrzsa et al. 55. It was found earlier that the pigmentation, serotonin content and immunoreactivity of the CNS and the serotonergic synaptic transmission of the metacerebral giant cell (MGC) showed seasonal variations 16"26"27'29. Therefore, the analysis of the effects of 5,6-DHT was carried out in autumn.
Electrophysiological experiments to monitor changes in the serotonergic synaptic transmission after injection of snails with 5,6-DHT
Neuroanatomical methods to follow ultrastructural changes after injection of snails with 5,6-DHT
Biochemical measurements to follow changes of serotonin (5-HT) and dopamine (DA) levels after injection with 5,6-DHT
Brains from 3 animals were dissected after 30 min, 1 h, 4 h, 24 h, as well as 2, 6, 12, 14, 21, 30 and 90 days following injection with the neurotoxin. The development of the pigmentation was tested on the serotonergic MGC in fresh wholemount preparations. For electron microscopy, the CNS was put into cold fixative containing 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) and kept in the fixative for 4 h at 4°C. The thick connective tissue sheath of the CNS was removed in the buffer and the individual ganglia of the CNS were separated and postfixed in 1.5% OsO4 buffered with 0.1 M s-collidine (pH 7.2) for 2 h at 4°C. After fix-
The monoamines were assayed by a Waters high-performance liquid chromatograph equipped with an electrochemical detector. Samples were collected from the pedal ganglia which contains most of the serotonergic neurons of the CNS, from the cerebral ganglia which contains a smaller number of the serotonergic neurons, and from the buccal ganglia which contains no serotonergic cell bodies, only a large number of serotonergic fibres26. These ganglia were dissected from 5 5,6-DHT-treated and control snails, respectively, 3 h, 24 h, 2, 3, 6, 9, 14, 17, 20 and 24 days after injection with vehicle or 5,6-DHT. In addition, from each animal 100 ml hae-
Electrophysiological experiments were carried out on isolated CNS preparations consisting of the ring ganglia and the buccal ganglia connected by the cerebro-buccal connectives. The serotonergic synaptic transmission was tested on the excitatory connection between the MGC and ipsilateral follower neurons A and M in the buccal ganglion, described by Cottrell and Macon ~6. The synaptic efficacy was tested daily in two preparations of both 5,6-DHT injected and vehicle injected animals during the first month after injection with 5,6-DHT. For another 2 months the synaptic efficacy was tested weekly in 3 preparations. For intracellular recording and stimulation conventional microelectrophysiological methods were used. All the experiments were carried out in normal Helix saline tr.
Figs. 1-7. Early structural alterations in the central nervous system (CNS) of Helix pomatia following injection of 5,6-dihydroxytryptamine (5,6-DHT) into the snails. Fig. 1. Thirty minutes after injection with the toxin the extracellular spaces are dilatated, predominantly in the fine neuropil areas (o). The glial elements are swollen (A). Fig. 2. Higher magnification picture of a swollen glial element ( A ) in the extraceUular space. Fig. 3. Thirty minutes after injection small dense deposits (small arrows) can be observed on the neuronal membranes. Fig. 4. Two days after injection with 5,6-DHT dense synaptic areas appear in the fine neuropil areas (o) of the ganglia. Fig. 5. Higher magnification picture of the fine neuropil (e) shows dense deposits (arrows) in the mitochondria. Fig. 6. Structural appearance of the neuropil area 6 days after the treatment. In the fibers and synaptic areas membranous structures appear (thick arrows). Fig. 7. At higher magnification, dense deposits (thin arrows) can still be detected in the mitochondria 6 days after injection with 5,6-DHT. The glial elements in the extracellular spaces are swollen (A). Original magnifications: Fig. 1, 5,000x; Fig. 2, 28,000x; Fig. 3, 48,000x; Fig. 4, 6,000×; Fig. 5, 16,000x; Fig. 6, 16,000x; Fig. 7, 28,000×. Bars = 1/~m,
223 molymph was used for assaying 5,6-DHT. The samples were homogenized in 0.1 M perchloric acid and centrifuged at 30,000×g
for 20 rain at 4°C. Aliquots of the clear supernatant were injected onto reverse phase C18 Nucleosil column (Whatman, 15 cm x 4.6
224
Figs. 8-12. Late structural alterations in the CNS of Helix pomatia following injection of 5,6-DHT into the snails. Fig. 8. Twelve days after injection, numerous fibres contain myelin-bodies which are frequently rolled up (arrows) and surrounded by gathered dense particles. Fig. 9. Similar rolled up myelin-bodies (arrows) can also be found in the synaptic areas of the CNS. Fig. 10. In some areas of the fine neuropil extremely dense synaptic areas with numerous empty vesicles can be observed, sometimes showing glomerular arrangement (A). Fig. 11. The structural appearance of the buccal neuropil 12 days after injection. The highly dense synaptic profiles with numerous small dense particles and empty vesicles are the most characteristic structural alterations (arrowheads). These profiles usually terminate on thick neuronal processes (ax). Fig. 12. Higher magnification picture from the same preparation showing dense particles (arrowheads) near the neuronal processes (ax). Original magnifications: Fig. 8, 10,000x; Fig. 9, 16,000x; Fig. 10, 17,500x; Fig. 11, 7,000x; Fig. 12, 17,500x. Bars = 1 ~m.
225 ram, 5/~m). The mobile phase contained 0.05 M sodium acetate, 0.2 M EDTA, 1 mM octanesulfonic acid, 10% methanol and the final pH was adjusted to 4.0 with citric acid. The flow rate was 1.0 ml/min and the column temperature was 4°C. The potential of the electrochemical detector was set at 0.7V. Biochemical measurements to follow changes of the uptake of serotonin (5-HT) and dopamine (DA) into neural tissue after injection with 5,6-DHT To investigate the direct pharmacological effect of 5,6-DHT on the in vitro uptake of [3H]5-HT and [3H]DA, the CNS were dissected from 30 uninjected animals and incubated for 30 min in
[3H]5-HT (n = 15) or [3H]DA n = 15) according to Osborne et al. 48. Radioactivity measurements were carried out on homogenized samples from 6 control and 24 experimental brains. The latter were incubated with the tritiated amines in the presence of 5,6DHT in the incubation medium at concentrations from 10 -6 t o 10 -3 M. The 50% inhibitory concentrations (IC50) of 5,6-DHT for the tritiated amines were determined graphically from log-probit plots. A detailed description of the measurement and evaluation procedure has been given by Kemenes et al. 41. To investigate the in vivo toxic effect of 5,6-DHT on the serotonin uptake mechanism, the [3H]5-HT uptake was also measured in ganglia dissected from vehicle (n = 5) and 5,6-DHT-injected (n = 5) animals on the 5th and 30th days following the injection. RESULTS
Time course of ultrastructural changes induced by 5,6DHT in the CNS of Helix In samples taken during the first day after the treatm e n t extreme dilatation of the extracellular spaces was the most conspicuous ultrastructural alteration in the CNS. This was most p r o m i n e n t in the d e e p fine neuropil areas of the ganglia. This effect was already seen within 30 min of the neurotoxin injection (Fig. 1). The glial elements in the CNS were also swollen (Fig. 2). Parallel with this p h e n o m e n o n , small dense deposits were seen scattered r a n d o m l y on the surface of the neuronal membrane (Fig. 3). Two days after injection neuronal fibres with increased diffuse density were observed in each ganglion. These fibers had a large n u m b e r of clear and a small n u m b e r of dense core vesicles (Fig. 4). In some terminals or
synaptic structures the mitochondria had small dense particles (Fig. 5). A t this time ultrastructural alterations in the perikarya of the serotonergic M G C were not yet observed. By the 6th day after injection with 5,6-DHT, m e m b r a nous structures a p p e a r e d in synaptic-like areas, and these contained small clear and dense-core vesicles (Figs. 6 and 7). O n the neuronal fibres swellings and irregularities were frequently observed (Figs. 6 and 7). O n the 12th day following the injection, m e m b r a n o u s structures such as myelin-bodies were already seen both in the synaptic-like areas and the neuronal fibres. These structures often had a rolled-up a p p e a r a n c e (Figs. 8 and 9). The affected synaptic-like areas did not possess true synaptic specializations such as thickening of the pre- or postsynaptic m e m b r a n e (Figs. 8, 9, 10 and 12). A r o u n d the myelin-bodies, electronlucent hollow and small dense particles were usually observed (Figs. 8 and 9). In some parts of the neuropil of the ganglia, the numerous nerve terminals showed increased osmiofilia and sometimes app e a r e d in a glomerular a r r a n g e m e n t (Fig. 10). In the buccal ganglia where there are no serotonergic cell bodies only axons 26, the nerve terminals with increased osmiofilia r e p r e s e n t e d the only toxin-induced structural alterations (Figs. 11 and 12). Following an increase in the n u m b e r of structures in the synaptic-like areas and axons, similar structures app e a r e d in the p e r i k a r y o n of the M G C by 14 days after the injection. These were first seen in the area of the axon hillock (Fig. 19). This was also the time when the first dark pigment granules were seen in the soma of the M G C (Fig. 18) in wholemount preparations. By the 21st day after the injection cytosome- and lipofuscin-like granules together with numerous myelinbodies started to a p p e a r in the p e f i k a r y o n of the serotonergic M G C . The n u m b e r of these a b n o r m a l structures then increased throughout the rest of the postinjection p e r i o d studied (Figs. 18 and 19). By the 30th day after
Figs. 13-15. Structural appearance of the neuropil of the CNS of Helix pomatia in the recovery phase following injection of 5,6-DHT into the snails. Fig. 13. By the 30th day after injection the dilatation of the extracellular spaces has disappeared, but the myelin-bodies and the gathered dense particles can be still detected in the synaptic areas (arrows). Fig. 14. Myelin-bodies and gathered dense particles are present even 90 days after treatment. Fig. 15. In the buccal ganglia the toxin-affected dense synaptic profiles show an inner clear area with scattered dense particles (curved arrows). Original magnifications: Fig. 13, 8,000x; Fig. 14, 20,000×; Fig. 15, 20,000×. Bars = 1/~m. Figs. 16-21. The appearance of different granular forms in the somata of metacerebral giant cell (MGC) at different times after the injection of the neurotoxin. Fig. 16. In wholemount preparations, in the control MGC dark pigment granules can not be observed, only natural pigments (arrowhead) are present. Fig. 17. In thin sections made from a control preparation only lipofuscine-like dense granules can be detected (arrows). Fig. 18. In wholemount preparations by 14 days after the injection dark pigment granules (arrowhead) appear in the MGC. Fig. 19. In thin sections made from a preparation 14 days after injection numerous rolled-up myelin-bodies (arrows) are present in the cytoplasm. Fig. 20. Thirty days after the injection the MGC is full of dark pigment granules (arrow) in the wholemount preparation. Fig. 21. In thin sections made from preparation 30 days after treatment different transient forms of the pigment granules can be seen. Besides the lipofuscine-like dense granules (arrows), various forms of membranous bodies (arrowheads) can be also be detected. Original magnifications: Fig. 16, 200x: Fig, 17, 15,000x: Fig. 18, 200x; Fig. 19, 16,000x; Fig. 20, 200x; Fig. 21, 12,000x. Bars = 1 pm on Figs. 17, 19 and 21; 100 /~m on Figs. 16, 18 and 20.
226
Figs. 13-21. For legend see p. 225.
227
Figs. 22-29. For legend see p. 228.
228 Figs, 22-29. Neurotoxin-induced alterations in horseradish peroxidase (HRP)-labelled and 5-HT immunostained neural elements of the Helix CNS. Fig. 22. Light microscopical appearance of a HRP-injected MGC from a control preparation. The unipolar MGC gives off two main branches, an ipsilateral (il) and a contralateral one (cL). Fig. 23. At higher magnification numerous side branches can be observed at the proximal segment of the contralateral branch of the HRP-filled MGC. The side branches run close to cell bodies and terminate near or on cell bodies (arrows). Fig. 24. Neurotoxin-induced structural alterations in a HRP-labelled serotonergic MGC 14 days after the injection of 5,6-DHT into the snail. In the perikaryon groups of rolled-up myelin-bodies can be seen (arrows) around the nucleus (N), Fig. 25. In the labelled neuronal processes myelin-bodies surrounded by dense particles can be detected (small arrows). The inset in Fig. 25 shows the area in the rectangle at higher magnification. Fig. 26. Neurotoxin-induced structural alterations in serotonin immunoreactive neuronal fibers 12 days after the injection. In the immunoreactive fibers large empty vesicles (A) and myelin-bodies (arrows) are present. Fig. 27. Higher magnification picture from the same preparation showing myelin-bodies also characteristic of 5,6-DHT-treated but not immunostained preparations 12 days after treatment (see Figs. 8 and 9). Figs. 28 and 29. In other sections from the same preparation gathered dense particles (o) can be identified in immunostained fibers. Original magnifications. Fig. 22, 120×; Fig. 23, 200x. Bars = 100 mm; Fig. 24, 8,000x; Fig. 25, 6,000x; insert, 15,000x; Fig. 26, 15,000x; Fig. 27, 42,000x; Fig. 28. 30,000x: Fig. 29, 20,000x. Bars = 1 pro.
the injection they became d o m i n a n t in the perikaryon and appeared as pigment granules in the wholemount preparations (Figs. 20 and 21). These structures were observed only in a low n u m b e r in the perikaryon of the control M G C (Figs. 16 and 17). By 30 days following the treatment, the n u m b e r of myelin-bodies increased considerably and they invaded the whole perikaryon (Figs. 20 and 21). By this time,
when the serotonergic cell bodies were already fully pigmented in w h o l e m o u n t preparations, the dilatation of the extracellular spaces had already decreased, The myelin-bodies were frequently surrounded by highly dense particles in the fibres and synaptic areas at the end of the first m o n t h after injection with 5 , 6 - D H T (Fig. 13). They were detected even 90 days after the injection, but less often than in the earlier periods following the treatment (Fig. 14). By the 90th day after the injection, in the neuropil of
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03 h 1 3 5 7 9 11 13 15 17 19 21 23 OAYS Fig. 30. Mean 5-hydroxytryptamine (5-HT), dopamine (DA) and 5,6-DHT levels in the cerebral (A), pedal (B) and buccal (C) ganglia at different times following injection of 5,6-DHT into snails. 5-HT and DA levels are expressed as a percentage of concentrations measured in vehicle-injected control snails. 5,6-DHT concentration is expressed in pmol/ganglion. The S.E.M.s were less than 14% of the mean values which are the means of 5 preparations at each point,
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Ultrastructural analysis of the 5,6-DHT-pigmented and HRP-labelled MGC In the perikaryon of the HRP-labelled MGC numerous myelin-bodies were seen on the 14th day following the injection of snails with 5,6-DHT (Fig. 24). In the processes of the cell, typical toxin-induced alterations, such as various myelin-bodies and gathered glycogen-like particles were observed. These were most prominent in the contralateral projection of the main axon and in its side branches (Fig. 25) which run close to the neuronal cell layer and terminate near or on neuronal cell bodies in the metacerebrum (Figs. 22 and 23). Distribution of serotonin immunoreactivity in neuronal elements of the CNS of 5,6-DHT-treated snails Serotonin immunoreactive fibers were observed in small groups in the neuropil areas. The immunoreactivity was found in neuronal processes and synaptic-like areas showing 5,6-DHT-induced morphological alteration affected appearance (Fig. 26). These labelled neuronal elements contained myelin-bodies surrounded by large electronlucent areas (Figs. 26 and 27), and dense particles (Figs. 28 and 29) that were typical in samples from 5,6-DHT treated snails. Frequently, the myelin-bodies showed immunoreactivity (Figs. 26 and 27). The effect of 5,6-DHT on serotonin (5-HT) and dopamine (DA) levels of the CNS In the cerebral and pedal ganglia 5,6-DHT increased
both 5-HT and D A levels within 3 h following injection of the snails with the toxin (Fig. 30A,B). The increase was more prominent in the pedal ganglia (Fig, 30B). In the buccal ganglia, an increase of the concentration of endogenous D A took place between 3 and 24 h after treatment but 5-HT levels started to drop. (Fig. 30C). In all 3 ganglia the concentration of both 5-HT and D A was lower during the first week after the injection in 5,6-DHT-treated animals than in controls. The concentrations reached a minimum at 50-70% of the control level by the 2nd or 3rd day after the application of the drug (Fig. 30). The decrease was smallest in the cerebral ganglia and largest in the buccal ganglia which has no serotonergic cell bodies, only serotonergic axons (Fig. 30). The DA concentration returned to the control level by the 7-8th days after injection and then had a slight overshoot. The 5-HT concentration returned to the control level only by the 19th-21st day following the injection and showed a slight overshoot later. On the day of the injection, 5,6-DHT was detected in high concentration in each ganglion of the CNS but it decreased to a low constant level by the 3rd-5th day. Some free toxin was still present even 30 days after the treatment (Fig. 30). In the haemolymph (not shown in Fig, 30) 5,6-DHT was detected in a very variable concentration during the first 3 days following the injection, but could not be assayed from the 5th to the 7th day following the treatment.
The in vitro pharmacological and the in vivo toxic effects into the CNS 5,6-DHT, when applied in vitro at concentrations from 10 -6 t o 10 -3 M significantly reduced the in vitro uptake of both [3H]5-HT and [3H]DA. The concentration of 5,6-DHT sufficient to reduce uptake by 50% (IC50) was 8.5)
