Snail neurons as a possible model for testing neurotoxic side effects of antitumor agents: paracrystal formation by Vinca alkaloids.

The suitability of neurons of the freshwater snail Lymnaea stagnalis as a test system for the neurotoxic side effects of antitumour Vinca alkaloids has been investigated, by studying the process of paracrystal induction by Vinca antitumour agents. Three Vinca alkaloids have been compared: the natural vinblastine and vincristine and the semisynthetic vindesine. They appear to induce two types of inclusion. The first type is paracrystalline and has a rod-like shape with a width of 0.3-2.5 micron and a length of 1-10 micron. It consists of hexagonally arranged tubules with a lattice constant of approximately 28 nm. The second type appears as ladder-like profiles with a periodicity of approximately 30 nm. It is proposed that the ladder-like profiles are in fact helical structures and are precursors of the paracrystals. Both types of inclusion may fill up large parts of the axons; they are rare in axon terminals and almost absent from the neuronal somata. It has been concluded that the process of paracrystal induction by Vinca alkaloids in Lymnaea neurons is very much the same as in mammalian neurons and may be largely responsible for the neurotoxic effects of the Vinca drugs, because it impairs axonal transport of neuronal secretory granules. Apparently, in this respect vindesine behaves in a similar way as the conventional vincristine and vinblastine drugs. Vincristine induces clearly more paracrystals than vindesine, whereas the least paracrystals occur in vinblastine-treated material. These differences correlate with the different clinical neurotoxicities of these drugs. Therefore, because Lymnaea neurons can be considered as excellent model systems for studies of the functioning of neurons in general, it is expected that counting the number of paracrystals in Lymnaea nervous tissue will prove to be a good method to predict the degree of clinical neurotoxicity of newly developed antitumor Vinca alkaloids.

Quantitative ultrastructural effects of cisplatin (Platinol), carboplatin (JM8), and iproplatin (JM9) on neurons of freshwater snail Lymnaea stagnalis.

Qualitative and quantitative ultrastructural effects of the platinum compounds cisplatin (Platinol), carboplatin (JM8), and iproplatin (JM9) were studied on two types of identified peptidergic neuron (caudodorsal cells, light green cells) in the pond snail Lymnaea stagnalis. Depending on the parameter under investigation, either one or both cell types were studied. Central nervous systems of the snail were incubated for 5 and 20 h in various identical and equitoxic drug concentrations. Cisplatin had the most severe effects. Platinol, i.e., cisplatin dissolved in NaCl solution with the addition of HCl (pH 2.0-3.0), as well as cisplatin dissolved in snail Ringer's solution (pH 7.8), caused swelling of axons and distensions of the intercellular spaces. This drug induced an increase in chromatin clump size in the caudodorsal cells (20-h incubation), while carboplatin and iproplatin induced the formation of many small chromatin clumps. Incubation in snail Ringer's solution (controls) and cisplatin affect the morphology of the nucleoli. At high dosages of cisplatin, the nucleoli of light green cells were transformed into homogeneous dense structures. The data indicate that platinum compounds react with nuclear and nucleolar DNA. All three drugs affected the activity and organization of the rough endoplasmic reticulum and the Golgi apparatus of the peptidergic neurons studied (qualitative observations). These effects, which point to a reduced neuropeptide synthesis, may be secondary, i.e., exerted via inhibition of RNA synthesis and ribosome formation (nucleoli). The fact that the number of neuropeptide granules in the cytoplasm of the cells remained constant (both cell types) may indicate that granule transport was also inhibited. Cisplatin and iproplatin induced an increase in the number of lysosomes in the light green cells. The number of lipid droplets in these cells was not affected by drug treatment. The results corroborate clinical data indicating that cisplatin is highly neurotoxic. Despite conflicting clinical data, observations on the snail neurons suggest that iproplatin is also neurotoxic, although less than cisplatin. Carboplatin is minimally neurotoxic, which is in accordance with clinical data. The central nervous system of Lymnaea is a suitable model for studying possible neurotoxic effects of platinum compounds.

Use of snail neurons in developing quantitative ultrastructural parameters for neurotoxic side effects of Vinca antitumor agents.

The central nervous system of the snail Lymnaea stagnalis was studied in order to develop a test system to predict the neurotoxic side effects of the three cytostatic Vinca alkaloids, vincristine (VCR), vindesine (VDS), and vinblastine (VLB). Vinca alkaloids appear to interfere with microtubule formation by the induction of paracrystalline inclusions. After in vitro incubation the numbers of these inclusions were counted in cross-sections of the cerebral commissure using electron microscopy. For each compound the number of paracrystalline profiles increases with increasing concentrations and incubation times. At equimolar concentrations (0.15 mM), VCR induces more paracrystals than VDS, and VDS induces more than VLB. These effects are clear after short periods of incubation (e.g., after 2 h, VCR:VDS:VLB = 5:2:1). Equitoxic concentrations of VCR, VDS, and VLB induce similar numbers of paracrystals. Furthermore, morphological changes in the cell bodies of identified neurons (light green cells) in the cerebral ganglia were observed. Quantitative analysis shows that at equimolar concentrations the surface area of nuclear chromatin of all Vinca alkaloid-treated cells is approximately 30% lower than that of the controls. The lamellae of the rough endoplasmic reticulum are swollen and have lost their regular arrangement. For VDS and VLB this swelling is accompanied by a strong increase (about 3-fold) in the total surface area of the rough endoplasmic reticulum. No increase was observed for VCR. The compounds do not affect the number of secretory granules. In contrast to the controls, all Vinca-treated cells show lipid droplets. After VCR treatment they are about 5-fold as numerous as after treatment with VDS or VLB. The total surface area of lysosomes increases about 1.3-fold by VDS and VLB treatment and about 3-fold by VCR treatment. From these quantitative data it is concluded that VCR is more neurotoxic than VDS and VLB. VDS appears to be more neurotoxic than VLB as judged from the data on paracrystal induction. On the basis of a comparison of these data with clinical data on Vinca-induced neurotoxicities, it is proposed that neurons of the snail L. stagnalis may be suitable for the development of a test system to predict the degree of clinical neurotoxicity induced by Vinca antitumor drugs.

Morphological and electrophysiological study of the effects of cisplatin and ORG.2766 on rat spinal ganglion neurons.

Eleven- to 12-wk-old rats were treated twice a week with cisplatin/saline or with cisplatin plus ORG.2766 during 12.5 wk. Cisplatin and ORG.2766 were administered at a final concentration of 0.04 mg/ml (i.p.) and 10 micrograms/ml (s.c.), respectively. Control animals were treated with saline. In this period the cisplatin-treated animals developed a peripheral neuropathy resulting in impairment of sensory functions. Estimates of the motor (MNCV) and sensory (SNCV) nerve conduction velocity were made after 0, 7.5, 10, and 12.5 wk. It appeared that the MNCV of the control, cisplatin-, and cisplatin plus ORG.2766-treated rats increased from 50 to 59 m/s. In contrast, the SNCV of the cisplatin-treated rats decreased significantly (P less than 0.001) from 63 to 56 m/s, whereas that of the control animals increased from 62 to 84 m/s. Rats which received cisplatin plus ORG.2766 showed an increase in SNCV up to control levels. After 12.5 wk the animals were perfused with a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M phosphate buffer. At the level of L5 and L6, 5 mm of spinal cord tissue and three dorsal root ganglia were removed and processed for electron microscopy. With the point-counting method the volume fraction (v/v) of somata and myelin in spinal ganglia was estimated. No significant change in the volume fraction of somata of the control (0.42), cisplatin (0.33)-, and cisplatin plus ORG.2766 (0.39)-treated rats was found. The same held true for the volume fraction of myelin of the control (0.53), cisplatin (0.59)-, and cisplatin plus ORG.2766 (0.58)-treated rats. In addition, the number of lysosomes per 100 microns 2 was estimated in spinal ganglion neurons and in spinal cord motor neurons of a total of 120 randomly chosen neurons. It was found that the number of lysosomes in the spinal ganglion neurons of the control animals was lower (10 per 100 microns 2) than in cisplatin-treated (30 per 100 microns 2) and in cisplatin plus ORG.2766-treated rats (28 per 100 microns 2) (P less than 0.05). No difference was observed in the number of lysosomes between cisplatin- and cisplatin plus ORG.2766-treated rats. The number of lysosomes in spinal cord tissue of cisplatin-treated rats (2.4 per 100 microns 2) did not differ from controls (0.1 per 100 microns 2) and from cisplatin plus ORG.2766-treated rats (0.8 per 100 microns 2).(ABSTRACT TRUNCATED AT 400 WORDS)

Target-dependent differentiation and development of molluscan neurons and neuroendocrine cells: use of parasitisation as a tool.

Specimens of the freshwater snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata show a strongly inhibited development of their reproductive tract. We hypothesised that the effects of the underdevelopment of targets are reflected at the level of the neuronal development of (i) the motor neurons innervating the male copulation organ and (ii) neuroendocrine cells regulating the gonad. We determined the state of neuronal development by measuring cell number, cell size and neuropeptide gene expression. Our results show that the neuronal development of both copulation controlling anterior lobe motor neurons of the right cerebral ganglion and neuroendocrine caudodorsal cells, which produce neuropeptides regulating ovulation, egg laying and accompanying behaviour, are affected in parasitised animals in which their respective target organs were not developed. The cell bodies were smaller and fewer cells were found to express neuropeptide genes compared to those in non-parasitised animals. These effects were not observed in the appropriate controls. Backfills and lesions of the penis nerve have shown that the inhibited development of central motor neurons in parasitised snails is target dependent; neighbouring neurons that have no connection with the male copulation organ are not affected. Our data suggest that this effect is established by target-derived neurotrophic factors that need this connection for being transported to the innervating motor neurons. We propose that the effect on the neuroendocrine caudodorsal cells is mediated by a humoral factor, since they have no known connection with their target. We have shown that the size and gene expression of motor neurons controlling copulation behaviour in the pond snail Lymnaea stagnalis are related to the size of their target, the copulation organ, and depend on the connection with this target.

The ACTH/MSH(4-9) analogue ORG 2766 stimulates microtubule formation in axons of central neurons of the snail Lymnaea stagnalis.

Central nervous systems of the pond snail Lymnaea stagnalis were incubated in vitro in different concentrations of ORG 2766 (10(-9)-2.5 x 10(-4) M) for 10 and 20 h. Quantitative ultrastructural study of cross sections of the cerebral commissure showed that the number of microtubules in large axons had increased after 10 h of incubation by approximately 50% (Experiment 1) and 30% (Experiment 2), respectively. No further increase was observed after 20 h of incubation. (The higher concentrations were studied.) Maximal stimulation was already found at a concentration of 10(-8) M. At a concentration of 10(-9) M control levels were observed. It is concluded that ORG 2766 stimulates microtubule formation already at very low concentrations. It is not clear whether the compound stimulates synthesis of tubulin, induces assembly of microtubules, or causes an increase in stability of microtubules. Nevertheless, ultrastructural data on the morphology of the glial cells indicate that these cells are activated by ORG 2766 treatment, which suggest that ORG 2766 has general trophic effects.

Differential trophic effects of ORG 2766, an ACTH(4-9)/MSH(4-9) analogue, on peptidergic neurons and glial cells in the snail Lymnaea stagnalis.

Cerebral ganglia of the pond snail Lymnaea stagnalis were incubated in vitro in 10(-6) M ORG 2766 for 10 and 20 h, with or without regular refreshment of the medium. Quantitative ultrastructural study of cross sections of the cerebral commissure showed that the number of microtubules in large axons of all ORG 2766-treated groups had increased after 10 h by approximately 40%. In a separate experiment, central nervous systems were incubated in ORG 2766 for only 15 min and then kept in Ringer's for 9 h and 45 min. Maximal stimulation (40% increase of microtubules) in these specimens was also observed. The results would seem to support the hypothesis that ORG 2766 binds to a receptor and initiates a long-lasting effect. It is argued that ORG 2766 stimulates novel synthesis of tubulin rather than being involved in the assembly of microtubules. Also, glial cells were found to be activated by ORG 2766. This was concluded from the fact that the number of heterochromatin clumps and the size of the clumps in these cells had decreased and the amount of glial tissue surrounding the axons had increased (approximately 50%). In contrast to the activating effects of ORG 2766 on glial tissue, this drug did not affect nucleoli, number, and size of the heterochromatin clumps and the Golgi apparatus in the neuropeptidergic caudodorsal cells. The data indicate that ORG 2766 exerts differential trophic effects.

Morphometric in vitro analysis of the control of the activity of the neurosecretory dark green cells in the freshwater snail Lymnaea stagnalis (L.)..

The neurosecretory Dark Green Cells (DGC) in the pleural and parietal ganglia of the freshwater snail Lymnaea stagnalis seem to be involved in osmoregulation. Previous experiments have indicated that changes of the osmolality of the environment induce activity changes of the DGC. Furthermore, it was shown that information on environmental osmolality reaches the DGC via the blood. In the present study right pleural and parietal ganglion complexes were cultured for 3 days in vitro under different osmotic conditions. Quantitative electron microscopy revealed that, compared with the control osmolality (130mOsm/kg H2O), osmolalities of 160 and 190 mOsm/kg H2O caused a reduced synthesis and an increased storage of neurohormone in the DGC. Apparently, the activity of the DGC depended on the osmotic pressure of the medium. It is proposed that in vivo the osmotic pressure of the blood (which is related to the osmolality of the environment) regulates DGC activity.

Ultrastructural dynamics of exocytosis in the ovulation-neurohormone producing caudo-dorsal cells of the freshwater snail Lymnaea stagnalis (L.).

The ultrastructural dynamics of exocytosis in the ovulation-stimulating neurosecretory Caudo-Dorsal Cells (CDC) of the freshwater snail L. stagnalis were studied after incubation of cerebral ganglia in Ringer's solutions with different concentrations of K+ and Ca2+. Detection of exocytosis was facilitated by the use of the tannic acid-glutaraldehyde fixation method (TAGO-method). In control Ringer (low K+) the frequency of exocytosis was rather low. Exocytosis mainly occurred as "terminal" exocytosis (TE); "intracellular" (ICE) and, particularly, "multiple" exocytosis (ME) took place infrequently. Incubation in high K+-containing Ringer strongly increased exocytotic activity. Compared to the controls the total frequency of exocytosis was 50 X as high, whereas TE, ICE and ME occurred 6 X, 47 X, and more than 300 X as frequently, respectively. In high K+/Ca2+-free Ringer the total frequency of exocytosis was only 2 X as high as in control Ringer. It is concluded that TE, ICE, and ME are normal, Ca2+-dependent exocytotic phenomena. The significance of their dynamics in response to K+-stimulation is discussed. The extremely high frequency of exocytosis, as well as the presence of "unaltered granule contents in transit", is explained by assuming that an exocytotic event in the CDC lasts rather long, viz. some minutes. The results may reflect the physiological mechanism by which the CDC release their ovulation hormone. The possible involvement of "clear" and "large" electron lucent vesicles in membrane reuptake after exocytosis is considered.

An ultrastructural in vitro study on the regulation of neurosecretory activity in the freshwater snail Lymnaea stagnalis (L.) with particular reference to caudo-dorsal cells.

The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis.

In vivo modulation of vincristine-induced neurotoxicity in Lymnaea stagnalis, by the ACTH(4-9) analogue Org 2766.

The use of the cytostatic agent vincristine (VCR) is limited by the occurrence of peripheral neuropathy. This side-effect is probably caused by interference with axonal microtubules. VCR depolymerizes microtubules and reacts with tubulin to form paracrystals. The potential of a neurotrophic ACTH(4-9) analogue, Org 2766, to counteract peripheral neuropathy caused by cytostatic agents is being investigated. In the present ultrastructural study, modulatory effects of Org 2766 on VCR-induced neurotoxicity were studied in vivo in neurons of the pond snail Lymnaea stagnalis, which has been shown previously to be a suitable test system to investigate neurotoxic side-effects of cytostatic agents. 24 h after treatment with VCR (25 microM), 68.4 +/- 34.7 paracrystals were counted per cross-section of the cerebral commissure and the number of microtubules in the axons had been lowered to 46% of the control level. After a survival period of two weeks all paracrystals had disappeared. By that time, no recovery of the axonal microtubular system could be observed. However, post-treatment with Org 2766 (10(-6) M) on day 6 after VCR treatment had induced a significant increase in the number of microtubules (+55%) on day 7. This beneficial effect lasted for the rest of the experimental period (14 days). These results suggest that post-treatment with Org 2766, i.e. after VCR clearance, can induce long-lasting beneficial effects on VCR-induced neurotoxicity in vivo.

Ultrastructural neuropathologic effects of Taxol on neurons of the freshwater snail Lymnaea stagnalis.

Cerebral ganglia of the freshwater snail Lymnaea stagnalis were incubated in vitro in 10 microM Taxol for 8 and 24 h. Cremophor EL (0.1%) was used as a diluant. The tissue was processed for electron microscopy. Various ultrastructural parameters were assessed quantitatively. Cremophor EL appeared to seriously affect the cell somata of the multipeptidergic caudodorsal cells. In the Cremophor-controls the mean area of Golgi zones, the percentage dense material (neuropeptides) in these zones, the number of large electron dense granules (these are involved in neuropeptide processing) and the mean nuclear heterochromatin clump size, were significantly smaller than in the Ringer-controls, whereas the number of lipid droplets was higher. All these parameters, except for the lipid droplets, were not different in the Cremophor-controls and the Taxol-treated specimens. After 24 h treatment, but not after 8 h, Cremophor EL furthermore induced an increase in the number of axonal microtubules. It is argued that the results might signify activation of the neurons by Cremophor EL. Taxol induced a significant increase in the number of microtubules in axons and cell somata. Furthermore an increase in the number of Golgi zones was observed, suggesting activated neuropeptide synthesis. In all groups immunostaining with antibodies to neuropeptides produced by the caudodorsal cells was normal. Release of neuropeptide (exocytosis) from axon endings was elevated after Taxol treatment, and exceptionally high in specimens cotreated with Taxol and Org 2766 (incubation time 22 h). The effect of Org 2766 and Taxol on the number of microtubules was cumulative.(ABSTRACT TRUNCATED AT 250 WORDS)