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)

Development of synapses on pyramidal and multipolar non-pyramidal neurons in the visual cortex of rabbits. A combined Golgi-electron microscope study.

A combined Golgi-electron microscope method was used to study the ultrastructural maturation of synapses on identified pyramidal and multipolar non-pyramidal neurons in the visual cortex of young and adult rabbits. In samples of 10 (time of eye opening), 14, 20 day old and 7 month old animals, fully impregnated pyramidal neurons within the layers II-V and multipolar non-pyramidal neurons mainly located in lower layer III and layer IV was studied. We found that synapses in 10 and 14 day old animals were occasionally immature in appearance. They were characterized by either a poorly defined postsynaptic band or equal rims of pre- and postsynaptic electron-dense material and could therefore not be classified as Gray type I or II. The distinction between both types of synapses was easier at day 20 and in the adults when the postsynaptic band of the asymmetrical (type I) synapses had become remarkably thicker. In pyramidal neurons the cytoplasmic organelles increased in number during development. Although a few symmetrical synapses were present on dendritic spines of pyramidal neurons in 14 and 20 day old animals, all pyramidal neurons exhibited the same types of synapses on specific sites of their neuronal surface. They received exclusively type II synapses on their somata, type I synapses on their dendritic spines and both types of synapses on their dendritic shafts. However, in the adult animals the frequency of occurrence of type II synapses, especially on basal dendritic shafts, had increased. In some cases only type II and no type I synapses were present. A striking finding in all young and adult animals was that synapses at the borderline between somata and apical dendritic shafts as well as on dendritic spines were frequently complex or interrupted. The characteristic ultrastructural features of adult spine-free and sparsely spiny multipolar non-pyramidal neurons e.g. the many cytoplasmic organelles and type I and II synapses on somata and on dendrites were already present at day 10. After day 10 the number of organelles and synapses increased prominently and in adult animals the different types of synapses on dendrites were located at relatively short intervals of about 4 microns. In contrast with the dendritic shafts of pyramidal neurons many asymmetrical synapses were observed on dendritic shafts of the non-pyramidal neurons analysed in the adult animals. Furthermore, it appeared that the number of synapses on these non-pyramidal neurons is about twice that on pyramidal neurons in day 20 old animals and about four times in adult animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptic characteristics of identified pyramidal and multipolar non-pyramidal neurons in the visual cortex of young and adult rabbits. A quantitative Golgi-electron microscope study.

The visual cortex of 20 day old rats and rabbits has been considered as mature on the basis of the observations that the dendritic arborization and the overall synaptic population have almost reached their adult stage in these animals. In the present study we have investigated the visual cortex of 20 day and 7 month old (adult) rabbits in order to determine whether this apparent adult appearance also holds for the synaptic organization of individual neurons. Neurons mainly located in layers III and IV of the primary visual cortex (area 17) were Golgi-impregnated, gold toned and deimpregnated and were then, after embedding in plastic, sectioned serially. The number and length of synaptic profiles, and the length of the neuronal boundaries were analysed in every tenth section. From these counts and measurements the size distribution of the synaptic discs, the number of synapses per 100 micron2 neuronal surface and the receptive surface expressed as the percentage of the total neuronal surface covered with synaptic contacts were estimated using stereological methods. At both ages studied, the density of synapses was significantly higher for the non-pyramidal neurons than for the pyramidal neurons. Differences in the amount of receptive surface were parallel to the differences observed for the number of synapses per 100 micron2. At day 20 the receptive surface of the non-pyramidal neurons was significantly larger than that of the pyramidal neurons. The receptive surface of the non-pyramidal neurons in the adult stage was not only larger than that of the pyramidal neurons in the adults, but also larger than that of the day 20 non-pyramidal neurons. From our results the following conclusions can be drawn: (1) The synaptic input received by the pyramidal neurons is mainly established at day 20 of postnatal life, i.e. prior to the establishment of adult visual behaviour. (2) The non-pyramidal neurons complete their maturation in a later stage than the pyramidal neurons. (3) Medium to large sized synaptic contacts are newly formed after day 20 and are mainly added to the synaptic population on dendrites of non-pyramidal neurons. (4) The specific increase in the number of synapses on non-pyramidal neurons is discussed in relation to intracortical inhibition which is thought to be important for the fine regulation of visual function during development.

Novel aspects of the ultrastructural organization of human corneal keratocytes.

PURPOSE: Proper functioning of the endothelium and proper structural organization of the keratocytes and collagen bundles are of ultimate importance for transparency of the cornea. The role of the endothelium has been investigated extensively, whereas the role of the keratocytes is still unclear. Detailed knowledge on the ultrastructural organization of keratocytes and the relationship between keratocytes and collagen bundles is as essential for understanding corneal transparency as is knowledge of endothelial functioning. METHODS: Thirty-five corneas (30 postmortem donor corneas and 5 fresh corneas from the operating theater; age range, 28 to 90 years) were used for light microscopy, transmission electron microscopy, and scanning electron microscopy. Serial frontal sections of the central stroma reaching from epithelium to endothelium and cross-sections were studied. At three levels, reconstructions of the mutual arrangement of keratocytes were made using semithin sections. RESULTS: Keratocytes have the appearance of highly active cells with an abundancy of organelles. Between the dendritic ramifications of these cells, large amounts of amorphous material is observed. One of the most remarkable observations is the presence of an extensive network of fenestrations along the surface of the keratocytes. Another important observation is the circular arrangement of keratocytes gradually turning clockwise like a corkscrew from epithelium to endothelium. CONCLUSIONS: From the current study, the following conclusions can be drawn: Keratocytes are not quiescent but are highly active cells probably involved in turnover of the extracellular matrix; fenestrations may be of functional relevance with respect to facilitation of diffusion and mechanical attachment of the collagen fibers to the keratocytes; the corkscrew organization of keratocytes suggests that they form completely closed sheets of communicating cells throughout the depth of the cornea, creating equal chances for all light rays to pass one or more keratocytes and thus minimizing variation in light scattering over the entire cornea.

Ultrastructural organization of human corneal nerves.

PURPOSE: Although the human cornea is densely innervated, observations of the nerve fiber distribution and ultrastructure are scarce. This study aimed to provide a detailed electron microscopic analysis of nerve fibers in the central and peripheral human cornea. METHODS: Samples from seven fresh corneas, obtained from the eyes of persons with melanoma, were processed for light and electron microscopic examinations. Both frontal and cross-sections were studied. Furthermore, serial ultrathin sections from the mid-epithelium to the anterior stroma were used. RESULTS: Unmyelinated nerve fiber bundles (as many as 30 nerve fibers and cross-section as large as 20 micrometers) run parallel to the stromal collagen fibers. Nerve fibers contain clear, dense cored and dense vesicles and are ensheathed by thin rims of Schwann cell protrusions and amorphic matrix. Some nerve fibers invaginate the cytoplasm of keratocytes. After passing through Bowman's membrane, bundles of straight fibers (cross-section 0.1 to 0.5 micrometers) and single-beaded nerve fibers, which both lack Schwann cell ensheathment, run parallel in an alternating manner. Beaded nerve fibers, containing many mitochondria and glycogen (cross-section as large as 2 micrometers), turn upward and invaginate both basal and wing cells. Except for the presence of myelinated nerve fibers in the peripheral stroma, no differences in the central cornea were observed. CONCLUSIONS: Nerve fibers invaginating epithelial cells and keratocytes suggest that both cell types are directly innervated. The presence of vesicles, mitochondria, and glycogen in stromal and epithelial nerve fibers suggest that classical and peptidergic transmitters, probably of sensory origin, innervate the human cornea. Peptidergic transmitters in nerve fibers may be involved in neuroimmunomodulation of the cornea.

Architecture of human corneal nerves.

PURPOSE: The corneal innervation, mainly analyzed in light microscopical studies, has been described as radially oriented stromal nerve bundles that ramify as leashes in the subbasal plexus. The current study aims to determine the orientation, the size, and the postmortem changes of the nerve fibers in the subbasal plexus of the human cornea. METHODS: Before processing for light and electron microscopy, the position of the corneas within the enucleated eyes of persons with melanoma and pairs of postmortem eyes was marked. The orientation and postmortem changes of the fibers were studied in serial "en face" semithin sections, and the size was determined in random, ultrathin cross-sections. RESULTS: Thirteen and a half hours after death, the majority of the nerve fibers were degenerated or gone. Nerve fiber bundles in the subbasal plexus run first in the 9-3 hours direction, then after bifurcation in the 12-3 hours direction and after a second bifurcation again in the 9-3 hours direction. From the main straight bundles, single-beaded fibers branch and run obliquely. Quantification of the nerve fibers shows an equally dense innervated central and central-peripheral cornea (mean fiber diameter, 0.4 micron) and a five to six times lower innervated peripheral cornea (mean fiber diameter, 0.67 micron). CONCLUSIONS: The nerve bundles in the subbasal plexus of the human cornea form a regular dense meshwork with equal density over a large central and central-peripheral area. Because of their size, the majority of the fibers can be classified as C-fibers.

Autosomal recessive cornea plana: in vivo corneal morphology and corneal sensitivity.

PURPOSE: Autosomal recessive corneal plana (RCP) is a rare corneal anomaly with unknown pathogenesis and a high incidence in Finland. The aim was to examine corneal sensitivity and the morphology of different corneal layers and subbasal nerves in RCP patients. METHODS: Three patients with a diagnosed autosomal recessive cornea plana were examined. Corneal sensitivity to different modalities of stimulation was tested in four corneas using noncontact esthesiometry. Tissue morphology of three corneas was evaluated, and in two corneas thickness of corneal layers was measured using in vivo confocal microscopy. RESULTS: Corneas of RCP patients appear to have mechanosensory, polymodal, and cold-sensitive nerve terminals. RCP patients had normal sensation thresholds for chemical, heat, and cold stimulation but a high threshold for mechanical stimulation. Their capacity to discriminate increasing intensities of stimulus was reduced, except for cold stimuli. Thickness of the epithelial layer was reduced, whereas total corneal and stromal thicknesses were slightly reduced or close to normal values. In all cases Bowman's layer was absent. Subbasal nerves had abnormal branching patterns. The arrangement of anterior keratocytes was altered, showing clustered and irregularly shaped nuclei. Increased backscattering of light in confocal microscopy through focusing (CMTF) profiles was observed throughout the stroma. Epithelial and endothelial cells appeared to be regular in shape. CONCLUSIONS: The present study revealed qualitative and quantitative alterations in corneal sensitivity, cellular morphology, and the thickness of corneal layers in RCP patients.

Corneal structure and sensitivity in type 1 diabetes mellitus.

PURPOSE: Corneal wound healing is impaired in diabetic cornea. The purpose of this study was to examine patients with type 1 diabetes mellitus for changes in corneal morphology and to correlate corneal sensitivity, subbasal nerve morphology, and degree of polyneuropathy with each other. METHODS: Forty-four eyes of 23 patients with diabetes and nine control eyes were included. Corneal sensitivity was tested with a Cochet-Bonnet esthesiometer (Luneau, Paris, France), and corneal morphology and epithelial and corneal thickness were determined by in vivo confocal microscopy. The density of subbasal nerves was evaluated by calculating the number of long subbasal nerve fiber bundles per confocal microscopic field. The degree of polyneuropathy was evaluated using the clinical part of the Michigan Neuropathy Screening Instrument (MNSI) classification, and retinopathy was evaluated using fundus photographs. RESULTS: A reduction of long nerve fiber bundles per image was noted to have occurred already in patients with mild to moderate neuropathy, but corneal mechanical sensitivity was reduced only in patients with severe neuropathy. Compared with control subjects the corneal thickness was increased in patients with diabetes without neuropathy. The epithelium of patients with diabetes with severe neuropathy was significantly thinner than that of patients with diabetes without neuropathy. CONCLUSIONS: Confocal microscopy appears to allow early detection of beginning neuropathy, because decreases in nerve fiber bundle counts precede impairment of corneal sensitivity. Apparently, the cornea becomes thicker in a relatively early stage of diabetes but does not further change with the degree of neuropathy. A reduction in neurotrophic stimuli in severe neuropathy may induce a thin epithelium that may lead to recurrent erosions.

Corneal morphology and sensitivity in lattice dystrophy type II (familial amyloidosis, Finnish type).

PURPOSE: To describe the corneal abnormalities and to measure different modalities of corneal sensitivity in corneal lattice dystrophy type II (familial amyloidosis, Finnish type, also known as gelsolin-related amyloidosis and originally as Meretoja syndrome). METHODS: Twenty eyes of 20 patients were examined by in vivo confocal microscopy and noncontact gas esthesiometry. RESULTS: Pleomorphism of, and dense deposits between or posterior to, the basal epithelial cells were frequently observed, as well as a reduction of long nerve fiber bundles in the subbasal nerve plexus. The anterior stroma was altered in most cases, with fibrosis and abnormal extracellular matrix. In 15 corneas, thick anterior and midstromal filaments, corresponding to lattice lines, and in 11 corneas, thin undulated structures were observed. The average mechanical sensitivity threshold of 12 subjects was increased, and in the remaining 8 subjects there was no response, even to the highest intensity of stimuli used. Three patients did not respond to CO(2), 11 to heat, and 2 to cold, but those patients who responded had normal thresholds. Patients with more long nerve fiber bundles per confocal microscopic image had better mechanical and cold sensitivity than patients with fewer nerve fiber bundles. CONCLUSIONS: Lattice lines seem to be related to amyloid material and not to corneal nerves. However, the subbasal nerve density appears reduced, which results mainly in a decrease in mechanical and, to a lesser extent, thermal sensitivity. The location of stromal filaments and undulated structures changes with increasing age.

Quantitative aspects of synapses on Golgi-impregnated neurons.

With the classical Golgi techniques, numerous types of neurons can be distinguished in the cerebral cortex, each with a specific dendritic geometry and pattern of axonal ramifications. In the present review we describe two techniques which allow quantification of synapses on identified neurons: (1) Golgi-rapid impregnation-gold toning-electron microscopy, and (2) Golgi-Kopsch impregnation-gold toning-electron microscopy in combination with staining of the tissue with ethanolic phosphotungstic acid (E-PTA). Both techniques were applied on neurons in the visual cortex of young and adult rabbits. By means of rotating and tilting specimens in the electron microscope, the nondistinctive ultrastructure of obliquely sectioned synapses can be circumvented, leading to precise estimates of asymmetrical vs. symmetrical synapses without complete reconstruction of the neuron.

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.

Efferent projections of the olivary pretectal nucleus in the albino rat subserving the pupillary light reflex and related reflexes. A light microscopic tracing study.

The olivary pretectal nucleus is a primary visual centre sensitive to luminance changes. It is involved in the pupillary light reflex, the consensual pupillary light reflex and related reflexes, such as the lid closure reflex whereby pupillary constriction takes place. Since the olivary pretectal nucleus is a small nucleus, previous studies using degeneration, horseradish peroxidase and radioactive amino acid tracing were limited regarding to the exclusiveness of the projections from the olivary pretectal nucleus. In the present study the position of the olivary pretectal nucleus in the rat was first localized by physiological recording of the neurons upon luminance stimulation. Subsequently, an anterograde tracer Phaseolus vulgaris leucoagglutinin was injected iontophoretically. This allows a much more precise localization of the olivary pretectal nucleus projections. Ascending and descending pathways originating from the olivary pretectal nucleus were observed. Ascending fibres project bilaterally to the intergeniculate leaflet, the ventral part of the lateral geniculate nucleus and ipsilaterally to the anterior pretectal nucleus. In addition, contralateral projections were observed to the zona incerta and the fields of Forel. Descending fibres project bilaterally to the periaqueductal gray, the nucleus of Darkschewitsch, the interstitial nucleus of Cajal, the Edinger-Westphal nucleus and the intermediate gray layer of the superior colliculus. Also a contralateral projection to the oculomotor nucleus and an ipsilateral projection to the pontine nucleus and the nucleus of the optic tract were found. Furthermore, the contralateral olivary pretectal nucleus received a small projection. Retrograde tracing experiments using two fluorescent dyes revealed that the fibres projecting to the contralateral olivary pretectal nucleus and to the contralateral interstitial nucleus of Cajal are collaterals. The projection from the olivary pretectal nucleus to the facial nucleus which has been described to receive an input in cats could not be confirmed for the rat. The fact that the Edinger-Westphal nucleus, the interstitial nucleus of Cajal and the superior colliculus receive an input from the olivary pretectal nucleus suggests that this primary visual centre is not only involved in the pupillary light reflex, but also in controlling eye and head position and saccadic eye movements. Although visual acuity largely depends on receptive field sizes of retinal ganglion cells and their central connections, the stronger sympathetic influence during the pupillary light reflex in animals with frontally placed eyes compared to animals with laterally placed eyes may also contribute to the higher visual acuity in animals with frontally placed eyes.

The specific architecture of the anterior stroma accounts for maintenance of corneal curvature.

AIM: To analyse the human corneal stroma in extreme hydration to discover if its structure is responsible for corneal stability. METHODS: Corneas in several hydration states were used: postmortem control corneas (PM; n=3), corneas left for 1 day in phosphate buffered saline (PBS; n=4), and corneas left for 1 day (n=4), 2 days (n=4), 3 days (n=2), and 4 days (n=4) in deionised water. All corneas were fixed under standardised conditions and processed for light and electron microscopy. In addition, two fresh corneas from the operating theatre were studied which were processed 6 months after storage in sodium cacodylate buffer. RESULTS: After 1 day in deionised water maximal stromal swelling was reached which did not change up to 4 days. The stroma of deionised water corneas (1400 microm) was much thicker than that of PBS corneas (650 microm) and PM corneas (450 microm). Deionised water treatment led to disappearance of all keratocytes leaving only remnants of nuclei and large interlamellar spaces. In these specimens the distance between the collagen fibres had increased significantly, but the diameter of the collagen fibres did not seem to be affected. A remarkable observation was that the most anterior part of the stroma (100-120 microm) in all deionised water specimens and those stored for 6 months in buffer was not swollen, indicating that the tightly interwoven anterior lamellae are resistant to extreme non-physiological hydration states. CONCLUSIONS: The rigidity of the most anterior part of the corneal stroma in extreme hydration states points to an important role in maintenance of corneal curvature. Since a large part of this rigid anterior part of the stroma is either removed (PRK) or intersected (LASIK), it is possible that in the long run patients who underwent refractive surgery may be confronted with optical problems.

Clearance of neurotransmitter from the cone synaptic cleft in goldfish retina.

Dendrites of bipolar and horizontal cells protrude deeply into the synaptic terminals of cones in goldfish retina. This arrangement gives the impression that the cone synaptic terminal surrounds a morphologically shielded compartment, the cone synaptic cleft, from which clearance of neurotransmitter by diffusion is limited. In this study the time constant of this clearance has been approached in two ways: (1) the morphological parameters determining the clearance (extracellular synaptic volume and leak area), were estimated using morphometric methods. These data were introduced into a diffusion model of the cone pedicle, yielding a time constant for the clearance of < 1 msec; (2) the time constant of the light onset response when the glutamate transporter in the cone was blocked with DL-threo-beta-hydroxyaspartate or dihydrokainate, was interpreted as the time constant of the clearance, yielding values of almost 650 msec compared to around 90 msec in control conditions. The decay time-constant of the Ca-dependent tail-currents in cones was used, as an approximation of the dynamics of the intracellular Ca-concentration and thus of the glutamate release by the cones. The decay time constant was about 800 msec. This suggests that the intracellular Ca-concentration in the synaptic terminal and hence the glutamate release by the cones drops with a similar large time constant. These results indicate that the cone pedicle in goldfish does not limit the clearance of neurotransmitters from the synaptic cleft and that the fast light onset response of horizontal cells under control conditions is due to activation of glutamate transporters by hyperpolarization of the cone membrane potential while the glutamate release drops slowly. The slow horizontal cell light onset response in beta-hydroxyaspartate or dihydrokainate may be due to a slow reduction of the glutamate release by the cones at light onset.

The effects of organ-culture on the density of keratocytes and collagen fibers in human corneas.

PURPOSE: Keratocytes are important in regaining corneal transparency during wound healing after surgery or trauma. Hitherto, there are still controversies concerning the effects of organ culture on the density and integrity of keratocytes and collagen fibers. The current study aimed at a systematic analysis of the effects of organ-culture on the morphology and density of keratocytes and collagen fibers. METHODS: Human corneas were organ-cultured in MEM for 7 (n = 17, 3 pairs), 14 (n = 18, 9 pairs) and 21 days (n = 18, 9 pairs). Of the pairs one cornea was processed in swollen condition and the fellow cornea after reversal of swelling in MEM plus Dextran. Eleven post-mortem corneas (PM) and 11 fresh corneas obtained from melanoma patients were used as controls. Stromal thickness, number of keratocyte profiles (corrected for swelling), number and diameter of collagen fibers were measured in light microscopical sections and electron micrographs. RESULTS: Stromal swelling due to organ-culture resulted in large keratocyte profiles with many vacuoles and large distances between collagen fibers in the posterior stroma. In contrast both keratocytes and distances between collagen fibers were not affected in the anterior stroma. After reversed-swelling the posterior corneal stroma was similar to that in fresh controls, indicating that the swelling process is largely reversible. The initial decrease in keratocyte density (18%) in the early post-mortem period did not progress during 21 days of organ culture. CONCLUSION: With respect to the morphology and density of keratocytes and collagen fibers it can be concluded that donor corneas remain suitable for transplantation up to at least 21 days after organ-culture.

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)