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Using the apomorphine-induced stereotyped gnawing response as a selection criterion, two distinct groups of rats can be distinguished, apomorphine-susceptible (APO-SUS) and apomorphine-unsusceptible (APO-UNSUS) rats. These two lines differ in several components of both striatal and extrastriatal areas. This study deals with the expression of neuropeptide Y (NPY)mRNA-expressing neurons in the nucleus accumbens, caudate putamen and cerebral cortex of both rat lines, using non-radioactive in situ hybridisation. The morphology of the neurons in the three regions is similar, viz. oblong, rectangular or triangular, with two or three processes. The neurons are homogeneously distributed in all regions, and in the nucleus accumbens they are particularly numerous ventrally to the anterior commissure. Using automated image analysis, the mean numerical density of NPYmRNA-positive neurons per brain region and the mean NPYmRNA expression level per neuron per brain region were determined. No differences appear in the numerical densities of NPYmRNA-containing neurons in the nucleus accumbens, caudate putamen and cortex between APO-SUS and APO-UNSUS rats. However, distinct differences between the rat lines are present in the level of NPYmRNA expression per neuron in the nucleus accumbens and in the caudate putamen, showing that NPY contributes to the differential neurochemical make-up of these rat lines that is responsible for their obvious differences in behaviour, physiology and immune competence. [ABSTRACT FROM AUTHOR]

The cerebral ganglia of the freshwater snail Lymnaea stagnalis contain two clusters of neurosecretory Caudo-Dorsal Cells (CDC). These cells produce a neurohormone which stimulates ovulation. Ganglion transplantation and quantitative electron microscopy show that neuronal isolation of the cerebral ganglia complex (CCC) results in an activation of the CDC. It was, therefore, concluded that the CDC are controlled by an inhibitory neuronal input originating outside the cerebral ganglia. Ultrastructural studies on synaptic degeneration in the CCC suggest that this input reaches the CDC via a special type of synapse-like structure, the type C-SLS. Furthermore, transplantation of CCC into acceptor snails leads to a reduced release and an increased intracellular breakdown of neurohormone in the CDC of the nervous system of the acceptors. It is supposed that these phenomena are caused by the release of an (unknown) factor from the transplanted CCC. Special attention was given to the formation and degradation of a peculiar type of neurohormone granule, the large electron dense granule. The physiological significance of the neuronal and non-neuronal control mechanisms which regulate CDC activity is discussed. [ABSTRACT FROM AUTHOR]

The process of neurosecretion in the Caudo-Dorsal Cells (CDC) of the freshwater snail Lymnaea stagnalis, which produce an ovulation hormone, shows a diurnal rhythmicity. Synthesis, transport and release of the neurosecretory material (NSM) is high during the evening and the early night and low during the rest of the day, while storage of NSM mainly occurs during the daytime. In the present study the role of the eyes in the regulation of the CDC-rhythm was investigated. During a 24-hr period, at time intervals of 6 hrs, cerebral ganglia, which contain CDC, of blinded and control snails (5 per group) were fixed and the CDC were studied with quantitative electron microscopical methods. The CDC of the controls showed a distinct diurnal rhythmicity. Blinding, on the other hand, clearly affected this rhythmicity. The results indicate that after blinding the circadian CDC-rhythms of individual snails are no longer synchronous with each other ('interanimal desynchronization'). It is suggested that the rhythm of CDC neurosecretory activity is synchronized by the natural light/dark cycle via the eyes. The information from the eyes probably reaches the CDC via a nervous pathway. True synapses and three types of synapse-like structures were found on the CDC. Their role in the regulation of CDC-activity is discussed. The effect of blinding is specific for the CDC; blinding does not influence the diurnal rhythmicity of another type of cerebral neurosecretory cells, the Light Green Cells (LGC). The CDC within a cluster act synchronously. This synchrony does not depend upon the presence of the eyes. Some structures which may be involved in establishing this synchrony, such as subsurface cisterns, desmosome-like structures and 'specific release sites', are described. [ABSTRACT FROM AUTHOR]

Using in situ hybridization with a pro-opiomelanocortin (POMC)-mRNA probe and immunocytochemistry with antisera to POMC and to various POMC-derived peptides, it is shown that melanotrope cells in the pars intermedia of the hypophysis of the South African aquatic toad Xenopus laevis contain POMC, α-melanophore-stimulating hormone (α-MSH), γ-MSH, acetylated and non-acetylated endorphins and adrenocorticotropic hormone (ACTH). With the exception of γ-MSH, these peptides are also found in the corticotrope cells in the rostral pars distalis. In the Xenopus brain, neuronal cell bodies in the ventral hypothalamic nucleus express POMC, α-MSH, γ-MSH, non-acetylated endorphins and ACTH, neurones in the anterior preoptic area reveal POMC, α-MSH, γ-MSH and non-acetylated endorphin, neurones in the suprachiasmatic nucleus contain α-MSH, non-acetylated endorphin and ACTH and neurones in the posterior tubercle show α-MSH, non-acetylated endorphin and ACTH immunoreactivities. In the locus coeruleus POMC and ACTH coexist, whereas α-MSH and non-acetylated endorphin occur together in the nucleus accumbens, the striatum and the nucleus of the paraventricular organ. Finally, α-MSH alone is present in the olfactory bulb, the medial septum, the medial and lateral parts of the amygdala, the ventromedial and posterior thalamic nuclei, the optic tectum and the anteroventral tegmental nucleus, and non-acetylated endorphin alone appears in the epiphysis. It is suggested that neurones that form POMC-derived peptides may play a direct or indirect role in the control of POMC-producing hypophyseal cells and/or in the physiological processes these endocrine cells regulate. This idea is supported by the fact that the suprachiasmatic nucleus and the locus coeruleus, both involved in melanotrope cell control, show POMC and POMC-peptide expression. A possible involvement in melanotrope and/or corticotrope control of the anterior preoptic and ventral hypothalamic nuclei, which both express POMC and various POMC-derived peptides, deserves future attention. [ABSTRACT FROM AUTHOR]

Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including α-MSH and N-acetylβ-endorphin. In this study three types of secretory granules, electron-dense(∼130 nm Ø), moderately electron-dense (∼ 160 nm Ø) and electronlucent (∼ 180 nm Ø), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl-α-MSH and N-acetyl-β-endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl-α-MSH and N-acetyl-β-endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti- N-acetyl-β-endorphin, probably by stimulating intragranular acetylation of β-endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl-α-MSH which takes place near the plasmalemma at the time of granule exocytosis. [ABSTRACT FROM AUTHOR]

Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIP-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIP- and anti-MIP-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurohormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes. [ABSTRACT FROM AUTHOR]

The cerebral caudodorsal cells of the pulmonate snail Lymnaea stagnalis control egg laying and egglaying behavior by releasing various peptides derived from two precursors. The biosynthesis, storage, intracellular breakdown and release of three caudodorsal cell peptides were studied by means of immuno-electron microscopy using antisera raised to fragments of these peptides: (1) Caudodorsal Cell Hormone-I (CDCH-I; derived from precursor I), (2) Caudodorsal Cell Hormone-II (CDCH-II; from precursor II), and (3) α-Caudodorsal Cell Peptide (α CDCP; from both precursors). After affinity purification of the antisera, the specificity of the sera was confirmed with dotting immunobinding assays. From the ultrastructural immunocytochemical data it has been concluded that the precursor molecules are cleaved at the level of the Golgi apparatus after which the C-terminal parts (containing α CDCP) and N-terminal parts (containing CDCH-I or CDCH-II) are sorted and preferentially packaged into large electron-dense granules (MD 150 nm), respectively. Very probably, the content of the large electron-dense granules is degraded within the cell body. The immunoreactivity of the secretory granules increases during discharge from the Golgi apparatus, indicating further processing. At least a portion of the secretory granules contains all three peptides, as shown by double and triple immunopositive stainings whereas other granules appear to contain only one or two of these peptides. The caudodorsal cells release multiple peptides via exocytosis from neurohemal axon terminals into the hemolymph and from blindly ending axon collaterals into the intercellular space of the cerebral commissure (nonsynaptic release). [ABSTRACT FROM AUTHOR]

The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20-36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive. The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules. [ABSTRACT FROM AUTHOR]

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O/1% CO, an O tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O/1% CO/79% N) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold ( p<0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a ∼1.9-fold ( p< 0.001) increase in number and a 1.2-fold ( p<0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, x 2.5; Mn-superoxide dismutase, x 2.1; catalase, x 4.0; glutathione peroxidase, x 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity. [ABSTRACT FROM AUTHOR]

The axon terminals of the neuroendocrine caudo-dorsal cells (CDC) of the freshwater snail Lymnaea stagnalis release an ovulationstimulating hormone by exocytosis in a calcium-dependent way. Ultrastructural studies of the terminals, involving the K-pyroantimonate method for the demonstration of calcium and control tests with EGTA, show that calcium occurs in mitochondria and in various types of vesicular structure. Quantitative investigations indicate that mitochondria accumulate calcium during a short period of high neurohormone-release activity ('active state'; ca. 45 min) and release it again into the axoplasm some hours later, during a period of low secretory activity ('resting state'). Probably, in this way mitochondria play an important role in the buffering of the axoplasmic calcium concentration during high hormone-release activity. HRP-incorporation studies strongly suggest that the calcium-containing vesicular structures constitute a mechanism of membrane sequestration by which the CDC axon terminals resorb, transform, and release parts of the axolemma after exocytotic hormone release. The results furthermore indicate that this mechanism also may be involved in the control of the calcium concentration of the axoplasm, by taking up calcium from the axoplasm and releasing it into the extracellular space. [ABSTRACT FROM AUTHOR]

Adenylate cyclase activity was demonstrated ultracytochemically in the neurohaemal area of the ovulation-neurohormone producing Caudo-Dorsal Cells (CDC) of the freshwater snail L. stagnalis. During electrical activity the axon terminals of the CDC release the hormone by exocytosis and show high adenylate cyclase activity on their plasma membranes. Electrically inactive terminals hardly show exocytosis and exhibit only little reaction product of adenylate cyclase. This result indicates that cAMP plays a role in the control of release of the CDC hormone. [ABSTRACT FROM AUTHOR]

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 Ca. 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); 'intercellular' (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 50x as high, whereas TE, ICE and ME occurred 6 x, 47x, and more than 300x as frequently, respectively. In high K/Ca-free Ringer the total frequency of exocytosis was only 2x as high as in control Ringer. It is concluded that TE, ICE, and ME are normal, Ca-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. [ABSTRACT FROM AUTHOR]

The release of neuropeptides and other proteinaceous hormones by exocytosis was studied in some detail by means of tannic acid in combination with glutaraldehyde (TAGO-method). This method strongly enhances the electron density of extracellular proteins including exocytosed secretory products, and therefore facilitates visualization of the release process in qualitative and quantitative respects. This study included a variety of neurosecretory cell types in the CNS of the freshwater snail L. stagnalis, the storage and glandular lobes of the corpora cardiaca of the locust L. migratoria, and the posterior pituitary of the rat. In addition, peptide secreting cells in the anterior pituitary were examined. The cytochemical specificity of the TAGO-method as well as the significance of 'frozen' contents of exocytosing secretory granules in mammotrophs and somatotrophs of the rat is discussed. [ABSTRACT FROM AUTHOR]

Two giant neurons (diameter 130 μm) were identified immunocytochemically by means of the unlabeled antibody enzyme technique with anti ACTH 1-39 and 1-24 in the pond snail Lymnaea stagnalis. The cells are located in the visceral and the right parietal ganglion, respectively. They contain moderately electron dense elementary granules (diameter 150-160 nm). By means of the intracellular horseradish peroxidase injection technique it was shown that the cells send fibres into the neuropiles of various ganglia and into nerves. Synapses occur on the fine fibre branches in the neuropile. Synapse-like structures were found on the cell bodies and on the major fibres. The giant neurons are electrotonically coupled. With toluidine blue staining for small peptides it was demonstrated that in the central nervous system of the pond snail numerous peptidergic neurons occur in addition to those identified with the classical staining methods for neurosecretion. [ABSTRACT FROM AUTHOR]

In the central nervous system (CNS) of the freshwater snail Lymnaea stagnalis 3 types of interneuronal contacts can be distinguished electron-microscopically, viz. true synapses, 'synapse-like structures' (SLS), and 'spinules'. Use of the electron microscope specimen tilting stage reveals numerous true synapses. Both 'terminal' and 'en passant' contacts occur on neurones and on glial cells. Furthermore 'bigeminal' synapses are present. Complex (combined) convergent and divergent synaptic arrangements are found. On the basis of the morphology of presynaptic vesicles 7 types of true synapses can be discerned. Histochemical data on the contents of the vesicles are lacking. However, vesicle morphology suggests that type IV is aminergic and type VII cholinergic. Terminal and en passant SLS may penetrate deeply into neuronal somata and large axons, and into glial cells. A cluster of synaptic vesicles is present in the presynapse-like element. Spinules (spine-coated 'evagination-invagination' specializations of the plasma membranes of 2 adjacent neuronal elements) are observed between somata, between axons, and between soma and axon. The neurosecretory Light Green Cells (LGC) and Caudo-Dorsal Cells (CDC) receive complex synaptic input. Type V true synapses, 2 types of SLS, and spinules contact the LGC. The complex morphology of the relationship between type A SLS and LGC, studied in serial sections, reveals that adjacent glial cells are also contacted by type A SLS. Type II true synapses, 3 types of SLS, and spinules are identified on the CDC. The validity of the methods of identification and classification of interneuronal contacts in the CNS of L. stagnalis, as well as the role of these contacts in the regulation of the activity of 'ordinary' neurones, neurosecretory cells, and glial cells is discussed. [ABSTRACT FROM AUTHOR]

The neurosecretory system of the freshwater snail Bulinus truncatus was investigated. With the Alcian blue-Alcian yellow (AB/AY) staining method at least 10 different types of neurosecretory cells (NSC) were distinguished in the ganglia of the central nervous system. The differences in staining properties of the NSC - with AB/AY the cells take on different shades of green and yellow - are borne out at the ultrastructural level: the NSC types contain different types of neurosecretory elementary granules. The neurosecretory system of B. truncatus is compared to that of Lymnaea stagnalis, the species which has received the most attention among the pulmonates. It appears from the comparison that the systems of both species show many similarities, although some differences are also apparent. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (130 mOsm/kg HO), osmolalities of 160 and 190 mOsm/kg HO 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. [ABSTRACT FROM AUTHOR]

In the rat pineal gland the mechanism of release of secretory material was studied ultrastructurally after incubating tissues in Ringer solution containing tannic acid. The results indicate that pinealocytes release the contents of secretory vesicles into the extracellular space via exocytosis, a phenomenon that has not been visualized previously in this cell type. This finding may reflect release of polypeptides by the pineal gland. [ABSTRACT FROM AUTHOR]