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It has been hypothesized that corticotropin-releasing factor (CRF) and its related neuropeptide urocortin 1 (Ucn1) play different roles in the initiation and adaptive phases of the stress response, which implies different temporal dynamics of these neuropeptides in response to stressors. We have tested the hypothesis that acute pain stress (APS) differentially changes the dynamics of CRF expression in the paraventricular nucleus of the hypothalamus (PVN), oval subdivision of the bed nucleus of the stria terminalis (BSTov) and central amygdala (CeA), and the dynamics of Ucn1 expression in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW). Thirty minutes after APS, induced by a formalin injection into the left hind paw, PVN, BSTov, CeA and npEW all showed a peak in cFos mRNA expression that was followed by a robust increase in cFos protein-immunoreactivity, indicating a rapid increase in (immediate early) gene expression in all four brain nuclei. CRF-dynamics, however, were affected by APS in a brain nucleus-specific way: in the PVN, CRF-immunoreactivity was minimal at 60 min after APS and concomitant with a marked increase in plasma corticosterone, whereas in the BSTov not CRF peptide but CRF mRNA peaked at 60 min, and in the CeA a surge of CRF peptide occurred as late as 240 min. The npEW differed from the other centers, as Ucn1 mRNA and Ucn1 peptide peaked at 120 min. These results support our hypothesis that each of the four brain centers responds to APS with CRF/Ucn1 dynamics that are specific as to nature and timing. In particular, we propose that CRF in the PVN plays a major role in the initiation phase, whereas Ucn1 in the npEW may act in the later, termination phase of the adaptation response to APS., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1/nucleobindin 2 (NUCB2) are assumed to play a role in feeding and adaptation to stress. Both peptides are highly expressed in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW), a center implicated in the regulation of stress adaptation and in the pathogenesis of stress-induced brain disorders, in a sex-specific manner. The present study was undertaken to test whether CART and nesfatin are involved in these actions of the npEW in the rat. Acute restraint and chronic variable mild stress were used. Following stress, physiological parameters (serum corticosterone levels, body, adrenal and thymus weights) were determined, CART and nesfatin-like immunoreactivity (LI) as well as mRNA expression were analyzed in the npEW nucleus. Our results depict the following changes: (1) Acute stress resulted in an increase in serum corticosterone levels that was higher in females; (2) In males, data on corticosterone and body weight gain and in females, data on body weight gain revealed an effect of chronic stress; (3) Both acute and chronic stress activated npEW neurons expressing CART and nesfatin-LI, as shown by increased cFos immunoreactivity; (4) Chronic, but not acute stress increased the amount of CART and nesfatin-LI in both males and females; (5) Neither acute nor chronic stress had an effect on CART and NUCB2 mRNA contents of npEW neurons in either sex. Taken together, our data suggest that CART and nesfatin are involved in the response of npEW neurons to chronic stress., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Leptin is critical for normal food intake and energy metabolism. While leptin receptor (ObR) function has been well studied in hypothalamic feeding circuitries, the functional relevance of ObR in extrahypothalamic areas is largely unknown. Central regulatory pathways involved in food intake utilize various neuropeptides, such as urocortin 1 (Ucn1), cocaine- and amphetamine-regulated transcript peptide (CART) and nesfatin-1. Ucn1 is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition to Ucn1, other satiety signals, such as CART and nesfatin-1, are highly expressed in neurons of the npEW. Using immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), we here show the presence of short and long forms of ObR in the rat npEW. Then, we tested our hypothesis that a change in plasma leptin will modulate the activity of npEW neurons containing Ucn1, CART and nesfatin-1. First, by double-labeling immunocytochemistry, we observed that almost all npEW neurons colocalizing Ucn1, CART and nesfatin-1 also contain ObR. Fasting rats for two days caused a marked body weight loss and reduced leptin plasma level in both genders. Ucn1 mRNA and CART mRNA were upregulated after fasting in males (3.3 and 2.4 times, respectively; P<0.05) but not in females. However, their peptide levels were not significantly changed. The peptide level and mRNA of nesfatin-1 were unaffected by fasting. We conclude that npEW-neurons containing Ucn1, CART and nesfatin-1 co-express ObR, and may be involved in leptin-mediated feeding control in male rats only.

Housekeeping gene (HKG) mRNAs are used to normalize expression data of genes of interest in quantitative reverse transcriptase polymerase chain reaction studies. Such normalization assumes constant HKG gene expression under all circumstances. Although sporadic evidence suggests that HKG expression may not always fulfill this requirement and, therefore, such normalization may lead readily to erroneous results, this fact is generally not sufficiently appreciated by investigators. Here, we have systematically analyzed the expression of three common HKGs, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunit 18S and beta-actin, in two different stress paradigms, in various brain areas, in male and in female rats. HKG expressions differed considerably with respect to brain area, type of stressor and gender, in an HKG-specific manner. Therefore, we conclude that before final experimentation, pilot expression studies are necessary to select an HKG which expression is unaffected by the experimental factor(s), allowing reliable interpretation of expression data of genes of interest.

In postmortem brains of patients with major depression, the expression of corticotrophin-releasing factor (CRF) is enhanced and that of brain-derived neurotrophic factor (BDNF) decreased. In mice over-expressing neuronal CRF (an animal model for depression) the expression of urocortin 1 (Ucn1) in the non-preganglionic Edinger-Westphal nucleus (npEW) is strongly down-regulated. Therefore, we hypothesized that an altered activity of Ucn1 neurons in the npEW would contribute to the pathogenesis of major depression. To test this hypothesis we measured Ucn1 mRNA and BDNF mRNA levels in the npEW of seven male and four female, drug-free suicide victims with major depression, and compared the data with those obtained from 10 male and seven female individuals without neurological and psychiatric disorders (controls). We show that compared with controls, the Ucn1-mRNA level in npEW neurons is about 9.12 times higher in male but unchanged in female suicide victims. Furthermore, BDNF mRNA expression in microdissections of npEW was 3.36 times lower in male suicide victims, but 5.27 times higher in female victims, compared with controls. Our data also show that male suicide victims had almost 11.47 times more Ucn1 and 4.26 times less BDNF mRNA in the npEW than female suicide victims. We discuss the significance of these data for npEW Ucn1 and BDNF, and propose that altered expressions of Ucn1 and BDNF in the npEW contribute to the pathogenesis of major depression and/or suicidality in a gender-specific manner.

Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.

Synaptosomal-associated protein of 25 kDa (SNAP-25) regulates various membrane fusion processes including exocytosis by endocrine and neural cells. To increase our understanding of the occurrence and regulation of SNAP-25 isoforms, we identified and characterized SNAP-25a and SNAP-25b mRNAs in the pituitary gland and brain of the amphibian Xenopus laevis. The proteins are strongly conserved and are resistant to botulinum neurotoxin A but not to botulinum neurotoxin E, as shown by Western blotting. The spatial distribution of the two SNAP-25 isoforms was assessed with in situ hybridization. Both SNAP-25a mRNA and SNAP-25b mRNA reside in cells in the pituitary distal lobe and, particularly, in the endocrine melanotrope cells in the pituitary intermediate lobe. The melanotrope cells are involved in the background adaptation process of the skin by releasing alpha-melanophore-stimulating hormone. Quantitation of the respective in situ hybridization signals in the Xenopus pars intermedia indicated a differential response, SNAP-25b mRNA being more highly expressed in black-adapted animals than SNAP-25a mRNA, and more than in white-adapted toads. This differential upregulation was also studied by real-time reverse transcriptase polymerase chain reaction, showing that in the intermediate pituitary lobe, both isoforms are physiologically controlled by the background light intensity stimulus, but with different intensities; in black-adapted animals SNAP-25b mRNA is upregulated by 3.33 times compared with white-adapted animals, but SNAP-25a only by 1.96 times. As to neural tissue, in situ hybridization showed that both isoforms coexist throughout the brain, sometimes with similar strengths, but in various areas either SNAP-25a mRNA or SNAP-25b mRNA expression is prevalent. It is speculated that each of the SNAP-25 isoforms in the Xenopus pituitary and brain has a distinct function in cellular fusion processes including secretion, and that their occurrence and regulation depend on the type of secreted neurotransmitter/hormone and/or the activity state of the cell.

In recent years a large body of evidence has emerged linking chronic stress with increased vulnerability for depression and anxiety disorders. As corticotropin-releasing factor (CRF) is hypersecreted under these psychological conditions, we used our CRF-overexpressing (CRF-OE) mouse line to study underlying brain mechanisms possibly causing these disorders. Urocortin (Ucn), a recently discovered member of the CRF peptide family may play a role in the pathophysiology of stress-induced disorders. Stressors recruit Ucn-immunoreactive neurons in the Edinger-Westphal nucleus (E-WN), which is the major site of Ucn expression. Furthermore, E-WN Ucn mRNA levels are upregulated in CRF-deficient mice. Based on these findings, we hypothesized the down-regulation of E-WN Ucn in CRF-OE mice and consequently, altered responsiveness to stressful stimuli. Our results support this hypothesis as we found weaker immunohistochemical labeling with anti-Ucn and a six times weaker Ucn mRNA signal in E-WN in CRF-OE mice. Moreover, E-WN Ucn-expressing neurons mounted a response to acute challenge in CRF-OE mice too. From these results it is concluded that the CRF and E-WN Ucn neuronal systems work in concert in response to acute challenges, but are inversely regulated in their activities during chronic hyperactivity of the hypothalamo-pituitary-adrenal axis.

To investigate the role of neurotransmitter secretion in the development and stabilization of synapses, the innervation of the diaphragm and intercostal muscles was studied in munc18-1 null mutant mice, which lack regulated secretion. We found that this mutant is completely devoid of both spontaneous and evoked neuromuscular transmission throughout embryonic development. At embryonic day (E) 14, axonal targeting and main branching of the phrenic nerve were normal in this mutant, but tertiary branches were elongated and no terminal branches were observed at this stage, in contrast to control littermates. Acetylcholinesterase staining was observed in the endplate region of mutant muscle from E14 onwards, but not as dense and confined to spots as in controls. Acetylcholine receptor staining was also present in the endplate region of the mutant muscle. In this case, the staining density and the concentration in spots (clusters) were similar to controls, but the distribution of these clusters was less organized. Starting at E15, some receptor clusters co-localized with nerve terminal staining, suggesting synapses, but most clusters remained a-neural. Electron microscopical analysis confirmed the presence of synaptic structures in the mutant. Between E14 and birth, the characteristic staining pattern of nerve branches gradually disappeared in the mutant until, at E18, an elaborate meshwork of nerve fibers with no apparent organization remained. In the same period, most of the motor neuronal cell bodies in the spinal cord degenerated. In contrast, sensory ganglia in the dorsal root showed no obvious degeneration. These data suggest that regulated secretion is not essential for initial axon path finding, clustering of acetylcholine receptors, acetylcholinesterase or the formation of synapses. However, in the absence of regulated secretion, the maintenance of the motor neuronal system, organization of nerve terminal branches and stabilization of synapses is impaired and a-neural postsynaptic elements persist.

The proteins munc18-1 and DOC2 are assumed to play a role in docking of synaptic vesicles in neurotransmitter exocytosis at the presynaptic junction. As the proteins are known to interact, they should co-exist within neurons. We have tested this hypothesis for exocytosis of both classical and peptidergic messengers, by investigating the distribution of the messenger RNAs of munc 18-1 and DOC2 homologues in the brain and pituitary gland of the clawed toad Xenopus laevis, using in situ hybridization. For this purpose we cloned a partial complementary DNA encoding Xenopus unc18 (xunc18) and used a corresponding RNA probe, together with an RNA probe for Xenopus DOC2. At the messenger RNA level DOC2 and xunc18 were found to be expressed throughout the Xenopus brain. All brain nuclei expressing DOC2-messenger RNA showed xunc18-messenger RNA expression as well. Co-expression was shown at the individual cell level in consecutive sections of large-sized neurons. A strong expression was demonstrated in the suprachiasmatic and magnocellular nuclei and in peptidergic endocrine cells in the intermediate and anterior lobes of the pituitary gland, suggesting roles of DOC2 and xunc18 in messenger release from peptidergic secretory systems. Combined in situ hybridization and immunocytochemical analyses show that neuropeptide Y-containing cells in the suprachiasmatic nucleus also express DOC2 and xunc18 messenger RNAs. Since these cells have a high secretory activity, controlling the activity of the pituitary pars intermedia, the levels of expression of DOC2 and xunc18 may be indicators for neuronal secretory activity. The present data represent the first evidence for the co-existence of DOC2 and munc18-1 and suggest co-ordinate action of these proteins at the level of brain nuclei, individual neurons and endocrine cells.

This review is concerned with recent literature on the neural control of the pituitary pars intermedia of the amphibian Xenopus laevis. This aquatic toad adapts skin colour to the light intensity of its environment, by releasing the proopiomelanocortin (POMC)-derived peptide alpha-MSH (alpha-melanophore-stimulating hormone) from melanotrope cells. The activity of these cells is controlled by brain centers of which the hypothalamic suprachiasmatic and magnocellular nuclei, respectively, inhibit and stimulate both biosynthesis and release of alpha-MSH. The suprachiasmatic nucleus secretes dopamine, GABA, and NPY from synaptic terminals on the melanotropes. The structure of the synapses depends on the adaptation state of the animal. The inhibitory transmitters act via cAMP. Under inhibition conditions, melanotropes actively export cAMP, which might have a first messenger action. The magnocellular nucleus produces CRH and TRH. CRH, acting via cAMP, and TRH stimulate POMC-biosynthesis and POMC-peptide release. ACh is produced by the melanotrope cell and acts in an autoexcitatory feedback on melanotrope M1 muscarinic receptors to activate secretory activity. POMC-peptide secretion is driven by oscillations of the [Ca2+]i, which are initiated by receptor-mediated stimulation of Ca2+ influx via N-type calcium channels. The hypothalamic neurotransmitters and ACh control Ca2+ oscillatory activity. The structural and functional aspects of the various neural and endocrine steps in the regulation of skin colour adaptation by Xenopus reveal a high degree of plasticity, enabling the animal to respond optimally to the external demands for physiological adaptation.

The amphibian Xenopus laevis adapts the colour of its skin to the colour of its background, by the release of the pro-opiomelanocortin-derived peptide alpha-melanophore-stimulating hormone from the pars intermedia of the pituitary gland. Suprachiasmatic neurons play an important role in adaptation to a light background as they produce the neurotransmitters GABA, dopamine and neuropeptide Y, which inhibit the release of alpha-melanophore-stimulating hormone. These factors are transported to axon varicosities contacting the melanotrope cells. In these varicosities GABA resides in electron-lucent vesicles and neuropeptide Y and dopamine coexist in electron-dense vesicles. In this study the effects of background adaptation on the morphology of the varicosities in the pars intermedia were studied, using immunoelectron microscopy and morphometry with freeze-substitution-fixed material. Varicosities were found singly and in clusters throughout the pars intermedia. Varicosities were identified by the presence of electron-dense and electron-lucent secretory vesicles, the latter being immunopositive for anti-GABA. Both varicosity types revealed active zones with exclusively GABA-containing vesicles in contact with the presynaptic membrane. When white- and black-adapted animals were compared, significant background effects were found with respect to the organization of the varicosities: the density of varicosity profiles was twice as high in white-adapted animals as in black-adapted ones, due to an increase in density of the clustered varicosities. Furthermore, in white-adapted animals varicosities were about twice as large as in black-adapted animals. With respect to vesicle types, single and clustered varicosities showed a differential effect. For both the population of electron-lucent and electron-dense vesicles, single varicosities showed equal numbers in white- and black-adapted animals, but clustered varicosities showed higher numbers of electron-lucent and electron-dense vesicles in black-adapted animals, indicating storage of neurotransmitters. Finally, in varicosities of white-adapted animals the number and size of the active zones and the number of electron-lucent vesicles attached to the active zones, were about twice as high as in black-adapted animals, indicating a stronger GABA release. It is concluded that the profound effects of environmental light conditions on synaptic structure and substructure in the Xenopus pars intermedia are related to a changed release activity of neurotransmitters inhibiting the activity of the melanotrope cells.

The amphibian Xenopus laevis is able to adapt the colour of its skin to the light intensity of the background, by releasing alpha-melanophore-stimulating hormone from the pars intermedia of the hypophysis. In this control various inhibitory (dopamine, gamma-aminobutyric acid, neuropeptide Y, noradrenaline) and stimulatory (thyrotropin-releasing hormone and corticotropin-releasing hormone) neural factors are involved. Dopamine, gamma-aminobutyric acid and neuropeptide Y are present in suprachiasmatic neurons and co-exist in synaptic contacts on the melanotrope cells in the pars intermedia, whereas noradrenaline occurs in the locus coeruleus and noradrenaline-containing fibres innervate the pars intermedia. Thyrotropin-releasing hormone and corticotropin-releasing hormone occur in axon terminals in the pars nervosa. In the present study, the neuronal origins of these factors have been identified using axonal tract tracing. Application of the tracers 1,1'dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine and horseradish peroxidase into the pars intermedia resulted in labelled neurons in two brain areas, which were immunocytochemically identified as the suprachiasmatic nucleus and the locus coeruleus, indicating that these areas are involved in neural inhibition of the melanotrope cells. Thyrotropin-releasing hormone and corticotropin-releasing hormone were demonstrated immunocytochemically in the magnocellular nucleus. This area appeared to be labelled upon tracer application into the pars nervosa. This finding is in line with the idea that corticotropin-releasing hormone and thyrotropin-releasing hormone stimulate melanotrope cell activity after diffusion from the neural lobe to the pars intermedia. After anterograde filling of the optic nerve with horseradish peroxidase, labelled axons were traced up to the suprachiasmatic area where they showed to be in contact with suprachiasmatic neurons. These neurons showed a positive reaction with anti-neuropeptide Y and the same held for staining with anti-tyrosine hydroxylase. It is suggested that a retino-suprachiasmatic pathway is involved in the control of the melanotrope cells during the process of background adaptation.

The amphibian Xenopus laevis is able to adapt to a dark background by releasing melanophore-stimulating hormone from the pars intermedia of the pituitary gland. The inhibition of melanophore-stimulating hormone release is accomplished by neuropeptide Y-containing axons innervating the pars intermedia. To determine the production site of neuropeptide Y involved in this inhibitory control, the distribution of neuropeptide Y in the brain has been investigated by immunocytochemistry and in situ hybridization. Immunoreactive cell bodies were visualized in, among others, the ventromedial and posterior thalamic nuclei, and the suprachiasmatic and ventral infundibular hypothalamic nuclei. A positive hybridization signal with a Xenopus-specific probe for preproneuropeptide Y-RNA was found in the diencephalic ventromedial thalamic nucleus and in the suprachiasmatic nucleus. With both immunocytochemistry and in situ hybridization, suprachiasmatic neurons appeared to be stained only in animals adapted to a white background; animals adapted to a black background showed no staining. Quantitative image analysis revealed that this effect of background adaptation is specific for suprachiasmatic neurons because no effect could be demonstrated of the background light condition on the ventral infundibular nucleus (immunocytochemistry) or the ventromedial thalamic nucleus (in situ hybridization). These results indicate that neurons in the suprachiasmatic nucleus enable the adaptation of X. laevis to a white background, by producing and releasing neuropeptide Y that inhibits the release of melanophore-stimulating hormone from the melanotrope cells in the pars intermedia of the pituitary gland.

The cerebral neuroendocrine Light Green Cells of the pulmonate snail Lymnaea stagnalis, which control body growth and associated processes, stain positively with an affinity-purified antiserum raised to a large part of the C-chain of pro-molluscan insulin-related peptides. At the ultrastructural level, the rough endoplasmic reticulum is immunonegative, the Golgi apparatus is slightly positive and secretory granules in the process of budding from the Golgi apparatus are strongly positive. These observations indicate that the Light Green Cells synthesize molluscan insulin-related peptides, which are processed before packing by the Golgi apparatus into secretory granules. The two morphologically distinct secretory granule types, i.e. with pale and dark contents, respectively, are equally immunoreactive with antiserum raised to the C-chain of molluscan insulin-related peptides. Secretory granules within lysosomal structures reveal various degrees of immunoreactivity, indicating their graded breakdown. The Light Green Cells release secretory material by the process of exocytosis into the haemolymph from neurohaemal axon terminals located in the periphery of the median lip nerve. The electron-dense (tannic acid method) released contents are clearly immunopositive. The same holds for secretory granule contents released from Light Green Cells axon profiles in the centre of the lip nerve. Some immunoreactivity is also present in the intercellular space between these axon profiles. It is concluded that molluscan insulin-related peptides may act in two ways, namely (1) as neurohormones via the haemolymph at peripheral targets and (2) in a non-synaptic (paracrine) fashion at targets within the central nervous system.

The neural innervation of the neurointermediate lobe of the pituitary of the amphibian Xenopus laevis has been studied at the light and electron microscopic level. In the pars intermedia melanotropes and stellate cells are abutted by varicosities originating from GABA- and neuropeptide Y-producing neurons. The varicosities contain two types of vesicle: electron-lucent vesicles (mean diameter 50 nm) which are immunopositive for GABA and larger (80 nm) electron-dense vesicles which are immunopositive for neuropeptide Y. Double immunogold labeling established that GABA and neuropeptide Y co-exist within the varicosities. In the pars nervosa similar varicosities, though low in number, occur. They are associated with neurosecretory nerve terminals, pituicytes and blood vessels. The possible significance of GABA and neuropeptide Y for the neural regulation of melanophore stimulating hormone-release from the pars intermedia is discussed.

1. Peptide release from the neurointermediate lobe of Xenopus laevis has been studied using dual pulse-chase incubation, superfusion and HPLC techniques. 2. Lobes release pulse-labelled material in two phases, the first phase lasting about 6 hr, the second persisting up to 14 hr. 3. In both phases similar, POMC-derived peptides are released. Their release can be inhibited by dopamine. 4. When release during the first phase is inhibited, newly synthesized peptides are shunted into the second release pathway. 5. It is concluded that the neurointermediate lobe contains two release compartments. The possible locations of these compartments within melanotrope cells have been discussed.

The fine structure of the axons of the cerebral, egg-laying stimulating caudodorsal cells of the snail Lymnaea stagnalis has been studied with various light and electron microscope techniques. Special attention was paid to exocytotic release of secretory material (demonstrated with the tanic acid method) from nonsynaptic release sites in the cerebral commissure. This phenomenon has been compared with neurohaemal release. The commissure consists of two morphological compartments, separated by a sheath of glial cells. The outer compartment is formed by the neurohaemal area of the caudodorsal cells, the inner consists of thousands of, mainly unidentified, axons. Furthermore, ventral caudodorsal cells send axons through the inner compartment. These give rise to collaterals, which divide into smaller collaterals, forming an extensive network ("collateral system") throughout the inner compartment. Eventually, collaterals end blindly within the inner compartment. They contain the same three morphological types of secretory granule as the neurohaemal axon terminals. The collaterals never form synaptic contacts; exocytotic release of the contents of secretory granules takes place at nonsynaptic release sites. These sites occur rather dispersed and do not face one particular type of neighbouring neural element. As in the neurohaemal area, both single and multiple exocytoses occur. Widened intercellular spaces, filled with flocculent, electron-dense material, occur near highly active nonsynaptic release sites. The spaces are often bordered by glial cells and may facilitate diffusion of released secretory material through the inner compartment. Apparently, a ventral caudodorsal cell releases secretory material in two fashions: from neurohaemal axon terminals into the haemolymph, and nonsynaptically, from the collaterals into the intercellular space of the central nervous system. Possible functions of the glial sheath between the neurohaemal area and the inner compartment are proposed. Most likely, the collateral system enables the caudodorsal cells to communicate with targets within the central nervous system in a nonsynaptic fashion. A possible target is the cerebral Ring Neuron, which sends an axon branch through the inner compartment and, as was previously shown neurophysiologically, is controlled by the caudodorsal cells in a nonsynaptic fashion.

The cerebral peptidergic caudodorsal cells of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying behaviour by releasing peptides into (1) the haemolymph, from neurohaemal axon terminals in the periphery of the cerebral commissure and (2) the intercellular space of the central nervous system, from collaterals in the inner compartment of this commissure. Recently, it was shown that collateral release occurs from nonsynaptic release sites, which lack the morphological specializations that are characteristic of classical synapses. Probably, these sites enable the caudodorsal cells to communicate with central neurons in a nonsynaptic ("paracrine", "diffuse", "hormone-like") fashion. The structural and ionic bases of nonsynaptic release were studied using the tannic acid-Ringer incubation-method for the detection of exocytotic release of secretory granule contents in vitro. Elevation of the extracellular potassium concentration strongly stimulates exocytotic activity in the collaterals. No stimulation was found in the absence of extracellular calcium ions. Similar results have been obtained for the neurohaemal axon terminals. Electron-dense material occurs apposed at the cytoplasmic side of the axolemma of collaterals (ethanolic phosphotungstic acid method). This material appears homologous with the presynaptic dense projections forming the "vesicular grid" in classical synapses. Such projections are also present in the neurohaemal axon terminals. It is concluded that secretion from nonsynaptic release sites in caudodorsal cell collaterals shares fundamental characteristics with secretion from conventional neuronal release sites (neurohaemal axon terminals and classical synapses); release occurs by exocytosis of secretory granules, is associated with a vesicular grid, is stimulated by membrane depolarization, and depends on the presence of extracellular calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of neuronal secretory products by exocytosis was studied ultrastructurally in the central nervous systems of three different species (the snail Lymnaea stagnalis, the cockroach Periplaneta americana and the rat). Tissues were fixed with: (1) a mixture of glutaraldehyde and osmium tetroxide, (2) the tannic acid-glutaraldehyde-osmium tetroxide (TAGO) method, and (3) the tannic acid-Ringer incubation (TARI) method. Especially after TARI-treatment, release of the contents of the secretory vesicles by exocytosis could be clearly demonstrated in: (1) synapses, (2) neurohaemal axon terminals (L. stagnalis), and (3) neuronal processes without morphological synaptic specializations (nonsynaptic release sites). Release from nonsynaptic release sites occurs in most cases over a large area of the plasma membrane of a neuronal process facing several neural elements. On the basis of the differences in morphology of the secretory vesicles at nonsynaptic release sites, it is proposed that various types of (peptidic) messenger are released from such sites. In some neurones of L. stagnalis nonsynaptic release sites have been found together with synapses, or with neurohaemal axon terminals (caudodorsal cells, light green cells and light yellow cells). The possibility that nonsynaptic release sites represent the morphological correlates of nonsynaptic communication in the central nervous system has been discussed.

Presence and structure of gap junctions have been studied at the ultrastructural level in the central nervous system of the freshwater snail Lymnaea stagnalis. Gap junctions are clearly visible in thin sections of glutaraldehyde-fixed and phosphotungstic acid-stained material as well as in freeze-fracture preparations. Various types can be distinguished on the basis of junctional size and shape, and numerical density, diameter, spacing and arrangement of junctional particles. Junctions are present between neurones and between glial cells. The neuroendocrine ovulation-hormone-producing caudodorsal cells show gap junctions at four sites, viz. between somata, between axons in the "loop area" and in the intercerebral commissure and between neurohaemal axon terminals. These junctions show common characteristics as to numerical density, diameter, spacing and arrangement of junctional particles, but morphometry reveals different mean values for junctional size and numerical density. Values are the highest for axons in the loop area, intermediate for axons crossing the commissure, and the lowest for somata and axon terminals. It is proposed that the gap junctions particularly play a role in electrotonic intercellular coupling. The results strongly suggest that the gap junctions between the caudodorsal cells--especially those in the loop area and between the crossing axons--are the morphological correlates of the previously demonstrated electrotonic coupling between these cells. This coupling may enable all cells of the network to act synchronously so that a large amount of ovulation-hormone can be released within a short period of electrical activity (the discharge).