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Flight activity of insects provides an attractive yet relatively simple model system for regulation of processes involved in energy metabolism. This is particularly highlighted during long-distance flight, for which the locust constitutes a well-accepted model insect. Peptide adipokinetic hormones (AKHs) are synthesized and stored by neurosecretory cells of the corpus cardiacum, a neuroendocrine gland connected with the insect brain. The actions of these hormones on their fat body target cells trigger a number of coordinated signal transduction processes which culminate in the mobilization of both carbohydrate (trehalose) and lipid (diacylglycerol). These substrates fulfill differential roles in energy metabolism of the contracting flight muscles. The molecular mechanism of diacylglycerol transport in insect blood involving a reversible conversion of lipoproteins (lipophorins) has revealed a novel concept for lipid transport in the circulatory system. In an integrative approach, recent advances are reviewed on the consecutive topics of biosynthesis, storage, and release of insect AKHs, AKH signal transduction mechanisms and metabolic responses in fat body cells, and the dynamics of reversible lipophorin conversions in the insect blood.

Rab3B is a monomeric GTPase that modulates norepinephrine secretion when expressed in PC12 neuroendocrine cells. In the present study we determined whether rab3B also regulates the organization of intercellular junctions, since this GTPase localizes to regions of cell contact in multiple cell types. The stable expression of rab3B, but not the closely related rab3A, led to two morphological phenotypes in PC12 cells: (i) reorganization of F-actin into long filopodia and (ii) redistribution of the junction-associated protein ZO-1. ZO-1 localization was not appreciably affected by the expression of a GTP binding mutant of rab3B (N135I) that stimulates norepinephrine secretion by PC12 cells. The apparent diversity of these rab3B phenotypes implies that this GTPase is capable of influencing cell signaling pathways that in turn modulate the cytoskeleton and junction organization. In support of this hypothesis we observed that rab3B expression also altered the profile of proteins that interact with the signaling molecule, phosphatidylinositol 3-kinase (PI3-kinase). The effect of rab3B on protein interactions with PI3-kinase was reversed by inhibitors of this kinase. Furthermore, PI3-kinase inhibitors virtually abolished ZO-1 localization at the surfaces of cells that express rab3B, but not rab3A, whereas these inhibitors had no effect on rab3B-dependent norepinephrine secretion. Our results indicate that rab3B can influence junctional protein targeting and secretion by distinct mechanisms.

A conglomerate of 8-12 neurons in the medulla externa of the crayfish eyestalk was explored in their reaction to a polyclonal antibody against the tyrosinated octapeptide Red Pigment Concentrating Hormone (Tyr-RPCH). These are large neurons with diameters within a range of 33-43 microns and they were all positively stained with neutral red. By intracellular staining with lucifer yellow, the neurons were found to branch extensively within the medulla externa and the lamina ganglionaris of the eyestalk. Each neurite bifurcates at about 40 microns from the soma. Both branches run to the medial edge of the eyestalk; one proceeds distally to the lamina ganglionaris, while the other runs proximally to the medulla interna. Both end freely in multiple arborizations, covering from the medial to the lateral edges of the eyestalk. No branches were found to the sinus gland, the main neurohaemal organ of the eyestalk. A group of 4 neurons in the conglomerate consistently rendered positive reaction to the anti-Tyr-RPCH antibody (A-RPCH). They are superficially located in the cluster, and at the electron microscope, they showed the usual features of a secretory cell, i.e., clear and dense granules, an active and well-developed Golgi apparatus, and rough endoplasmic reticulum. The dense granules were larger (mean diameter: 101.5 nm) than the clear granules (mean diameter: 90.3 nm). The immunopositive reaction at the electron microscope was found to be largely confined to the dense-cored granules.

The caudodorsal cells of Lymnaea and the bag cells of Aplysia are neuroendocrine cells whose peptide products have homologous functions, i.e., regulation of egg deposition. One of the Lymnaea products, calfluxin, increases cytosolic and hence mitochondrial calcium concentrations in secretory cells of the albumen gland, an exocrine organ that secretes perivitellin fluid around the egg cells during packaging; the changes can be visualized at the ultrastructural level for quantification with the pyroantimonate precipitation technique and are correlated with changes in the secretory and biosynthetic activity in the gland. Comparable studies have now been carried out with Aplysia and indicate that the bag cells of A. californica and A. brasiliana also contain a factor with calfluxin-related activity, and that the factor is not the egg-laying hormone (ELH). The bag cell factor does not affect mitochondrial calcium levels in the Lymnaea albumen gland, and synthetic calfluxin does not affect the Aplysia gland. Thus, although the bag cell and caudodorsal cell peptides have the same activity in their respective genera, the sequences have diverged sufficiently during the course of evolution to preclude cross-reactivity. Calfluxin-related activity was also detected in the atrial gland of A. californica and the atrial gland-like epithelium of A. brasiliana, two exocrine organs in the oviduct that express genes structurally related to the bag cell ELH gene. It is postulated that the active atrial gland factors may be peptides A and B.

Cells with rhabdomeric structures characteristic of photoreceptor cells occur within the corpus cardiacum of Melanogryllus desertus. These cells are few in number and are larger than intrinsic secretory cells. Each has numerous rhabdomeric microvilli forming a rhabdome within the cell. The arrangement of large mitochondria under the rhabdomeric microvilli around the dilated endoplasmic reticulum membranes and multivesiculated bodies is distinctive. Rhabdome cells have synaptic contacts with the numerous neurosecretory axons that surround them. Other neurosecretory axons come in close contact with the membranes of microvilli.

Ultrastructural comparison of different types of perisympathetic organs (POs) in three species of Coleoptera (Chrysocarabus auronitens, Oryctes rhinoceros, and Tenebrio molitor) showed that the structure of these organs was not related to their morphological types but to their topography. Two kinds of PO structure may be distinguished: compact median and diffuse lateral. They were similar in that both were surrounded by thin neural lamellae and exhibited numerous glial cells originating in the perineurium (type I perineurial cells) as well as abundant neurosecretory endings. They were different in as much as in median POs, the neurosecretory endings were generally surrounded by perineurial processes but in transverse POs, these endings were sheathless. Only one type of neurosecretory axon was distinguished in the median organs but three or four in the transverse. The nature of the processes by which neurosecretory granules are released may depend on the type of neurosecretory axon. For instance, exocytosis always occurred for dense spherical granules, and granule fragmentation was visualized for granules of smaller size.

Synaptophysin (protein p38) immunoreactivity has been detected immunohistochemically in neuroendocrine cells of the human adrenal medulla, carotid body, skin, pituitary, thyroid, lung, pancreas and gastrointestinal mucosa as well as in 87 out of 93 neuroendocrine tumours investigated, including pheochromocytomas, chromaffin and non-chromaffin paragangliomas, ganglioneuromas, pituitary adenomas, thyroid medullary carcinomas, parathyroid adenomas, lung carcinoids and neuroendocrine carcinomas, pancreatic and gut endocrine tumours and cutaneous merkelomas. Parallel ultrastructural investigation of synaptophysin-reactive cells and tumours revealed the presence, in addition to dense-cored, secretory granules, of a population of pleomorphic, small, clear vesicles resembling synaptic vesicles of nerve terminals as well as the synaptophysin immunoreactive vesicles already described in rat adrenal medullary and pituitary cells. Synaptophysin immunoreactivity showed several differences in its distribution among tumour and non-tumour endocrine cells when compared to chromogranin A immunoreactivity, a well known marker of the core of endocrine granules. Synaptophysin represents a reliable general marker of neuroendocrine cells and tumours, which may be useful in diagnostic histopathology.

The clinical, histopathologic, and ultrastructural features of a primary cutaneous vulvar neuroendocrine neoplasm (Merkel cell carcinoma) are presented. This recently described tumor arises in the dermis and is often aggressive with metastasis to regional lymph nodes. Ultrastructural study of this case reveals morphologic similarities to normal cutaneous Merkel cells, including peripherally located dense-core neurosecondary-like granules, and immunohistochemical studies revealed ACTH within neoplastic cells. By light microscopy this tumor is readily confused with other primary or metastatic cutaneous neoplasms; therefore, the importance of electron microscopic examination is emphasized for definitive diagnosis of this unusual tumor.

The ultrastructural study of perisympathetic organs (POs) in Roscius elongatus revealed the existence of two opposite neurohemal structures. Distal POs formed a network at the end of each of the first pro-, meso-, metathoracic, and abdominal nerves. They display a thin branched structure favoring contact between axon endings and hemolymph. The distal metathoracic POs contained two types of neurosecretory ending, and the distal abdominal POs three types. On the contrary, "subjacent" POs, strangely located laterally in the abdominal area of the ganglionic nerve mass, display a dense compact structure, containing only one type of neurosecretory ending. These characters demonstrate that the "subjacent" POs, earlier classified as transverse POs because they are symmetrically located, are in fact median POs. In transverse POs, signs of extrusion by exocytosis were observed only in one type of neurosecretory ending. In "subjacent" POs unusual pictures were found indicating abundant release by single or compound exocytosis. Such pictures prove that "subjacent" POs have a neurohemal function despite their dense structure and internal localization. Physiological implications of these opposite structures are discussed.

A combination of Golgi, Phaseolus vulgaris leucoagglutinin, and transmission electron microscopic (EM) techniques was used to investigate the morphology of neurosecretory axons and their endings in the neurohypophysis of the rat. Light microscopy indicated that the neurosecretory processes are very tortuous, varicose, and branched, often running in close association with blood vessels. EM, as well as reconstruction from serial thin sections, demonstrated that the combination of synaptoid membrane specializations, dense core vesicles, and accumulations of microvesicles, thought to indicate areas of preferential hormone release, can occur anywhere that the axons contact the basal lamina (BL) lining the perivascular space. Usually, but not always, this was accompanied by some degree of axonal dilation. Individual neurosecretory axons frequently entwined around, or ran adjacent to, blood vessels, a short length of axon forming multiple "endings." Thin glial (pituicyte) processes were interposed between these endings. Axonal processes were also seen to end blindly as end-bulbs suspended in the perivascular space where they were often surrounded by pituicyte processes. The morphology observed suggests that reductions in the contact length of individual nerve terminals could be mediated through increased pituicyte coverage along BL. Conversely, a reduction of pituicyte coverage along the BL could lead to more areas of axonal contact with the BL inducing or allowing the formation of more endings, such as has been reported to occur during lactation or prolonged elevation of plasma testosterone levels.

Neurons containing immunoreactivity for melanin-concentrating hormone (MCH) were located in the brain of the teleost Poecilia latipinna by light microscopic (peroxidase antiperoxidase) and electron microscopic (immunogold) methods. Neuronal cell bodies were found in the tuberal hypothalamus, mostly within the nucleus lateralis tuberis, pars lateralis, containing MCH-immunoreactive granules up to 150 nm in diameter. From here bundles of immunoreactive fibers could be traced through the preoptic area as far forward as the olfactory bulb, and through the posterior hypothalamus up into the pretectal thalamus and midbrain. The main projection was, however, to the neurohypophysis, where MCH fibers were observed to form contacts with pituicytes, basement membranes around blood vessels, and the endocrine cells of the pars intermedia. Occasionally MCH-immunoreactive terminals were also seen near the corticotrophs of the rostral pars distalis. These results support the hypothesis that MCH may act as a systemic hormone, a central neurotransmitter, and a modulator of pituitary function.

Neurosecretory granules (NSG) were isolated by density-gradient centrifugation from homogenates of the intercerebral commissure (COM) and the median lip nerves (LN) of the freshwater snail Lymnaea stagnalis. COM and LN are rich in axon terminals of the ovulation hormone-producing caudodorsal cells (CDC) and of the growth hormone-producing light green cells (LGC), respectively. Electron microscopy of COM isolates showed that a single fraction contained large numbers of NSG, which were identical to NSG of CDC axon terminals of the intact COM. High ovulation hormone activity was associated with this fraction. LN NSG were also isolated in large numbers in a single fraction. The isolated LN NSG were identical to NSG of intact LGC axon terminals. They appeared to have a higher specific gravity than CDC NSG.

Using immunofluorescent techniques, mesotocin (MT) and vasotocin (VT) neurosecretory systems were identified in the brain of different lizards: one temperate species living in southern France and three desert species coming from southern Algeria. In the four species, MT and VT were shown to be synthesized in specific neurons located in the anterior preoptic area (POA), the supraoptic (SON), paraventricular (PVN), and ventromedial (VMN) nuclei. The neurosecretory axons of the POA neurons terminated in the vicinity of the lamina terminalis; in the three desert species, several additional VT fibers extended more rostrally, going into the olfactory bulb. The axons originating in SON, PVN, and VMN ended either in the external zone of the rostral median eminence (ME) near the adenohypophyseal portal vessels, or in the neural lobe of the hypophysis. A short exposure to cold (4 degrees) in some specimens of Lacerta muralis induced a differential response in the dorsal and ventral parts of the PVN. Whereas the dorsal part remained unchanged, the ventral part instead appeared to be essentially composed of small neurons void of secretory granules. Cold exposure also led to a marked accumulation of both MT and VT in the internal zone of the ME, but of only VT in the external ME zone. On the other hand, a conspicuous amount of MT appeared in adenohypophyseal cells.

A comparative ultrastructural study of insects was made whose primitive perisympathetic organs consisted either of a single median neurohemal formation per segment (Periplaneta americana) or of two transverse formations (Carausius morosus), or again, of three--one median and two transverse--as in Locusta migratoria. The results showed that the perisympathetic organs always comprised two types of structure (alpha and beta), even when a single formation exists. In Periplaneta and Carausius, both types were seen to coexist in the same neurohemal formation. In Locusta, however, they were separated, the alpha structure constituting the median organ and the beta structure, the two transverse organs. Each of the two structures have special features: the beta structure displays the usual characteristics of a loose neurohemal organ, i.e., it is penetrated by sinuses and has neurosecretory endings devoid of a glial coat; the alpha structure, on the contrary, forms dense compact organs whose endings are covered with a continuous glial layer. During the evolution, this last structure gradually separated from the other neurohemal formations. It was observed to correspond to a single type of neurosecretory cell, distinct from all other cell types by the particular mode of its release. These results for primitive perisympathetic organs confirm earlier findings for advanced organs. They indicate that both the alpha and beta structures are generally present in insects, thus showing their importance in the physiology of these animals.

By use of an rabbit antiserum raised against the crustacean hyperglycemic hormone (CHH) of the shore crab, Carcinus maenas, CHH-producing perikarya were detected in the brain of the isopod, Porcellio dilatatus, by the double-antibody immunofluorescence technique. The course of the secretory axons to the neurohemal organ, the sinus gland, could be traced. The sinus gland also exhibited strong fluorescence. The reaction was very specific, no other structures gave appreciable immunofluorescence. There are two CHH cells in each hemisphere of the brain, located in the medio-rostral part of the protocerebrum. This area appears to correspond to the medulla terminalis of decapods. The cells are aldehyde fuchsin-positive and have been previously described as beta 1 cells. The immunofluorescence could be inhibited by preabsorption of the antiserum with pure Carcinus-CHH.

A study of the yellow fluorescent neurons (M-NSC) of the subesophageal ganglion of the earthworm Octolasium complanatum has been made by using alternate semithin and thin sections in a correlate fluorescence and electron microscopy technique and it results that elementary neurosecretory granules are present where the yellow fluorescent substance is. These granules are similar in size and morphology to the granules of monoamine type. The intimate contact of the capillaries with the granular cytoplasmic area of the M-NSC provides evidence of a probable release of the secretion from the cell body. These results support the hypothesis of the endocrine role of the M-NSC.

Neurosecretory granules (NSG) were isolated by differential centrifugation from a homogenate of the corpora cardiaca-corpora allata complex of newly emerged, adult, female Blaberus discoidalis cockroaches. By centrifuging at 11,000 g, the procedure yielded a final pellet that was enriched in small, membrane-bound vesicles (103 nm mean diameter). This pellet contained 61% of the cytochromogenic activity present in the original homogenate at an 11-fold increase in concentration. A pellet formed at 4000 g contained a mixture of mitochondria and larger NSG (146 nm mean diameter) and most of the hindgut-stimulating hormone activity. This procedure provided a quick, simple method for purifying neurosecretory granules and separating them into two categories of size and endocrine content.

The optic gland in terrestrial pulmonates secretes gonadotropic hormone. The present study investigates the fine structure of the optic gland to clarify the control of secretion. Animals used were the slug, Limax marginatus, and the snail, Euhadra peliomphala. The optic gland cell has a process and the cytoplasm is filled with large granules about 750-1300 nm in diameter with a thin cortex in L. marginatus and 800-1700 nm delimited by a thick peripheral layer in E. peliomphala. These gland cells are characterized by well-developed granular endoplasmic reticulum. Granule formation was seen in the region of the Golgi apparatus. During the breeding season, the medial neurosecretory cells of the brain are active in the production and release of secretory materials. Although no neurosecretory cell bodies occur in the tentacular ganglion, neurosecretory axons penetrate into the optic gland cells. Cobalt filling reveals that axons of the medial neurosecretory cells project to the tentacular ganglion, near the optic gland. These results suggest that the optic gland is controlled by a neurohormone originating from the medial neurosecretory cells of the brain.

In a light and electron microscope study, neurosecretory cells morphologically homologous with the egg-laying hormone-producing caudodorsal cells (CDC) and growth hormone-producing dorsal cells (DC, light green cells) of the freshwater basommatophoran snail Lymnaea stagnalis have been found in five genera (seven species) of Basommatophora, viz. in Lymnaea palustris and Lymnaea ovata, in Planorbis planorbis and Planorbis vortex, in Planorbarius corneus, in Bulinus truncatus, and in Biomphalaria glabrata. It is concluded that the functions of these cells are homologous as well. The homologies of the respective neuron types regard their locations in the cerebral ganglia, their clustering in groups, location of their neurohemal area (CDC: cerebral commissure; DC: median lip nerves), and ultrastructural characteristics (e.g., abundance of rough endoplasmic reticulum, well-developed Golgi apparatus, presence of two types of neuron-specific secretory granules, and release of granule contents by exocytosis into the hemolymph). In addition, CDC show large electron-dense granules and DC reveal infoldings of the plasma membrane at the abaxonal side of the soma as well as synaptic input. On the other hand, each neuron type shows species-specific characteristics, particularly with regard to the number of cells and the structure of the neurohemal area. Furthermore, the CDC show marked differences between genera in the morphology (especially the mean diameter) of type 2 and, particularly, type 1 secretory granules. The morphology of the two types of secretory granules in the DC differs strongly between species. The possible relation between the morphology and the chemical contents of secretory granules has been discussed.

The versatility of horseradish peroxidase is its usefulness both as an antigenic marker and as a probe molecule. We have demonstrated in the neuroendocrine cell that an HRP-bound antibody offers a high order of resolution for determining in which cellular compartment an antigen is located and where it is not. When native peroxidase is applied as an intracellular probe, it labels organelles associated with endocytosis in retrograde axonal transport and with the lysosomal system in both retrograde and orthograde axonal transport. The investigation that remains is the application of lectin-bound HRP to determine the pathways of membrane flow at the time when the neuroendocrine cell is stimulated to synthesize, transport, and secrete its peptide. For example, we are interested to know (1) whether internalized axon terminal membrane tagged with wheat germ agglutinin-HRP is channeled to all Golgi saccules engaged in the production of secretory granules in salt stimulated supraoptic neurons; and (2) if internalized cell membrane of the supraoptic cell body is tagged with wheat germ agglutinin-HRP and channeled to GERL, will this membrane be transferred from GERL to secretory granules, lysosomes in the cell body and axon, the axonal endoplasmic reticulum, and to autophagic/crinophagic vacuoles in axon terminals of salt-stressed supraoptic neurons? These additional studies should provide a more comprehensive, morphological picture of membrane flow in a neuroendocrine cell that is responding to the metabolic demands placed upon it.