Microapocrine Secretion Research Articles

The Evolution, Gene Expression Profile, and Secretion of Digestive Peptidases in Lepidoptera Species

Serine peptidases (SPs) are responsible for most primary protein digestion in Lepidoptera species. An expansion of the number of genes encoding trypsin and chymotrypsin enzymes and the ability to upregulate the expression of some of these genes in response to peptidase inhibitor (PI) ingestion have been associated with the adaptation of Noctuidae moths to herbivory. To investigate whether these gene family expansion events are common to other Lepidoptera groups, we searched for all genes encoding putative trypsin and chymotrypsin enzymes in 23 publicly available genomes from this taxon. Phylogenetic analysis showed that several gene family expansion events may have occurred in the taxon’s evolutionary history and that these events gave rise to a very diverse group of enzymes, including proteins lacking the canonical SP catalytic triad. The expression profile of these enzymes along the midgut and the secretion mechanisms by which these enzymes enter the luminal content were also analyzed in Spodoptera frugiperda larvae using RNA-seq and proteomics. These results support the proposal of a midgut countercurrent flux responsible for the direction of these proteins to the anterior portion of the midgut and show that these enzymes reach the midgut lumen via both exocytosis and microapocrine secretion mechanisms.

Enzymatic activities in the digestive tract of spirostreptid and spirobolid millipedes (Diplopoda: Spirostreptida and Spirobolida)

Millipedes represent a model for the study of organic matter transformation, animal-microbial interactions, and compartmentalisation of digestion. The activity of saccharidases (amylase, laminarinase, cellulase, xylanase, chitinase, maltase, cellobiase, and trehalase) and protease were measured in the midgut and hindgut contents and walls of the millipedes Archispirostreptus gigas and Epibolus pulchripes. Assays done at pH 4 and 7 confirmed activities of all enzymes except xylanase. Hydrolysing of starch and laminarin prevailed. The hindgut of E. pulchripes was shorter, less differentiated. Micro-apocrine secretion was observed only in the midgut of A. gigas. Merocrine secretion was present in midgut and hindgut of E. pulchripes, and in the pyloric valve and anterior hindgut of A. gigas. Alpha-polysaccharidases were mostly active in the midgut content and walls, with higher activity at pH 4. The low activity of amylase (A. gigas) and laminarinase (E. pulchripes) in midgut tissue may indicate their synthesis in salivary glands. Cellulases were found in midgut. Chitinases, found in midgut content and tissue (E. pulchripes) or concentrated in the midgut wall (A. gigas), were more active at an acidic pH. Polysaccharidases were low in hindguts. Protease shows midgut origin and alkaline activity extending to the hindgut in E. pulchripes, whereas in A. gigas it is of salivary gland origin and acid activity restricted to the midgut. Some disaccharidases, with more alkaline activity, showed less apparent midgut-hindgut differences. It may indicate an axial separating of the primary and secondary digestion along the intestinal pH gradient or the presence of enzymes of hindgut parasites.

Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion.

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.

Morphology and ultrastructure of the midgut gland ("hepatopancreas") during ontogeny in the common spider crab Maja brachydactyla Balss, 1922 (Brachyura, Majidae)

We studied the anatomy and cytology of the midgut gland (MGl) of the common spider crab Maja brachydactyla Balss, 1922 at several life stages (zoea, megalopa, first juvenile, and adult) using dissection, histology, electron microscopy, computed tomography, and micro-computed tomography (micro-CT). In newly hatched larvae, 14 blind-end tubules form the MGl. The length of the tubules increases during the larval development. In the late megalopa, the number of tubules also increases. In adults, 35,000 to 60,000 blind-ending tubules comprise the MGl. In all life stages, a square-net network of muscle fibers surround the tubules. We describe five cell types in the MGl in all larval stages, which have a similar location, histology, and ultrastructure in larvae and adults: embryonary (E-) cells, resorptive (R-) cells, fibrillar (F-) cells, blister-like (B-) cells, and midget (M-) cells. Major difference between larval and adult cells is the larger size of the adult cells. Microapocrine secretion occurs from the microvilli of the B-cells. No ultrastructural changes were observed during larval development, which suggests that the function of each cell type might be similar in all life stages. The role of each epithelial cell type in larvae and adults is discussed.

The study of the calpain and caspase-1 expression in ultrastructural dynamics of Ehrlich ascites carcinoma necrosis

An expression of calpain and caspase-1 as well as the concomitant ultrastructural alterations were investigated during necrosis of the mouse Ehrlich ascites carcinoma. The calpain expression was registered at 0 h and 1 h although caspase-1 did not induce any signals during these time periods. The rise of the cytoplasmic lytic zones contacted by calpain antibodies was identified as a morphologic event corresponding to the expression of calpain. Lytic zone's distribution followed by the appearance of the calpain/caspase-1 clusters assigned for lysis of the Golgi vesicles and ER. Also, the microapocrine secretion of the vesicles containing the calpain/caspase-1 clusters was detected. Further, the lysis of the plasma membrane occurred due to progression of intracellular lysis. Rupture of the plasma membrane resulted in the termination of secretion and dissemination of cell contents. The nuclei still had their normal shape. Nuclear lysis continued to rise with intranuclear lytic zones, of which the progression was accompanied with the presence of calpain/caspase-1 clusters. The data contribute to the concept of the initial role of calpain for tumor cell destruction, provide first evidence of the calpain/caspase-1 pathway in tumor cells, and highlight microapocrine secretion as a possible tumor cell death signalling mechanism.

Fine structure of the midgut epithelium in the millipede Telodeinopus aoutii (Myriapoda, Diplopoda) with special emphasis on epithelial regeneration

The midgut of millipedes is composed of a simple epithelium that rests on a basal lamina, which is surrounded by visceral muscles and hepatic cells. As the material for our studies, we chose Telodeinopus aoutii (Demange, 1971) (Kenyan millipede) (Diplopoda, Spirostreptida), which lives in the rain forests of Central Africa. This commonly reared species is easy to obtain from local breeders and easy to culture in the laboratory. During our studies, we used transmission and scanning electron microscopes and light and fluorescent microscopes. The midgut epithelium of the species examined here shares similarities to the structure of the millipedes analyzed to date. The midgut epithelium is composed of three types of cells-digestive, secretory, and regenerative cells. Evidence of three types of secretion have been observed in the midgut epithelium: merocrine, apocrine, and microapocrine secretion. The regenerative cells of the midgut epithelium in millipedes fulfill the role of midgut stem cells because of their main functions: self-renewal (the ability to divide mitotically and to maintain in an undifferentiated state) and potency (ability to differentiate into digestive cells). We also confirmed that spot desmosomes are common intercellular junctions between the regenerative and digestive cells in millipedes.

Gelsolin role in microapocrine secretion.

A role of gelsolin in opening the way along the microvilli for secretory vesicles during microapocrine secretion is proposed here. Data obtained with different techniques showed that many digestive enzymes are released by microapocrine secretion in insects. Proteins that might be involved in the machinery of microapocrine secretion were selected from our transcriptomes and literature searches. The proteins were annexin, Complex actin-related proteins 2 and 3 (ARP 2/3) cofilin, fimbrin, gelsolin 1, gelsolin 2, moesin, myosin 1, myosin 6, protein disulphide isomerase 1 (PDI 1), PDI 2 and profilin. The cDNAs coding for annexin, fimbrin, gelsolin 1, myosin 1, PDI 1 and PDI 2 were cloned and their sequences deposited in GenBank. Only gelsolin 1 and myosin 1 are expressed exclusively in the midgut (semiquantitative reverse transcriptase PCR). As myosin 1 may have a structural role in microvilli, gelsolin 1 is the best guess to be involved in the secretory machinery. A truncated recombinant gelsolin 1 was used to generate antibodies with which it was shown labelling inside and around midgut cell microvilli shown in an electron microscope, reinforcing a microvillar role for gelsolin 1. Suppression of gelsolin 1 synthesis by RNA interference prevents secretory vesicles from advancing inside the microvilli, in agreement with its putative role in severing the actin filaments to free the way for the vesicles.

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida, Phasmatidae): A morphological, physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.

Midgut proteins released by microapocrine secretion in Spodoptera frugiperda

Microapocrine vesicles bud from the lepidopteran midgut microvilli as double membrane vesicles. To identify the proteins secreted by this process, antibodies raised against isolated microapocrine vesicles from Spodoptera frugiperda were used for screening a midgut cDNA expression library. Positive clones were sequenced, assembled and N blasted against S. frugiperda sequences obtained by pyrosequencing midgut mRNA. This procedure led to the extension of microapocrine sequences that were annotated. A similar procedure was used to identify midgut microvillar proteins that necessarily are part of the microapocrine vesicle. Forty-eight proteins were associated with microvillar membranes. They pertain to 8 functional groups: digestive enzymes, peritrophic membrane, protection, transporters, receptors, secretory machinery, cytoskeleton and signaling, and unknown. Twenty-eight proteins are putatively secreted by microapocrine secretion. Most of them are digestive enzymes, but the list also includes proteins involved in protection and in peritrophic membrane formation. Among the identified digestive enzymes, aminopeptidases are typically microvillar and group into the classes 1, 2, 3, 5, and 6. There are two amylases secreted by microapocrine secretion: one is a digestive enzyme and the other is a transporter-like amylase with no clear function. One lipase has a predicted transmembrane loop, whereas the others are supposed to be secreted by microapocrine secretion and be digestive. Trypsin is membrane bound and is delivered by microapocrine secretion, but has no predicted features to bind membranes. It may remain bound through the signal peptide till be delivered into the midgut lumen. Proteins supposed to be involved in the microapocrine secretory machinery were: calmodulin, annexin, myosin 7a, and gelsolin 1. Their putative roles are discussed, but more research is necessary to settle this subject.

Egg hull formation in Callochiton dentatus (Mollusca, Polyplacophora): the contribution of microapocrine secretion

Abstract. Egg hull formation during oogenesis in the chiton Callochiton dentatus does not follow the typical model of merocrine secretion involving Golgi vesicle exocytosis. Instead, microapocrine secretions are primarily responsible for egg hull formation, although merocrine secretions contribute “areolae” and the vitelline layer. Microapocrine secretion mechanisms are poorly understood, involving a different cellular pathway than is typical. Egg hull formation in C. dentatus involves two types of microapocrine secretions released by the oocyte, one of which is described here for the first time. The plesiomorphic jelly‐like egg hull of chitons, as exemplified by the eggs of members of the basal order Lepidopleurida and present also in eggs of C. dentatus (Chitonida: Callichitonidae), may have evolved solely as an oocyte secretion, whereas members of some other families in the order Chitonida form their egg hulls with considerable secretory input from the follicle cells as well.

Ultrastructure of hull formation during oogenesis in Rhyssoplax tulipa (=Chiton tulipa) (Chitonidae: Chitoninae)

Egg hull formation is described in detail for the first time in Rhyssoplax tulipa from South Africa. Although previous descriptions of hull formation in chitons provide some basis for comparison, the mechanisms we have discovered are totally different. The process begins with the release of clear vacuoles from the oocyte into the intercellular space, which separates it from a layer of follicle cells. Materials contributing to the layers of the hull are formed primarily by micro-apocrine secretion processes, in which extensions of the follicle cells and oocyte are budded off into the intercellular space, rather than by formation and release of vesicles from the Golgi bodies, as previously thought. Later in oogenesis, these secretions become organized into three functional layers, each with a characteristic substructure. Follicle cells mold the shape of the hull spines even down to the petalloid tips that are characteristic of this group. Late in oogenesis the oocyte secretes the vitelline layer beneath the hull and the follicle cells break down and are sloughed off. Differences in the mechanisms of hull formation among chitons are providing a new set of morphological characters that may yield insight into chiton phylogeny. To our knowledge, this is the first description of micro-apocrine secretions during oogenesis in a mollusc, although it has been reported in other phyla.

Identification of midgut microvillar proteins from Tenebrio molitor and Spodoptera frugiperda by cDNA library screenings with antibodies

The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, α-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins.

Ultrastructure of the Salivary Glands of Pachycondyla(=Neoponera) villosa(Fabricius)(Formicidae: Ponerinae): Functional Changes during the Last Larval Instar.

The cells of secretory region of the salivary glands of Pachycondyla (=Neoponera) villosa at the time of enzyme production presents the basal cellular membranes profusely folded and the intercellular junctional membranes present a few enlarged spaces. The rough endoplasmic reticulum and the Golgi bodies shift from being flat and small vesicular cisternae to enlarged vesicular cisternae according to the cell physiological state and characterize an asychronic cell cycle. Enzymes are released into the lumen by microapocrine secretion. The stage of silk production is detected after a behavioral act, when the nurse worker separates the mature larva. At this time, the salivary gland cells present only one physiological state (synchronized secretory cycle): this state was characterized by basal cellular membrane poorly folded, intercellular junctions presenting some small spaces, rough endoplasmic reticulum compounded by flat cistenae, enlarged Golgi bodies with fibrous material inside and a few secretory vesicles containing silk, which undergo exocytosis. The silk in the lumen shows a forms: tactoid and flocculent material.

The peritrophic membrane of Spodoptera frugiperda: secretion of peritrophins and role in immobilization and recycling digestive enzymes.

A peritrophin from the Spodoptera frugiperda peritrophic membrane (PM) and microvillar proteins from S. frugiperda anterior midgut cells were isolated and used to raise antibodies in a rabbit. These antibodies, as well as a Tenebrio molitor amylase antibody that cross-reacts with S. frugiperda amylases, and wheat-germ aglutinin were used in immunolocalization experiments performed with the aid of confocal fluorescence and immunogold techniques. The results showed that the peritrophin was secreted by anterior midgut columnar cells in vesicles pinched-off the microvilli (microapocrine secretion). The resulting double membrane vesicles become single membrane vesicles by membrane fusion, releasing peritrophin and part of the amylase and trypsin. The remaining membranes still containing microvillar proteins and membrane-bound amylase and trypsin are incorporated into a jelly-like material associated with PM. Calcofluor-treated larvae lacking a PM were shown to lose the decreasing gradient of trypsin and chymotrypsin observed along the midgut of control larvae. This gradient is thought to be formed by a countercurrent flux of fluid (in the space between PM and midgut cells) that powers enzyme recycling.

Annual changes in fine structure of inner epithelial lining of the ovary of a marine sculpin, Alcichthys alcicornis (Teleostei: Scorpaeniformes), with internal gametic association.

Alcichthys alciocornis has a viscous ovarian fluid in the ovarian cavity, which plays an important role in its unique mode of reproduction called internal gametic association (i.e., internal insemination and sperm-egg association but a delay in the physiological fertilization until spawning). Seasonal changes in fine structure of the inner epithelial lining and capillary endothelium of the ovary revealed that ovarian fluid originated as a result of the secretory activity of the tissues. The ovarian cavity of A. alcicornis is lined with an ovigerous lamella epithelium and an ovarian wall epithelium. During the spawning period, both epithelia actively secreted proteinaceous substances which seemed to constitute the ovarian fluid. The substances appear to be synthesized in the rough-surfaced endoplasmic reticulum from the material which was transported from the blood capillary, taken into the epithelial cells by endocytosis, accumulated in secretory vesicles via Golgi apparatus in the cells, and finally released into the ovarian cavity by exocytosis. Microapocrine secretion was also observed to occur in both epithelia. Secretory activity of both epithelia by exocytosis and microapocrine secretion showed distinct seasonal changes. Active exocytosis and microapocrine secretion were observed during the spawning period (April-May). These activities slightly declined during the degeneration period (May-June) and were lost during the early recovery period (July). During the mid to late recovery period (October-March), there was some exocytosis but no microapocrine secretion. © 1995 Wiley-Liss, Inc.

Observation of the internal configuration of rat incisor odontoblasts by scanning electron microscopy using the AODO method.

The internal configuration of rat incisor odontoblasts was studied mainly by scanning electron microscopy (SEM) using the AODO method (low concentration aldehyde prefixation, osmium tetroxide postfixation, dimethyl sulfoxide (DMSO) freeze-fracture, osmium tetroxide maceration). The present SEM findings were compared with the results obtained by conventional transmission electron microscopy (TEM) of epon-embedded specimens. The following results were obtained: 1) Functioning odontoblasts were characterized by a concentric, laminar rough endoplasmic reticulum (rER) with many long mitochondria interposed. 2) A network of tubular smooth endoplasmic reticulum (sER) was observed in the odontoblast process and distal portion of both functioning and resting odontoblasts. 3) The tubulo-vesicular elements which have been found to present a modified Golgi-GERL organelle with secretory and absorptive functions were demonstrated in both the functioning and resting odontoblasts. Structurally they consist of the sER network and strings of granules and vesicles. 4) Various types of cytoplasmic bodies, e.g., lysosomes, cytosomes and multivesicular bodies, related to the sER were also noted in both functioning and resting odontoblasts. 5) Microapocrine secretion of membranous vesicles of various sizes into the predentin and along the lateral branchings of odontoblast processes in the circumpulpal dentin was observed during the matrix apposition stage of the odontoblasts. The present morphological study revealed the three-dimensional configuration of the intra- and extra-cellular structures related to dentinogenesis by odontoblasts.

The effects of thermal stimulation on the ultrastructure of the human atrichial sweat gland. I. The fundus.

Ultrastructural examination of sweat glands from the human loin before and during heat-induced activity indicated that the sweat is formed from the contents of disrupted cells as well as from the products of secretion. The principal secretory processes appear to be fluid transport and exocytosis of vesicles. However, configurations suggesting microapocrine secretion were also observed. It is concluded that the mechanisms involved in sweat production in man are fundamentally similar to those in animals and the terms 'apocrine' and 'eccrine' should be discarded. The myoepithelial cells which were contracted at the onset of sweating appeared to be under less tension after 3 h of continuous activity.

The surface structure of the human nasal mucosa

This study is part of a larger investigation, which includes morphometric and histopathologic studies, with the common objective of obtaining a comprehensive description of normal and altered nasal mucosa. Specimens from normal young subjects of both sexes (4 girls and 10 boys, 4-14 years of age) and middle-aged male persons (40-60 years of age) were investigated. Squamous metaplasia starts at an early age and becomes more prominent with increasing years. Pseudostratified epithelium is replaced by fully developed stratified squamous epithelium through a sequence of epithelial alterations starting with stratified cuboidal epithelium followed by mixed stratified cuboidal/stratified squamous epithelium. Each stage during this transformation can be readily recognized by its fine structural surface pattern. The changes in surface features during metaplastic transformation include (i) an increase in number and shortening of the microvilli, (ii) the appearance of microridges in squamous epithelium and (iii) alterations in mucus secretion, changing from macroapocrine in pseudostratified epithelium to microapocrine secretion in metaplasia. Metaplasia usually appeared in sharply demarcated areas, and each type is always surrounded by the directly preceding or succeeding variety of epithelium.

Electron-microscopic study of the secretion of the ependymal cells in the domestic cat (ependymin-β cells)

We have studied, by electron microscopy, the ultrastructural aspects of secretion (neurosecretion) of the ependyma of the third ventricle of the domestic cat. We have found cytoplasmic protrusions and isolated masses of cytoplasm, some with homogeneous cytoplasm and others with very dense granulation (protein-beta?). Axons, synaptic terminals and free secretory granules in the ventricular lumen were also seen. The existence of ependymin-beta cells (ependymocyte-beta) and axohormonal buttons is suggested. The ependymal cells are classified into seven types: (1) covering ependymocytes, (2) tanycyt ependymocytes, (3) secretory ependymocytes, (4) ependymocytes-beta, (5) neurosecretory ependymocytes, (6) neurosensorial ependymocytes (crown-like) and (7) supraependymal microgial ependymocytes. A neurohormonal hypothesis and the possible existence of one or more cerebral hormones (neurohormones) are suggested. These hormones would flow into the CSF through some of the ependymal cells (by microapocrine secretion, liberation of neurosecretion granules, or by axohormonal buttons): this could be the most important link in the endocrine system, assuring the functional unity throughout the ventricular system of the cerebrospinal axis which it winds through, although its basic influence is exercised) on the hypophysis level as a vertex of the classical endocrine system.

Fine structures of the cerebrospinal fluid-contacting neurons in the hypothalamus of the lamprey, Lampetra japonica.

In the preoptic, infundibular and posterior recesses of the lamprey, Lampetra japonica cerebrospinal fluid (CSF)-contacting neurons are distributed much more abundantly than in higher vertebrates. They are classified into three different types on the basis of their granules size or electron density: type 1 containing round dense granules of large size (180--230 nm in diameter), type 2 containing variously shaped dense granules of medium size (150--200 nm) and type 3 containing variously shaped granules of small size (100--150 nm). The neurons of the third type may be further classified into two subtypes: type 3a possessing dense granules and type 3b showing less dense granules. The intraventricular process of CSF-contacting neurons contains Golgi cisternae that are forming new secretory granules and well developed granular endoplasmic reticulum with dilated cisternae. It is suggested that secretory granules might be formed in the intraventricular process in addition to the perikaryon. Besides the small cytoplasmic protrusions presumably involved in microapocrine secretion, intraventricular processes occupied with many empty granules are detected which might represent a process of diacrine secretion. The intraventricular processes further contain multivesicular bodies which incorporate old or overproduced secretory granules, suggesting a process (crinophagy) and large lipid droplets likely corresponding to the final digestive product. The CSF-contacting neurons of all types are usually supplied with axo-somatic synapses on the perikaryon and subsurface cisternae are sometimes observed beneath the postsynaptic membrane. Axon terminals are also found forming synapses on the intraventricular process of type 2 and 3 neurons.