Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.     

Pre- and post-synaptic structures in insect CNS: Intramembranous features and sites of α-bungarotoxin binding

The central neuropile of thoracic ganglia in the central nervous system (CNS) of the cockroach Periplaneta americana contains synapses with characteristic pre- and post-synaptic membrane specializations and associated structures. These include dense pre-synaptic T-bars surrounded by synaptic vesicles, together with post-synaptic densities of varying electron opacity. Exocytotic release of synaptic vesicles is observed only rarely near presynaptic densities, but coated pits are seen at variable distances from them, and may be involved in membrane retrieval. After freeze-fracture, paralinear arrays of intramembranous particles (IMPs) are detected on the P face of many presynaptic terminals, with associated dimples indicative of vesicular release. The E face of these membranes exhibits protuberances complementary to the P face dimples, as well as scattered larger IMPs. Post-synaptic membranes possess dense IMP aggregates on the P face, some of which may represent receptor molecules. Electrophysiological studies with biotinylated α-bungarotoxin reveal that biotinylation does not inhibit the pharmacological effectiveness of the toxin in blocking acetylcholine receptors on an identified motoneurone in the metathoracic ganglion. Preliminary thin section ultrastructural analysis of this tissue post-treated with avidin-HRP or avidin-ferritin indicates that α-bungarotoxin-binding sites are localized at certain synapses in these insect thoracic ganglia.     

Pre- and post-synaptic structures in insect CNS: intramembranous features and sites of alpha-bungarotoxin binding

The central neuropile of thoracic ganglia in the central nervous system (CNS) of the cockroach Periplaneta americana contains synapses with characteristic pre- and post-synaptic membrane specializations and associated structures. These include dense pre-synaptic T-bars surrounded by synaptic vesicles, together with post-synaptic densities of varying electron opacity. Exocytotic release of synaptic vesicles is observed only rarely near presynaptic densities, but coated pits are seen at variable distances from them, and may be involved in membrane retrieval. After freeze-fracture, paralinear arrays of intramembranous articles (IMPs) are detected on the P face of many presynaptic terminals, with associated dimples indicative of vesicular release. The E face of these membranes exhibits protuberances complementary to the P face dimples, as well as scattered larger IMPs. Post-synaptic membranes possess dense IMP aggregates on the P face, some of which may represent receptor molecules. Electrophysiological studies with biotinylated alpha-bungarotoxin reveal that biotinylation does not inhibit the pharmacological effectiveness of the toxin in blocking acetylcholine receptors on an identified motoneurone in the metathoracic ganglion. Preliminary thin section ultrastructural analysis of this tissue post-treated with avidin-HRP or avidin-ferritin indicates that alpha-bungarotoxin-binding sites are localized at certain synapses in these insect thoracic ganglia.     

Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits

Freeze-fracture analysis of the neural connections in the outer plexiform layer of the retina of primates (Macaca mulatta and Macaca arctoides) demonstrates a remarkable diversity in the internal structure of the synaptic membranes. In the invaginating synapses of cone pedicles, the plasma membrane of the photoreceptor ending contains an aggregate of A-face particles, a hexagonal array of synaptic vesicle sites, and rows of coated vesicle sites, which are deployed in sequence from apex to base of the synaptic ridge. The horizontal cell dendrites lack vesicle sites and have two aggregates of intramembrane A-face particles, one at the interface with the apex of the synaptic ridge, the other opposite the tip of the invaginating midget bipolar dendrite. Furthermore, the horizontal cell dendrites are interconnected by a novel type of specialized junction, characterized by: (a) enlarged intercellular cleft, bisected by a dense plate and traversed by uniformly spaced crossbars; (b) symmetrical arrays of B-face particles arranged in parallel rows within the junctional membranes; and (c) a layer of dense material on the cytoplasmic surface of the membranes. The plasmalemma of the invaginating midget bipolar dendrite is unspecialized. At the contact region between the basal surface of cone pedicles and the dendrites of the flat midget and diffuse cone bipolar cells, the pedicle membrane has moderately clustered A-face particles, but no vesicle sites, whereas the adjoining membrane of the bipolar dendrites contains an aggregate of B-face particles. The invaginating synapse of rod spherules differs from that of cone pedicles, because the membrane of the axonal endings of the horizontal cells only has an A-face particle aggregate opposite the apex of the synaptic ridge. Specialized junctions between horizontal cell processes, characterized by symmetrical arrays of intramembrane B-face particles, are also present in the neuropil underlying the photoreceptor endings. Small gap junctions connect the processes of the horizontal cells; other gap junctions probably connect the bipolar cell dendrites which make contact with each cone pedicle. Most of the junctional specializations typical of the primate outer plexiform layer are also found in the rabbit retina. The fact that specialized contacts between different types of neurons interacting in the outer plexiform layer have specific arrangements of intramembrane particles strongly suggests that the internal structure of the synaptic membranes is intimately correlated with synaptic function.     

Orthogonal arrays of intramembrane particles in the supporting cells of the guinea-pig vestibular sensory epithelium

Membrane specializations of the contact region between afferent nerve endings and supporting cells of the sensory epithelia of guinea-pig vestibular endorgans were examined by thin-section and freeze-fracture electron microscopy. The calyx-type nerve endings (C-endings) are separated from supporting cells (SC) by a 25-30 nm space. At irregular intervals along the upper lateral surface of supporting cells, the intercellular space narrows markedly to form special close contacts between the C-ending and SC plasma membranes. Freeze-fracture replicas reveal membrane specializations--orthogonal arrays of particulate units--in the region where the close intercellular contacts were found in sections. Orthogonal arrays consisting of from 5 to 20 units were observed on the cytoplasmic (P) fracture face of the lateral SC plasma membrane. These particulate units from a 12 x 12-nm square, and each unit is composed of four 6-nm subunits. Possible roles of the orthogonal arrays are discussed.     

Square arrays and their role in ridge formation in human lens fibers

Square arrays in human lens fibers were studied with freeze-fracture and thin-section TEM. In superficial fibers a number of patches of square array particles in the P face and pits in the E face are found in the smooth membrane. In the deeper cortex and the nucleus, fiber cells have undulating membranes and many ridges. Numerous patches of the particles (P face) are distributed in the concave regions, and the pits (E face) in the convex areas of the bumpy membrane. In most ridges, patches of the particles occur at regular intervals in the "valley" portion, while the pits are on the "crest" portion of ridges. Also, continuous square arrays having the same "valley" location as the regularly arranged patches are found in areas with extensive ridge patterns. The overlapping of the outer portions of two adjacent square arrays is found on the sides between the "crest" and the "valley" of the ridges. Structurally, square arrays are located in a nonjunctional part of the membrane; in an orthogonal crystalline arrangement; and with a particle size of about 6 nm and center-center spacing about 6.4 nm. They are structurally different from gap junctions found in the lens fibers. Thin-section studies reveal two types of cellular contacts: thin pentalamellar structures (about 12-13 nm in overall thickness) associated with the ridge patterns are believed to be square arrays; thick heptalamellar structures (about 16-17 nm in overall thickness) with a narrow gap in between the two central laminae are believed to be gap junctions. This study strongly suggests that square arrays are specifically involved in ridge formation in human lens fibers.     

Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada     

Filipin-cholesterol complexes in plasma membranes and cell junctions of Tenebrio molitor epidermis

The polyene antibiotic filipin combines with cholesterol in membranes to form complexes that are readily identifiable in the electron microscope. The distribution of filipin-cholesterol (FC) complexes is most easily studied by freeze-fracture. Larval epidermis of Tenebrio molitor (Insecta, Coleoptera) was maintained in vitro for 48 hr, since the electrophysiological properties of the cells are best characterized under these conditions. The cells were fixed in buffered 3.0% glutaraldehyde at RT for 15 min, transferred to fresh fixative containing 1% DMSO and filipin (final concentration; 0.5 mg/ml) for 3 hr RT. Control cells were treated in fixative containing 1% DMSO only. In freeze fracture replicas, FC complexes appear on the plasma membrane as large circular protrusions measuring 26.5 +/- 6.8 nm (x +/- s.d.) n = 50, in diameter and 17.1 +/- 2.8 nm, n = 50, in height and 11.7 +/- 2.6 nm, n = 25, in depth. Protrusions are about two times more frequent on the E face while pits are several times more frequent on the P face. FC complexes are most abundant (greater than 50/mu m2) on the basal membrane surface of the cells but are excluded from regions of hemidesmosomal plaques that anchor the cells to the basal lamina. FC complexes are also abundant on the apical surfaces of the cells where cuticle secretion occurs. In the lateral regions below the junctional belt, FC complexes are less numerous but often appear to increase in frequency in a graded fashion away from the junctional region. The septate junctions are relatively free of FC complexes except in regions where they open to form islands. These islands often contain gap junctions but the FC complexes rarely invade the particle domains of the gap junctions. Single FC complexes were seen in three out of a total of 97 gap junctions. Exposure of the epidermis to 20-hydroxyecdysone for 24 hr in vitro did not induce the appearance of FC complexes within the cell junctions.     

Cell to cell relationships in the seminiferous epithelium in the mouse embryo

Summary Germ cells and Sertoli cells in embryonic mouse testes (day 14 to 20 of gestation) were examined by sectioning and freeze-fracture. Intercellular cytoplasmic bridges between the germ cells are observed in day 14 and older embryos. Membrane specializations with dense fuzzy material similar to the socalled desmosome-like structures are found between Sertoli cells and germ cells. A cell contact area with dense opposed membranes is also found between adjacent germ cells. Asymmetrical dense fuzzy lining of both Sertoli and germ cell membranes is noted. Pinocytotic pits or caveolae are frequently found in the Sertoli cell membrane. Between adjacent Sertoli cells, gap junctions of various sizes and focal meshworks of the occluding junctions are found. Most of the occluding junctional particles are located in the center of the grooves in the E face, and are similar to those in postnatal and adult Sertoli cell junctions. In addition, on both fractured faces there are ridges and grooves devoid of particles which are continuous with occluding junctions with particles, suggesting an initial stage in the formation of occluding junctions of the Sertoli cells. Particles gathered at the site of desmosome-like structures are present on the P face of the Sertoli cell.This work is supported by the Japanese Ministry of Education     

Freeze-Fracture Study of the Bloodstream Form of Trypanosoma brucei gambiense

The ultrastructure of Trypanosoma brucei gambiense was investigated by the freeze-fracture method. Three different regions of the continuous plasma membrane; cell body proper, flagellar pocket, and flagellum were compared in density and distribution of the intramembranous particles (IMP's). The IMP-density was highest in the flagellar pocket membrane and lowest in flagellum. Intra membranous particles of the cell body membrane were distributed uniformly on both the protoplasmic (P) and exoplasmic (E) faces. On the P face of the flagellar membrane, a single row of IMP-clusters was seen along the juncture of the flagllum to the cell body. Since the spacing of the IMP-clusters was almost equal to the spacing of the paired rivet structures observed in thin section, these clusters likely are related to the junction of flagellum and cell body. At the neck of the flagellar pocket, several linear arrays of IMP's were found on the P face of the flagellar membrane, while on the E face rows of depressions were seen. At the flagellar base, the clusters of IMP's were only seen on the P face. On the flagellar pocket membrane, particle-rich depressions and linear particle arrays were also found on the P face, while on the E face such special particle arrangements were not recognized. These particle-rich depressions may correspond to the sites of pinocytosis of the bloodstream forms which have been demonstrated in thin sections.     

The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis

What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.     

Structural diversity of gap junctions. A review.

Gap junctions are plasma membrane specializations characterized as aggregates of intramembranous particles in two apposed membranes meeting particle-to-particle in the 2-4 nm intermembrane 'gap'. Recent thin-section and freeze-fracture evidence has revealed significant structural variations of gap junctional structure at various stages of development and from different organisms and tissues. It is suggested that a comparative analysis of these differences may provide clues to the specific biological functions(s) of these ubiquitous organelles.     

Intercellular junctions in myriapods

Tissue from the intestinal tract of myriapods, including millipedes, centipedes and pauropods were examined in tracer-impregnated sections and freeze-fracture replicas. The foregut and hindgut of all three classes exhibit pleated septate junctions; these display undulating intercellular ribbons in thin sections. In replicas they show discrete intramembranous particle (IMP) arrays aligned in rows in parallel; with one another. The tissues of the hindgut also possess scalariform junctions, characterized by cross-striated intercellular clefts in sections and IMP-enriched membranes in replicas. Gap junctions occur in all groups, but they are atypical in replicas in that their component IMPs do not always fracture onto the E face, as is characteristic of other arthropods; some IMPs cleave to the P face and others to the E face. The midgut of these organisms exhibits smooth septate junctions with conventional straight septal ribbons and occasional interseptal columns. However the intramembranous appearance in replicas is variable, particularly in centipedes, in that the rows of IMPs in chemically-unfixed propanecryofixed tissues, are prominent and adhere preferentially to the E face, with complementary P face grooves, while in fixed tissues the IMPs are much less distinct and fracture to either P face or E face. They tend not to protrude far beyond the mid-plane of the membrane bilayer and lie in rows which commonly take on the form of a network. Individual rows of the network sometimes curve to run beside a second row, over a short distance, before bending away into another part of the network. The aligned particle rows, which are much more prominent in millipedes, where they frequently lie in close parallel appositions, do not fuse into ridges as often occurs in insect tissues. The myriapod junctions, therefore, are of the same general kind as are found in the gut tract of other arthropod groups, but differ with respect to the subtleties of their intramembranous organization and disposition.     

The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis.

Special occluding junctions between Sertoli cells near the base of the seminiferous epithelium are the structural basis of the blood-testis permeability barrier. In micrographs of thin sections, multiple punctate pentalaminar contacts between apposed membranes are observed in the junctional regions.In freeze-fractured mature testis, the junctional membranes exhibit up to 40 parallel circumferentially oriented rows of intramembrane particles preferentially associated with the B-fracture face, but with complementary shallow grooves on the A-face. Short rows of particles may remain with the A-face resulting in discontinuities in the B-face particle rows. In addition, elongate aggregations of particles of uniform size (～70 A) arranged in one or more closely packed rows are occasionally found adjacent to the linear depressions on the A-face of the Sertoli junction. These are interpreted as atypical gap junctions.In immature testis, occluding junctions are absent but typical gap junctions are common. These gradually disappear. In the second postnatal week, linear arrays of particles appear on the B-face. Initially meandering and highly variable in direction, these gradually adopt a consistent orientation parallel to the cell base. The establishment of the blood-testis barrier appears to be correlated with this reorganization of the intramembrane particle rows. Sertoli junctions were shown to be resistant to hypertonic solutions that rapidly dissociate junctions of other epithelia.Sertoli junctions thus differ from other occluding junctions in their (1) basal location, (2) large number of parallel particle rows, (3) absence of anastomosis between rows, (4) preferential association of the particles with the B-face, (5) intercalation of atypical gap junctions, (6) unusual resistance to dissociation by hypertonic solutions.     

Membrane specializations in the peripheral retina of the housefly Musca domestica L.

Membrane specializations of the peripheral retina of the housefly (Musca domestica) are revealed in thin sections and freeze fracture/etch replicas. Septate junctions are abundant in corner areas of the pseudocone enclosure bonding: between homologous corneal pigment cells (CPC); between homologous large pigment cells (LPC); between CPC-LPC; between Semper cells (SC); between SC-CPC. Spot desmosomes are present between Semper cells. It is likely that septate junctions function as strengthening adhesions in this area. A new membrane specialization similar to a continuous junction was observed between retinular cells of the same or adjacent ommatidium. This junction has indistinct septa in the 115 A intermembrane cleft and is intermittent in character. When this junction is absent, the apposed cells gape apart. In freeze fracture studies, this junction is characterized by bridges composed of fused membrane particles and randomly arranged particles on the P face, and noncorresponding grooves on the E face. The ridges are elongate and roughly parallel and sometimes they form enclosures. Mitochondria line up along these junctions, often within 90 A of the unit membrane. This membrane specialization has characteristics of tight and continuous junctions. In line with previous findings, we suggest that this junction assists in retinular cell orientation, possibly in enforcing the ommatidial twist and in maintaining localized ionic concentration gradients between retinular cells.     

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system

Summary Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6–7 nm particles soley near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions) contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia.The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neural lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.     

Fine structure of the palisade zone of the rat median eminence as revealed by freeze-fracturing

Summary Nerve terminals in the palisade zone of the rat median eminence were investigated with freeze-fracture electron microscopy. Fracture face P of the specific terminals showed two populations of intramembranous particles (IMP): a large and a small variety. The large IMP-s often formed small irregular clusters. In nerve terminals the total number of both populations of IMP-s was considerably less than that found on P membrane faces of ependymal feet. On fracture face E of the nerve terminals, the number of IMP-s was about a quarter of that seen on fracture face P.On both fracture faces of most terminals a few small round impressions (or elevations respectively) were found which may be interpreted as broken necks of either exo- or endocytotic vesicles. Neither gap nor tight junctions occurred at lateral membranes of the specific axon terminals. Similarly, no membrane specializations were observed with freeze-fracturing on membrane areas adjacent to the basement membrane. The findings are discussed in relation to a possible exocytosis mechanism of the hypothalamic releasing and inhibiting hormones.     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.