Stanley D. Carlson

Temperature-Sensitive Paralytic Mutations Demonstrate that Synaptic Exocytosis Requires SNARE Complex Assembly and Disassembly

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

The fine structure of neuroglia in the lamina ganglionaris of the housefly,Musca domestica L.

SummarySix morphologically distinct glial cell layers are described in the housefly lamina ganglionaris, a region previously thought to be composed of only three. 1. The external glial layer abuts the basement membrane of the retina. The cells of this layer have a highly involuted surface membrane and an abundance of ribosomes and rough endoplasmic reticulum (ER) throughout their cytoplasm. They envelop the traversing photoreceptor and mechanoreceptor axons as well as the large tracheoblast cells of the fenestrated layer. They are referred to as thefenestrated layer glia. 2. The second glial layer is composed of large, horizontally elongated cells with large elongate nuclei. They contain large membrane-bounded vacuoles and extensive arrays of parallel-running microtubules and smooth ER. These glia invest the photoreceptor axons through much of the multiple chiasmatic (pseudocartridge) region and are thus designated as thepseudocartridge glia. 3–4.Satellite glia comprise the third and fourth glial layers. Thin cytoplasmic processes of these multipolar glia intervene between the tightly packed monopolar neuron somata and the photoreceptor axons of the nuclear layer. The satellite glia are distinguished into two sub-groups: distal and proximal. The distal satellite glia are exclusively responsible for the large glial invaginations of the type I monopolar cell bodies. Multilaminated processes of the proximal layer of satellite glia surround the photoreceptor axons and the neurite neck of the monopolar neurons prior to their entry into the plexiform layer. The proximal satellite glia also contain prominent lipid deposits. 5.Epithelial glia are columnar cells that occupy the plexiform layer. They envelop the optic cartridges of the neuropil and are the substrate for two characteristic glial-neuronal invaginations; i.e. the capitate projection and the ‘gnarl’. The cytoplasm of the epithelial glia is electron dense and contains numerous stacked arrays of infolded membrane. 6.Marginal glia form the proximal boundary of the optic neuropil. They invest the axons entering or leaving through the base of the lamina ganglionaris. Marginal glia contain large numbers of parallel microtubules and numerous polyribosomes. Fine structural evidence is presented relevant to the role of these six glial layers in the maintenance of ionic and metabolic homeostasis across the retina-lamina barrier.

Ultrastructure of capitate projections in the optic neuropil of Diptera

SummaryPhotoreceptor axons in the first optic neuropil of the dipteran flies Musca domestica and Drosophila melanogaster were examined with electron microscopy. The objective was to determine ultrastructure, persistence and glial source of the capitate projections found within these neurons. Capitate projections are simple or compound processes of epithelial glial cells which profusely insert into form-fitting folds of axon terminals of the peripheral retinular cells (R1–6) in the synaptic plexus portion of the first optic neuropil. These neuro-glial junctions may be simple indentations, have a head with a single stalk, or possess a single, circular stalk from which 3 or 4 bulbous (glial) heads are elaborated. Using serial thick sections of Drosophila neuropil for HVEM we were able to observe that the stalks connecting nearly all capitate projections led directly to a glial cell. Thus no disembodied heads were found suspended in axoplasm. Capitate projections appeared to be persistent structures, present in young as well as senescent adults. No evolution of form was found; thus 3 distinct expressions of these glial processes (without transitional forms) are present. From freeze-fracture replicas and serial HVEM sections it was determined that there were approximately 3 capitate projections per μm2 in Drosophila and Musca, respectively. About 800 capitate projections exist per Musca axon terminal or about 5 times the number of chemical synapses. Cps were slightly larger in Drosophila than in Musca, although the Musca retinular axon has twice the diameter and length of that of the fruit fly. The evidence was reviewed in light of the likely supportive function of capitate projections on the R1–6 terminals.

Glial membrane specializations and the compartmentalization of the lamina ganglionaris of the housefly compound eye

SummaryMembrane specializations in the lamina ganglionaris of the housefly are investigated using conventional thin-section EM, freeze-fracture replication and the diffusion of colloidal lanthanum. All glial cells in the lamina are coupled by gap junctions. Desmosomes also link all glia except the epithelial glia. Extensive glia-glial and glia-neuronal septate junctions are present in the pseudocartridge zone and nuclear layer. Septate junctions in the nuclear layer intermingle with bands of interglial and glia-neuronal tight junctions. Tight junctions are also found between satellite and epithelial glia at the border of the nuclear and plexiform layers, between adjacent epithelial glial cells in the plexiform layer, between epithelial and marginal glia at the proximal boundary of the optic neuropil, between marginal glial cells, and between marginal glia and axons. Colloidal lanthanum, introduced through an incision in the cornea, penetrates the retina but is occluded from the neuropil by septate junctions in the pseudocartridge zone. The disposition of tight and septate junctions is described in relation to the compartmentalization of the lamina. Two major compartments are delineated. The first represents the nuclear layer and contains the cell bodies of second-order visual neurons (monopolar neurons). The second compartment constitutes the plexiform layer of the lamina. Within the plexiform layer, each optic cartridge is partitioned into a separate subcompartment. Also, tracheoles and axons of long visual fibres are isolated from the optic cartridges by glial tight junctions. Morphological evidence for compartmentalization is correlated with previously established electrical properties of the insect lamina ganglionaris.

The Glial Cells of Insects

To state that neuroglial cells are those ubiquitous companions of neurons is a generality not too helpful in characterizing insect neuroglia. We now know that in insect nervous systems, glial cells are not “ubiquitous” and that naked axons do exist (e.g., Trujillo-Cenoz, 1962). In addition, some types of insect glia are so specialized or evolved that they do not directly contact the neuron, but rather overlie another kind of glial cell, the latter being the true contiguous associate of the neuron.

Ultrastructure and blood–nerve barrier of chordotonal organs in the Drosophila embryo

Chordotonal organs of Drosophila embryos have become models for studies of developmental biology and molecular genetics due to their consistent segmental placement and mutability. Our first goal was to find the origin and anatomical correlate of the blood–nerve barrier of this PNS proprioreceptor in wild type embryos. The concept of a blood–nerve barrier for the PNS of the Drosophila embryo is new, and the present data are the first in this regard. A second goal was to reveal the ultrastructure of these four-celled stretch receptors, focusing particularly on the ‘core’ of this organ: the bipolar neuron enclosed by a scolopale cell. These latter data have resulted in a graphic reconstruction of the chordotonal organ which reveals how the four consistent cells fit together. At Stage 13 we first observed a clearly recognizable scolopale cell with an enclosed neuron. Surprisingly, an operative blood–nerve barrier, comprised of occlusive pleated-sheet septate junctions, exists at this relatively early stage. A blood–brain barrier is not yet functioning in the CNS during this same stage, as the perineurium is not present until Stage 17. Cross-sectional views of a more mature chordotonal organ show that the neuron’s inner segment has a ‘tongue-in-groove’ formation which fits the dendrite into the scolopale cell. Other newly discovered fine structural features are: hemidesmosomes linking individual scolopale rod bundles to the inner dendrite, and a cap cell matrix bonding with the tip of the ciliary dendrite. Functional aspects of these findings are discussed.

Synaptic vesicle activity in stimulated and unstimulated photoreceptor axons in the housefly. A freeze-fracture study

SummaryLight-stimulated and unstimulated photoreceptor (retinular) axon terminals in the lamina ganglionaris (first optic neuropil) of the housefly are examined using freeze-fracture replication. The presence of numerous, cross-fractured capitate projections permits unmistakable identification of the retinular axon terminal membrane. Regardless of the conditions of illumination, the protoplasmic face (P-face) of the terminal membrane contains numerous bowtie-shaped particle clusters (active zones) which resemble theen face form and disposition of the presynaptic ribbon found in thin sections. Estimates from freeze-fractured material indicate that each retinular axon possesses at least 175 such active zones. In eyes fixed during illumination, active zones are surrounded by many membrane dimples indicative of vesicle fusion sites. Such synaptic vesicle sites are seldom encountered in terminals which are dark-adapted and fixed in the dark. Results from light-adapted eyes placed in the dark following the onset of fixation suggest that endocytosis may occur in the extrasynaptic regions of this inhibitory synapse. P-face particles are uniformly distributed throughout the extrasynaptic regions of unstimulated terminals. Particle density increases in areas peripheral to the active zones in stimulated eyes, particularly within the regions presumed to be undergoing active endocytosis. These structural findings are discussed in the context of the Heuser-Reese model of vesicle exocytosis and recycling.

Membrane specializations in the first optic neuropil of the housefly,Musca domestica L. II. Junctions between glial cells

SummaryThin section and freeze-fracture replicas of the first optic neuropil (lamina ganglionaris) of the flyMusca were studied to determine the types, extent and location of membrane specializations between neurons. Five junctional types are found, exclusive of chemical synapses. These are gap, tight and septate junctions, close appositions between retinular (R) axons and capitate projections (in which an epithelial glial cell invaginates into an R axon). Junctional types and their cellular associations follow: gap junctions, between lamina (L) interneurons, L1–L2; tight junctions, between L1–L2; L3–L4; L4-epithelial glial cell; and R7–R8. Septate junctions, between L1–L2, L3–L4, L3-β, L4-β, α-β, and an unidentified fibre making septate junctions with L1 and L2. Close appositions are found between R axons in the distal portion of the optic cartridges of this neuropil prior to extensive R chemical synapses with L1, L2. These loci (seen in freeze-fracture replicas) have rhomboidal patches of hexagonally arrayed P face particles.Intermembranous clefts between R axons are about 50 Å and are invariably electron lucent. These points of near contact between R terminals are probably the sites of low electrical resistance measured by Shaw (1979). Capitate projections are for the first time revealed in freeze fracture surfaces. Here epithelial glia send many, short, mushroom-shaped processes invaginating into R axons forming a tenacious structural bond. All four membrane leaflets (P and E faces of R axon and glial membrane) in the capitate projection possess particles in higher densities than in the surrounding nonspecialized regions. The known, general functions of each membrane specialization were correlated with the functional capacities of those lamina neurons possessing them in an effort to interpret better the integrative capacity of this neuropil. These data provide some fine structural bases for a putative ‘blood-brain’ barrier between lamina and haemolymph, between lamina and peripheral retina, and possibly between lamina and second optic neuropil.

Close apposition of photoreceptor cell axons in the house fly

Abstract Transmission electron microscopy of the first optic ganglion of the house fly Musca domestica reveals that retinular (R) axons branch and interdigitate throughout the external plexiform layer. Axonal membranes of neighboring cells are in close apposition to each other, usually without junctional modifications. In some cases R axonal membranes exhibit greater electron density in the areas of contiguity. Adjacent axons may show a near confluency of axoplasm at points where membrane boundaries are interrupted or obscured. Physiological implications of these ultrastructural findings are discussed.

Analog of vertebrate anionic sites in blood-brain interface of larval Drosophila

The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a “charge-selective barrier” for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.