Slowly contracting muscles power the rapid jumping of planthopper insects (Hemiptera, Issidae).

The planthopper insect Issus produces one of the fastest and most powerful jumps of any insect. The jump is powered by large muscles that are found in its thorax and that, in other insects, contribute to both flying and walking movements. These muscles were therefore analysed by transmission electron microscopy to determine whether they have the properties of fast-acting muscle used in flying or those of more slowly acting muscle used in walking. The muscle fibres are arranged in a parallel bundle that inserts onto an umbrella-shaped tendon. The individual fibres have a diameter of about 70 µm and are subdivided into myofibrils a few micrometres in diameter. No variation in ultrastructure was observed in various fibres taken from different parts of the muscle. The sarcomeres are about 15 µm long and the A bands about 10 µm long. The Z lines are poorly aligned within a myofibril. Mitochondrial profiles are sparse and are close to the Z lines. Each thick filament is surrounded by 10-12 thin filaments and the registration of these arrays of filaments is irregular. Synaptic boutons from the two excitatory motor neurons to the muscle fibres are characterised by accumulations of ~60 translucent 40-nm-diameter vesicle profiles per section, corresponding to an estimated 220 vesicles, within a 0.5-µm hemisphere at a presynaptic density. All ultrastructural features conform to those of slow muscle and thus suggest that the muscle is capable of slow sustained contractions in keeping with its known actions during jumping. A fast and powerful movement is thus generated by a slow muscle.

Drosophila ABC transporter mutants white, brown and scarlet have altered contents and distribution of biogenic amines in the brain.

Monoamines such as dopamine, histamine and serotonin (5-HT) are widely distributed throughout the brain of the fruit fly Drosophila melanogaster, where many of their actions have been investigated. For example, histamine is released from photoreceptor synapses in the lamina neuropile of the visual system. Mutations of the genes white, an important eye pigmentation marker in fly genetics that encodes an ABC transporter, and its binding partner brown, cause neural phenotypes not readily reconciled solely with actions in eye pigmentation. We find that flies mutant for these genes, and another binding partner, scarlet, have about half the wild-type amount of histamine in the head, as well as reduced 5-HT and dopamine. These differences parallel reductions in immunoreactivity to the corresponding biogenic amines. They also correlate with the amine content of fractions after differential centrifugation of head homogenates. Thus, most of the amine is found in the vesicle-rich fraction of wild-type head homogenates, whereas it is found in the supernatant fractions from white, brown and scarlet flies. White co-expresses in lamina epithelial glia with Ebony, which conjugates histamine to beta-alanine. Histamine is then released when the conjugate is hydrolyzed in photoreceptors, by Tan. Mutant white ameliorates the effects of tan on head histamine whereas it exacerbates the effects of ebony. Our results are consistent with the proposal that histamine uptake by the epithelial glia may be white dependent. Behavioral abnormalities in white, brown and scarlet mutants could arise because aminergic neurons in the Drosophila brain have reduced amine for release.

GABAergic synaptic transmission modulates swimming in the ascidian larva.

To examine the role of the amino acid GABA in the locomotion of basal chordates, we investigated the pharmacology of swimming and the morphology of GABA-immunopositive neurones in tadpole larvae of the ascidians Ciona intestinalis and Ciona savignyi. We verified that electrical recording from the tail reflects alternating muscle activity during swimming by correlating electrical signals with tail beats using high-speed video recording. GABA reversibly reduced swimming periods to single tail twitches, while picrotoxin increased the frequency and duration of electrical activity associated with spontaneous swimming periods. Immunocytochemistry for GABA revealed extensive labelling throughout the larval central nervous system. Two strongly labelled regions on either side of the sensory vesicle were connected by an arc of labelled fibres, from which fibre tracts extended caudally into the visceral ganglion. Fibre tracts extended ventrally from a third, more medial region in the posterior sensory vesicle. Two rows of immunoreactive cell bodies in the visceral ganglion extended neurites into the nerve cord, where varicosities were seen. Thus, presumed GABAergic neurones form a network that could release GABA during swimming that is involved in modulating the time course and frequency of periods of spontaneous swimming. GABAergic and motor neurones in the visceral ganglion could interact at the level of their cell bodies and/or through the presumed GABAergic fibres that enter the nerve cord. The larval swimming network appears to possess some of the properties of spinal networks in vertebrates, while at the same time possibly showing a type of peripheral innervation resembling that in some protostomes.

Histamine compartments of the Drosophila brain with an estimate of the quantum content at the photoreceptor synapse.

Reliable estimates of the quantum size in histaminergic neurons are not available. We have exploited two unusual opportunities in the fly's (Drosophila melanogaster) visual system to make such determinations for histaminergic photoreceptor synapses: 1) the possibility to microdissect successively from whole fly heads freeze-dried in acetone: the compound eyes; the first optic neuropils, or lamina; and the rest of the brain; and 2) the uniform sheaves of lamina synaptic terminals of photoreceptors R1-R6. We used this organization to count scrupulously the numbers of 30-nm synaptic vesicles from electron micrographs of R1-R6 profiles, and from microdissections we determined the regional contents of histamine in the compound eye, lamina, and central brain. Total head histamine averages 1.98 ng of which 9% was lost after freeze-drying in acetone and a further 28% after the brain was microdissected. Of the remainder, 71% was in the eye and lamina. Assuming that histamine loss from the tissue occurred mostly by diffusion evenly distributed among all regions, the overall lamina content of the head would be 0.1935 ng before dissection. From published values for the volumes of the brain's compartments, the computed regional concentrations of histamine are highest in the lamina (4.35 mM) because of the terminals of R1-R6. The concentration in the retina is approximately 13% that in the lamina, suggesting that most histamine is vesicular. There are approximately 43,500 +/- 7,400 (SD) synaptic vesicles per terminal and, if all histamine is allocated equally and exclusively among these, the vesicle contents would be 858 +/- 304 x 10(-21) moles or approximately 5,000 +/- 1,800 (SD) molecules at an approximate concentration of 670 mM. These values are compared with the vesicle contents at synapses using acetylcholine and catecholamines.

Involvement of V-ATPase in the regulation of cell size in the fly's visual system.

In the fly's visual system, two classes of lamina interneuron, L1 and L2, cyclically change both their size and shape in a rhythm that is circadian. Several neurotransmitters and the lamina's glial cells are known to be involved in regulating these rhythms. Moreover, vacuolar-type H+-ATPase (V-ATPase) in the optic lobe is thought also to participate in such regulation. We have detected V-ATPase-like immunoreactivity in the heads of both Drosophilla melanogaster and Musca domestica using antibodies raised against either the B- or H-subunits of V-ATPase from D. melanogaster or against the B-subunit from two other insect species Culex quinquefasciatus and Manduca sexta. In the visual systems of both fly species V-ATPase was localized immunocytochemically to the compound eye photoreceptors. In D. melanogaster immunoreactivity oscillated during the day and night and under constant darkness the signal was stronger during the subjective night than the subjective day. In turn, blocking V-ATPase by injecting a V-ATPase blocker, bafilomycin, in M. domestica increased the axon sizes of L1 and L2, but only when bafilomycin was applied during the night. As a result bafilomycin abolished the day/night difference in axon size in L1 and L2, their sizes being similar during the day and night.

The regulation of circadian rhythms in the fly's visual system: involvement of FMRFamide-like neuropeptides and their relationship to pigment dispersing factor in Musca domestica and Drosophila melanogaster.

The cross-sectional area of axon profiles in two classes of interneuron, L1 and L2, in the fly's lamina, exhibits a circadian rhythm of swelling and shrinking; axon caliber also changes after microinjecting putative lamina neurotransmitters. Among these, the neuropeptide pigment-dispersing factor, PDF, is proposed to transmit circadian information from the housefly's (Musca domestica) clock to L1 and L2, increasing axon caliber during the day. Testing whether other neurotransmitters may modulate this effect we have: (1) examined optic lobe cell immunoreactivity to FMRFamide peptides and its co-immunolocalization to PDF in M. domestica and Drosophila melanogaster, and to the product of the circadian clock gene PER in D. melanogaster; and (2) made microinjections of FMRFamide and related neuropeptides into the second neuropil, or medulla. In M. domestica, nine groups of optic lobe cells, several cells in the lateral and dorsal protocerebrum, and in the subesophageal ganglion, together contribute dense FMRFamide immunoreactive arborizations in almost all central brain and optic lobe neuropils. In D. melanogaster a similar pattern of labeling arises from fewer cells. Daytime microinjections show that another neuropeptide, similar to molluscan FMRFamide, shrinks M. domestica's L1 and L2 axons, thus opposing the action of PDF. We discuss evidence for a medulla site of action for a released FMRFamide-like peptide, either from: MeRF2 cells, acting directly on L1 and L2's medulla terminals; or MeRF1 cells, acting indirectly via medulla centrifugal cells C2 and C3.

Mitochondria are redistributed in Drosophila photoreceptors lacking milton, a kinesin-associated protein.

Photoreceptors are richly supplied with mitochondria, where they are required to meet the energetic demands, in the soma, of phototransduction and, in the terminal, of neurotransmitter release. Compromising the latter, we have made photoreceptors R1-R6 in Drosophila ommatidia homozygous for either of two alleles, milt(186) and milt(92), of milton in whole-eye mosaics. Such mutant photoreceptors fail to target mitochondria to their terminals. We show from quantitative electron microscopy (EM) that mitochondria are totally lacking at the terminal but nevertheless abundant and present throughout the soma, where their distribution differs from that of control ommatidia, however, being more heavily concentrated in the nuclear region. Mitochondria are sparse at the basalmost level of mutant ommatidia, and are lacking beneath the basement membrane, in the axons and terminals of these cells. The absence of mitochondria from R1-R6 terminals and concommitant reductions in synaptic vesicle packing density, previously reported, we show here are accompanied by reduced immunoreactivity to the photoreceptor transmitter histamine but not by any change in total head histamine content, as determined by high-performance liquid chromatography. Mutant terminals also contain vesicle profiles with a wider range of sizes. These two phenotypes suggest that the reduced availability of ATP when mutant terminals lack a mitochondrial supply compromises their ability to pump histamine into synaptic vesicles and perturbs membrane distribution within the terminal. In addition, a band of somata in the lamina cortex, at least some of which are postsynaptic neurons not homozygous for milton, also shows altered mitochondrial targeting, with abnormal clusters of mitochondria, as visualized by immunolabeling with anti-hsp and by serial EM. Within the lamina, terminals of mutant photoreceptors are penetrated by neighboring cells with invaginations that frequently contain mitochondria, suggesting that a mechanism exists for intercellular metabolic support. Our findings indicate the direct and compensatory responses in a population of neurons when mitochondria are not correctly targeted to their synaptic terminals.

Tracking particles in four dimensions with in-line holographic microscopy.

We describe a simple holographic method that has enabled us to capture as a single data set the trajectories of micrometer-sized objects suspended in water. By subtracting consecutive holograms of a particle suspension and then adding these difference holograms, we constructed a final data set that contains the time evolution of the particle trajectories free from spurious background interference effects. The method is illustrated by a recording of the motion of 5-10-microm diameter algae in water.

Digital in-line holography of microspheres.

We have used digital in-line holography (DIH) with numerical reconstruction to image micrometer-sized latex spheres as well as ferrimagnetic beads suspended in gelatin. We have examined in detail theoretically and experimentally the conditions necessary to achieve submicrometer resolution of holographic reconstructions. We found that both transparent and opaque particles could be imaged with a resolution that was limited only by the wavelength of the light used. Simple inspection of intensity profiles through a particle allowed an estimate to be made of the particle's three position coordinates within an accuracy of a few hundred nanometers. When the derivative of a second-order polynomial fitted to the intensity profiles was taken, the X, Y, Z position coordinates of particles could be determined within +/-50 nm. More-accurate positional resolution should be possible with the help of more-advanced computer averaging techniques. Because a single hologram can give information about a large collection of distributed particles, DIH offers the prospect of a powerful new tool for three-dimensional tracking of particles.

Digital in-line holography for biological applications.

Digital in-line holography with numerical reconstruction has been developed into a new tool, specifically for biological applications, that routinely achieves both lateral and depth resolution, at least at the micron level, in three-dimensional imaging. The experimental and numerical procedures have been incorporated into a program package with a very fast reconstruction algorithm that is now capable of real-time reconstruction. This capability is demonstrated for diverse objects, such as suspension of microspheres and biological samples (diatom, the head of Drosophila melanogaster), and the advantages are discussed by comparing holographic reconstructions with images taken by using conventional compound light microscopy.

Neuronal form in the central nervous system of the tadpole larva of the ascidian Ciona intestinalis.

The dorsal tubular central nervous system (CNS) of the ascidian tadpole larva is a diagnostic feature by which the chordate affinities of this group, as a whole, are recognized. We have used two methods to identify larval neurons of Ciona intestinalis. The first is serial electron microscopy (EM), as part of a dedicated study of the visceral ganglion (1), and the second is the transient transfection of neural plate progeny with green fluorescent protein (GFP) (2), to visualize the soma and its neurites of individual neurons in whole-mounted larvae of C. intestinalis. Our observations reveal that ascidian larval neurons are simple inform, with a single axonal neurite arising from a soma that is either monopolar or has only very few, relatively simple neurites arising from it, as part of a presumed dendritic arbor. Somata in the visceral ganglion giving rise to axons descending in the caudal nerve cord are presumed to be those of motor neurons.

The larval ascidian nervous system: the chordate brain from its small beginnings.

The body plan of the tadpole larva of ascidians, or sea-squirts, is widely presumed to be close to that of the hypothetical ancestor of all chordate animal groups, including vertebrates. This is nowhere more obvious than in the organization and development of the dorsal tubular nervous system. Several recent developments advocate this model neural system for studies on neurobiology and neurogenesis. These include advances in our understanding of development in ascidian embryos and of differentiation among the cellular progeny of its neural plate; the application of transgenic and mutant approaches to studies on ascidian larval neurones; and the prospect of advances in genomic analyses. In addition to providing ways to study a working chordate brain in miniature, all these offer insights into the ancestral condition of the developing vertebrate brain.

Visualization of synaptic markers in the optic neuropils of Drosophila using a new constrained deconvolution method.

The fruitfly Drosophila melanogaster offers compelling genetic advantages for the analysis of its nervous system, but cell size precludes immunocytochemical analysis of wild-type structure and mutant phenotypes beyond the level of neuronal arborizations. For many antibodies, especially when immunoelectron microscopy is not feasible, it would therefore be desirable to extend the resolution limit of confocal microscopy as far as possible. Because high-resolution confocal microscopy suffers from considerable blurring, so-called deconvolution algorithms are needed to remove, at least partially, the blur introduced by the microscope and by the specimen itself. Here, we present the establishment and application of a new deconvolution method to visualize synaptic markers in Drosophila optic neuropils at the resolution limit of light. We ascertained all necessary parameters experimentally and verified them by deconvolving injected fluorescent microspheres in immunostained optic lobe tissue. The resulting deconvolution method was used to analyze colocalization between the synaptic vesicle marker neuronal synaptobrevin and synaptic and putative synaptic markers in photoreceptor terminals. We report differential localization of these near the resolution limit of light, which could not be distinguished without deconvolution.

The central nervous system, its cellular organisation and development, in the tadpole larva of the ascidian Ciona intestinalis.

From its numerical composition, the central nervous system (CNS) of the ascidian larva is one of the simplest known nervous systems having a chordate plan. Fewer than 350 cells together constitute a caudal nerve cord, an interposed visceral ganglion containing motor circuits for swimming and, rostrally, an expanded sensory vesicle containing major sensory and interneuron regions of the CNS. Some cells are ependymal, with ciliated surfaces lining the neural canal, while others are clearly either sensory receptors or motoneurons, but most are distinguishable only on cytological grounds. Although reassignments between categories are still being made, there is evidence for determinancy of total cell number. We have made three-dimensional cell maps either from serial semithin sections, or from confocal image stacks of whole-mounted embryos and larvae stained with nuclear markers. Comparisons between the maps of neural tubes in embryos of successive ages, that is, between cells in one map and their progeny in older maps, enable us to follow the line of mitotic descent through successive maps, at least for the caudal neural tube. Details are clear for the lateral cell rows in the neural tube, at least until the latter contains approximately 320 cells, and somewhat for the dorsal cell row, but the ventral row is more complex. In the hatched larva, serial-EM reconstructions of the visceral ganglion reveal two ventrolateral fibre bundles at the caudalmost end, each of 10-12 axons. These tracts include at least five pairs of presumed motor axons running into the caudal nerve cord. Two pairs of axons decussate. Complementing this vertebrate feature in the CNS of the larval form of Ciona, we confirm that synapses form upon the somata and dendrites of its neurons, and that its motor tracts are ventral.

The determination of histamine in the Drosophila head.

Histamine is a neurotransmitter at arthropod photoreceptors. Even though the fruit fly, Drosophila melanogaster, is a widely used model in neuroscience research, the histamine content of its nervous system has not so far been reported. We have developed a high performance liquid chromatography (HPLC) method with pre-column o-phtaldialdehyde-mercaptoethanol (OPA-ME) derivatization and electrochemical detection, to determine this amine in Drosophila. The histamine content of the fly's head averages about 2.0 ng per head. In heads of the mutant hdc(JK910), a presumed null for the gene encoding the enzyme that synthesizes histamine, histamine was not detected in measurable amounts. In heads of the mutant sine oculis, which lacks compound eyes, only 28% of this amine was found compared with wild type flies, so histamine is mainly present in the compound eye photoreceptors. Also observed in histamine-deficient mutants was a decrease in the peak which contains a substance having the same retention time as carcinine (beta-alanyl-histamine). Our method was not able to detect compounds previously reported as histamine metabolites in insects. In spite of this, the method we have developed enables the fast and accurate measurement of histamine in the heads of Drosophila, suitable for screening mutants.

Neurite morphogenesis of identified visual interneurons and its relationship to photoreceptor synaptogenesis in the flies, Musca domestica and Drosophila melanogaster.

The first neuropile, or lamina, of the fly's optic lobe comprises a model set of identified neurons that are arrayed in cylindrical modules, called cartridges. The cartridge acquires adult form only in the second half of the fly's pupal life. All cells are by then correctly located within each of the lamina's cartridges (Drosophila, Musca), becoming invested by glial cells after 75% of pupal development (P + 75%). In adult cartridges, two lamina cells, L1 and L2, receive input from photoreceptor terminals R1-R6, at so-called tetrad synapses that form in the pupa when these cells' dendrites contact R1-R6. Single-section electron microscopy (EM, Drosophila) and serial-EM reconstructions of L1 and L2 (Musca) reveal relationships between the morphogenesis of L1/L2 dendrites and the formation of tetrads. Neurite outgrowth is initially (P + 55%) random and neurites are unbranched; many neurites invaginate surrounding terminals of R1-R6 but, later, embrace the outer surfaces of these. The maximum profusion of neurites at P + 74% coincides with peak numbers of nascent tetrads; neurites then branch vertically, in the lamina's depth. Later, neurites failing to reach R1-R6's outer surfaces regress. Down the length of their axons, L1 and L2's neurites initially form a random sequence, L1 partnering L1 as often as L2, etc., but beginning at P + 74%, L1 partners L2, and L2 partners L1, with progressive strictness. L1 has more neurites overall than L2. These observations are consistent with the following hypotheses: a neurite only survives if it contacts a presynaptic site; a synapse only survives if it progressively acquires the appropriate number and combination of postsynaptic neurites, culminating in a tetrad; an interaction exists between the neurites of L1 and L2, so that the growth of one respects the pattern of growth of the other.

Organization of efferent peripheral synapses at mechanosensory neurons in spiders.

The mechanosensory neurons of arachnids receive diverse synaptic inputs in the periphery. The function of most of these synapses, however, is unknown. We have carried out detailed electron microscopic investigations of the peripheral synapses at sensory neurons in the compound slit sense organ VS-3 of the spider Cupiennius salei. Based on the localization of discrete presynaptic vesicle populations, it is possible to discriminate at least four different synapse types, containing either: (1) small round, electron-lucent vesicles 32 nm in diameter; (2) large round, clear 42-nm vesicles; (3) a mixture of small and large clear, round vesicles, similar in size to those in Type 1 and Type 2 synapses, respectively, and granular and dense-core vesicles; or (4) clear, round 37- to 65-nm vesicles. Combined immunocytochemical labeling at the light and the electron microscopic level suggests that gamma-aminobutyric acid (GABA) is the transmitter in many of the 32-nm vesicle synapses, and glutamate in many of the 42-nm ones. Based on vesicle type and particular synaptic configuration, various forms of presumed efferent synaptic contacts are distinguishable with the sensory neurons, the surrounding glia, and between the putative efferent fibers themselves. These include simple unidirectional synapses, reciprocal synapses, serial synapses, and convergent as well as divergent dyads. These various synaptic microcircuits are suited to serve a variety of functions. Among these are direct postsynaptic inhibition or excitation of the mechanosensory neurons, and disinhibition or sensitization via presynaptic inhibition or excitation. The observed synaptic configurations are compared with those at the crustacean muscle receptor organ. They reveal a remarkable complexity of synaptic microcircuits at spider sensilla and suggest manifold possibilities for subtle, efferent control of sensory activity.

Circadian rhythms in light-evoked responses of the fly's compound eye, and the effects of neuromodulators 5-HT and the peptide PDF.

Two sets of wide-field neurons extend neurites into the fly's optic lamina, where monopolar cells receive photoreceptor input. They exhibit immunoreactivity to antibodies raised against either 5-hydroxytryptamine or the crustacean peptide PDH, respectively. Both are proposed whole-field neuromodulators of vision, apparently regulating a circadian rhythm of monopolar cell size. Seeking functional correlates, we have re-examined the electroretinogram for circadian rhythmicity, and for responses to locally injected 5-hydroxytryptamine and peptide. Long-term electroretinogram recordings from Calliphora entrained to a light/dark cycle and then transferred to constant darkness, uncovered a gradual, modest increase during the subjective night in the electroretinogram's ON- and OFF-transients, from the lamina's monopolar cells. Five to twenty nl of 5-hydroxytryptamine (10(-3) mol.1(-1)) injected into the head haemolymph strongly enhanced the electroretinogram transients, an action reversed by 5-hydroxytryptamine antagonists. Injected into the eye, 5-hydroxytryptamine (10(-4) mol.1(-1)) had the opposite effect; the rapid onset there suggests direct action, whilst the opposing effect from haemolymph injection suggests a different receptor site. Pigment-dispersing hormone (2.2 x 10(-5) mol.1(-1)) injected into the haemolymph increased the electroretinogram transients along a biphasic course, with a slow partial recovery; injected into the eye, it lacked effect.