Calliphora Erythrocephala Research Articles

Cyclic 3',5'-nucleotide phosphodiesterase: cytochemical localization in photoreceptor cells of the fly Calliphora erythrocephala.

The distribution of cyclic 3',5'-nucleotide phosphodiesterase activity was determined in photoreceptor cells of the fly Calliphora erythrocephala. With cAMP as substrate, staining was most intense within the phototransducing region of these cells, the rhabdomeral microvilli and also in the extracellular space surrounding the microvilli and in the mitochondria. With cGMP as substrate, the intensity within the rhabdomeres was less marked, while their extracellular surroundings were stained heavily. Thus, compared to cGMP, cAMP is the better substrate for the phosphodiesterase in the rhabdomeres of the fly. For comparison, the same cytochemical method was used to localize the well-known phosphodiesterase activity in retinal tissue of the mouse. Under the same conditions as used for fly photoreceptors, a very intense reaction product was obtained in rod outer segments. With regard to the conflicting reports concerning the light-stimulated changes of cyclic nucleotides in invertebrate photoreceptor cells, the results presented here further argue for an important role of a cyclic nucleotide in the process of phototransduction of invertebrates.

An arrestin homolog of blowfly photoreceptors stimulates visual-pigment phosphorylation by activating a membrane-associated protein kinase.

An arrestin homolog (Arr2, 49-kDa protein) of blowfly (Calliphora erythrocephala) retinae undergoes light-dependent reversible binding to the photoreceptor membrane. In order to characterize this arrestin homolog and to study its function in a well-defined experimental system, we developed a purification scheme which used microvillar photoreceptor membranes as an affinity binding matrix. Additional purification steps included ammonium sulfate precipitation, gel filtration and binding to heparin-agarose. The molecular mass of purified Arr2, as judged by SDS/PAGE, is in the range 45-49 kDa. The isoelectric point, as judged by gel isoelectric focussing, is 8.7. Arr2 is specific to the retina, where it is subject to phosphorylation at multiple sites. Binding of purified Arr2 to isolated photoreceptor membranes efficiently activates the light-induced phosphorylation of visual pigment. Since the assay system used is deficient in rhodopsin phosphatase activity, the arrestin-stimulated phosphate incorporation into rhodopsin results solely from the activation of a protein kinase. Phosphorylation experiments with highly purified membrane preparations indicate that rhodopsin kinase is tightly associated with the rhabdomeric membrane or the microvillar cytoskeleton. Rhodopsin kinase is released from the membrane or inactivated upon treatment with urea. It is concluded that this arrestin is a regulator protein that controls visual-pigment phosphorylation by affecting the interaction of metarhodopsin and rhodopsin (metarhodopsin) kinase.

Serotonin-immunoreactivity and serotonin binding sites in the brain of the blowfly calliphora erythrocephala: A combined immunohistochemical and autoradiographic study

1. The serotonergic system in the brain of the blowfly, Calliphora erythrocephala, was analysed by two different methods. 2. Serotonergic neurones were visualized by immunocytochemistry with an antibody against serotonin. 3. Putative serotonin receptors were localized by quantitative autoradiography of [ 3H]serotonin binding. 4. We found regions with a colocalization of immunoreactive neurones and binding sites, and regions with a mismatch as well. 5. In regions of overlap, which include the optic lobes and the central body complex, serotonin might act as a neurotransmitter. 6. In areas of mismatch, as found e.g. in the lateral parts of the protocerebrum, serotonin might have a modulatory action.

A soluble calcium-binding protein (scbp) present in drosophila melanogaster and calliphora erythrocephala muscle cells

1. 1. Soluble calcium binding proteins (SCBP) were isolated from homogenates of whole flies, from the thorax and from muscles of Drosophila melanogaster and Calliphora erythrocephala. 2. 2. Crude preparations were obtained by extraction at low ionic strength, acid and heat treatment. The Drosophila protein was purified by gel filtration, hydrophobic interaction and ion exchange chromatography. In contrast to calmodulin the Drosophila SCBP did not bind to phenyl-Sepharose in a Ca 2+-dependent way. 3. 3. Both the Drosophila and Calliphora protein revealed identical properties. 4. 4. The apparent molecular mass of the SCBP is 24 kDa. Separation in urea-PAGE demonstrated the existence of two isoforms. 5. 5. The calcium-binding property was assured by a calcium dependent electrophoretic mobility shift and autoradiography of 45Ca 2+-incubated Western blots. 6. 6. The proteins are abundant in the thorax and were even detectable in crude extracts of various mascles (leg muscles and the extracoxal depressor). In contrast, in power muscles and in the thoracic ganglion the proteins could not be observed.

The localization of acid phosphatase as a prelude to cell death in the blow-fly calliphora erythrocephala

The localization of acid phosphatase as a prelude to cell death in the blow-fly calliphora erythrocephala

Dual peptidergic innervation of the bloffly hindgut: A light- and electron microscopic study of FMRFamide and proctolin immunoreactive fibers

The innervation of the hindgut, rectal valve, rectum and rectal papillae of the adult blowfly, Calliphora erythrocephala, was studied by means of light and electron microscopic immunocytochemistry using antibodies against the neuropeptides proctolin and FMRFamide. 2. Branches from the abdominal nerves reaching the posterior portion of the gut were found to contain mostly neurosecretory type axons and to innervate the muscle coat of all hindgut structures studied. 3. Some of the axons found in these nerve branches innervating the gut display proctolin- others FMRFamide-like immunoreactivity. Both types of peptidergic axons were found to have abundant terminals in the muscle coat of the hindgut, rectum and rectal valve and in the medulla of the rectal papillae. 4. It is clear that two separate peptidergic systems derived from the abdominal ganglion are supplying the hindgut structures, and, possibly, they use proctolin- and FMRFamide-like peptides respectively as their trassmitters or modulators.

Ommochrome pigment granules: A calcium reservoir in the dipteran eyes

1. 1. Ultrastructure and Ca-sequestering properties of eye-color pigment granules (PGs) were studied in the eyes of wild type and mutant flies Drosophila melanogaster, Calliphora erythrocephala and Musca domestica. 2. 2. New type of ommochrome PGs localized in primary pigment cells and basal terminals of the processes of Semper cells was found in Drosophila eye. 3. 3. At room temperature these PGs exhibit OsO 4-dependent structural kbility. X-ray microanalysis revealed a less Ca-binding ability of the PGs as compared to those in secondary pigment cells. 4. 4. In the eyes of Musca and Calliphora ommochrome PGs were found to be more stable in respect to Ca removal as compared to Drosophila. 5. 5. Ommochromes provide much more Ca-sequestering ability to PGs than pterins do.

Temporal resolving power of blowfly visual system: effects of decamethonium and hyperpolarization on responses of laminar monopolar neurons

1. The temporal resolving power of blowfly (Calliphora erythrocephala) photoreceptors and laminar monopolar neurons was investigated by stimulating the eye with sinusoidally modulated light of different intensities. Temporal resolving power was measured as -3 dB values of the maximal amplitude. 2. -3 dB values of photoreceptors increased only slightly with intensity from 25 to 35 Hz within the intensity range studied. -3 dB values of monopolar neurons increased sigmoidally from 30 Hz at low light intensities to 100 Hz at high intensities. 3. With regard to dark membrane potential, responses of monopolar neurons to stimulation with sinusoidally modulated light were formed by depolarizing and hyperpolarizing components. With iontophoretic application of decamethonium and hyperpolarizing current it was possible to reduce the depolarizing and hyperpolarizing components, respectively. 4. Monopolar neurons, whose depolarizing components were reduced by decamethonium, showed a constantly low temporal resolving power (30-40 Hz), similar to that of photoreceptors. The intensity dependence of cells with reduced hyperpolarizing components had nearly the same sigmoidal shape as that of untreated cells. However, the s-3 dB values were shifted 30–40 Hz to lower values. 5. These results suggest that the hyperpolarizing components of monopolar neurons' responses to sinusoidally modulated light are mainly responsible for a basic level of the temporal resolving power, whereas the depolarizing components produce the increase of temporal resolving power with increasing light intensity.

Actin and α-tubulin immuno-EM labelings reveal differences in intra versus interphotoreceptor matrix

The photoreceptor cells in the compound eyes of higher diptera are clustered in groups (ommatidia) of eight receptor cells. The cells from six adjacent ommatidia are organized into optical units, neuro-ommatia sharing the same visual field. In those ommatidia the optical axes of the photopigment containing structures (rhabdomeres) are parallel. The rhabdomeres of the photoreceptor cells are separated from each other by an interstitial i.e innerommatidial space (IOS). In the photoreceptor cell body, besides of the normal cell organelles, a cellular matrix is a structurally apparent component. Similar kind of reticular formation is also found in the IOS containing some unidentified filamentary substance, of which composition and functional significance for optical properties of vision is the aim of this report.The prefixed (2% PA + 0.2% GA in 0.1-n phosphate buffer, pH 7.4, for 1h), frozen section blocks of the compound eye of the blowfly (Calliphora erythrocephala) were prepared by immuno-cryo-techniques. The ultrathin cryosections were incubated with antibodies of monoclonal α-tubulin and polyclonal smooth muscle actin. Control labelings of excess of antigen, non-immune serum and non-present antibody were perforated.

Lanthanum mimicks the trp photoreceptor mutant of Drosophila in the blowfly Calliphora

The effect of lanthanum on the light response of blowfly (Calliphora erythrocephala) photoreceptors was studied. The electrophysiological behaviour of the photoreceptors in the presence of La can be summarized as follows: 1. Upon long stimulation the photoreceptors responded with a 'transient receptor potential', i.e. the cells depolarized at the onset of the stimulus and then repolarized to (or below) the resting potential. This effect was dependent on stimulus intensity and occurred only at high intensities. During illumination membrane noise was reduced. 2. The light-induced changes in membrane potential were paralleled by changes in membrane resistance. 3. The time course of the receptor response was slowed down. 4. Light adaptation led to an increase in response latency. 5. The recovery of the receptor response after light adaptation was slowed down. 6. The sensitivity of the receptor cells measured by the response to short light stimuli was reduced. In summary, the electrophysiological behaviour of Calliphora photoreceptors in the presence of La was very similar to that of the photoreceptors of the trp (transient receptor potential) mutant of Drosophila melanogaster. This result suggests that La and trp mutation affect the same cellular processes in the photoreceptors.

Neural circuits mediating visual flight control in flies. I. Quantitative comparison of neural and behavioral response characteristics

The motion-sensitive horizontal cells in the lobula plate of the fly are assumed to play a key role in the sensory control of yaw torque generated by the flying animal during course-stabilization maneuvers and the fixation of objects. This inference results from comparisons of electrophysiological data obtained from blowflies (Calliphora erythrocephala) and behavioral data obtained mainly from houseflies (Musca domestica) and fruitflies (Drosophila melanogaster). Apart from few exceptions, the compatibility of these physiological and behavioral data has not been critically tested. In the present study, the responses of the equatorial horizontal cell HSE of Calliphora and the yaw torque responses of Calliphora and Musca were recorded under identical visual stimulation with moving periodic gratings. The goal of the experiments was to obtain electrophysiological and behavioral data on Calliphora, on the one hand, and behavioral data on Calliphora and Musca, on the other hand, that allow direct comparisons between the physiological properties of the HSE and the visually induced torque responses in both species. The dependence of the HSE responses and the yaw torque responses on the direction, contrast frequency, and brightness of a moving periodic grating were evaluated quantitatively. The results of the electrophysiological recordings and torque measurements are in close agreement and thus represent strong evidence that the horizontal cells are, in fact, involved in yaw torque control in both species. Measurements of the cellular and behavioral responses as function of the stimulus position in the visual field, however, reveal differences between the spatial sensitivity of the horizontal cells and the sensory input to the motor system.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of elementary movement detectors in the flyCalliphora erythrocephala

Experiments, involving sequential micro-stimulation of two (or more) adjacent neuroommatidia in the compound eye of the blowfly,Calliphora erythrocephala (Mg.), are presented. These experiments, using brief flashes of 3 ms duration with low intensities in the order of 8·10−3 Cd/m2, are performed to isolate individual response contributions of single Elementary Movement Detectors in the input micro-circuitry of the motion-sensitive directionally-selective H1-neuron (Figs. 4 through 6). A two-dimensional mapping of single EMD contributions to the overall response will be presented for the dark adapted eye (Fig. 7). It is concluded that under such low illumination levels (when compared to normal daylight situations, where illumination typically varies between 1 and 200 Cd/m2), contributions from EMD's with sampling bases up to 8Δ φ h, oriented along the horizontal sensitivity axis of the neuron, contribute to the total response of the neuron. In addition, results will be presented for similar experiments, in which various backgrounds with different sizes and intensities are superimposed on the sequence of stimulus flashes (Fig. 8). The dependence of the relative contributions for the smallest four sampling bases (i.e. 2, ..., 8 timesΔ φ h) on background size, background intensity and distance of the background field to the projections of the flashes, will be discussed. Supplementary experiments (Figs. 9 through 12) will be presented, which indicate that when in addition to an ongoing sequence in (for instance) the null direction, thus inhibiting the activity of the neuron, a second sequence is presented somewhere in the receptive field of the H1-neuron, the total response is a non-linear combination of both individual responses. Interpretation in terms of a pooling correlation scheme, which, over a limited target region, sums the activities of the EMD's in a highly non-linear fashion, provides a qualitative explanation.

Sequence of the Calliphora erythrocephala 3B55 satellite: a sequence which is proportionately replicated and transcribed in germ-line polyploid nuclei.

Sequence of the Calliphora erythrocephala 3B55 satellite: a sequence which is proportionately replicated and transcribed in germ-line polyploid nuclei.

Morphological and functional characteristics of arpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala)

1. We have discovered a previously unreported visual mutant of the blowfly,Calliphora erythrocephala. It shows a reduced or absent visual performance, e.g., in escape and optomotor behavior. The effects of this mutation on the ultrastructure were studied by electron microscopy (Figs. 3–8) and on electrophysiological function, by intracellular recordings (Figs. 1 and 2). 2. The genetic basis of this spontaneous mutation was studied by test crosses of mutant and wild-type flies. The defect appears to be in an autosomal recessive gene (Table 1). 3. Of the mutant stock studied soon after eclosion (n = 18) 35% shows optomotor reactions, whereas only 6% studied in later life (n = 240) shows any optomotor behavior. 4. The absence of the receptor potentials in photoreceptor cells is not directly associated with structural disorders in the early life of these mutant flies, but several types of degenerative changes are manifested in the retinular cells later on. The optomotorically blind specimens have normal (about −60 mV) resting membrane voltages but no detectable receptor cell voltage response to light, indicating a block in phototransduction. The spectral and polarization sensitivities of optomotor-positive flies are normal (Fig. 2). 5. At the beginning of degeneration the number of lysosomes in the receptor cells is increased compared with normal flies, but their number as well as that of other components of the cell interior decrease later on. During the progression of the degeneration, the rhabdomeres shrink while the mitochondria swell and disintegrate (Figs. 6–8). 6. The blocking of phototransduction is proposed to lead to disturbance of the turnover of the rhabdomeres and finally to degeneration of the receptor cells.

Dye Transport Across the Retinal Basement Membrane of the Blowfly Calliphora Erythrocephala

ABSTRACT In the blowfly, Calliphora erythrocephala, transport of dye into or out of the retina, following injection into the eye or thorax, was investigated, mainly by microspectrophotometry and fluorimetry. After injection into the eye, Phenol Red, Trypan Blue, Lucifer Yellow and 9-amino-acridine were transported out of the retina; Procion Yellow and Rhodamine-123 stayed in it. The time constants of this transport process were in the range 45–80 min at 23°C, depending on the dye. When Lucifer Yellow was injected into the thorax, it was transported into the retina. The transport of these dyes across the retina was inhibited by nitrogen, ouabain and low temperature, indicating that active processes are involved.

Light and electron microscopic immunocytochemistry of neurons in the blowfly optic lobe reacting with antisera to rfamide and fmrfamide

Different antisera to the molluscan cardioexcitatory peptide FMRFamide, and its fragment, RFamide (Arg-Phe-NH 2), label a distinct population of neurons in the opitic lobe of the blowfly, Calliphora erythrocephala. Seven morphological types of RFamide/FMRFamide-like immunoreactive neurons could be distinguished in the optic lobes based on the locations of their cell bodies, their axonal projections and the distribution of their processes. Of these, two types could be resolved in their entire extent, the others were labeled only in their cell bodies and terminal processes or were partly obscured by other immunoreactive processes. The RF-like immunoreactive neurons in the optic lobes are of two main classes: (1) two types of large field projection neurons and (2) five types of local neurons. One type of projection neurons (five in each lobe) connects the entire projected retinal mosaic of the medulla and lobula in the optic lobe with protocerebral centres associated with the mushroom body calyx. The other type (2–3 invading each lobe) has cell bodies in the protocerebrum and contralateral processes invading optic lobes. Of the class of local neurons there are two amacrine RF-like immunoreactive neurons in each medulla. Each of these amacrines supplies the entire mosaic with fine processes. The remaining local RF-like immunoreactive neurons are present in relatively large numbers (one type in more than 2000 copies in each medulla) and supply the medulla, lobula and lobula plate neuropils with fine varicose processes. In the medulla the RF-like immunoreactive processes are arranged in strict layers whereas in the lobula complex the distribution is diffuse. Electron microscopic immunocytochemistry, using both pre-embedding immuno peroxidase antiperoxidase and post-embedding protein A-gold labeling, was employed for analysis of cytology and synaptic connections of RF-like immunoreactive neurons in the medulla. The varicosities of the processes of the large field projection neurons were not found to make chemical synapses with other neurons in the medulla. The spines of the RF-like immunoreactive processes of the large medulla amacrines, however, make pre- and postsynaptic contacts with other neural elements. Our findings indicate that an RFamide/FMRFamide-like substance may be used as a neurotransmitter or neuromodulator by optic lobe neurons of different types. The local and projection RF-like immunoreactive pathways probably play different roles in visual processing.

Light- and electron-microscopic immunocytochemistry of peptidergic neurons innervating thoracico-abdominal neurohaemal areas in the blowfly.

Ventral thoracic neurosecretory cells (VTNCs) of the blowflies, Calliphora erythrocephala and C. vomitoria, innervating thoracic neuropil and the dorsal neural sheath of the thoracico-abdominal ganglion have been shown to be immunoreactive to a variety of mammalian peptide antisera. In the neural sheath the VTNC terminals form an extensive neurohaemal network that is especially dense over the abdominal ganglia. The same areas are invaded by separate, but overlapping serotonin-immunoreactive (5-HT-IR) projections derived from neuronal cell bodies in the suboesophageal ganglion. Immunocytochemical studies with different antisera, applied to adjacent sections at the light-microscopic level, combined with extensive cross-absorption tests, suggest that the perikarya of the VTNCs contain co-localized peptides related to gastrin/cholecystokinin (CCK), bovine pancreatic polypeptide (PP), Met- and Leu-enkephalin and Met-enk-Arg6-Phe7 (Met-enk-RF). Electron-microscopic immunogold-labeling shows that some of the terminals in the dorsal sheath react with several of the individual peptide antisera, whilst others with similar cytology are non-immunoreactive. In the same region, separate terminals with different cytological characteristics contain 5-HT-IR. Both 5-HT-IR and peptidergic terminals are localized outside the cellular perineurium beneath the acellular permeable sheath adjacent to the haemocoel. Hence, we propose that various bioactive substances may be released from thoracic neurosecretory neurons into the circulating haemolymph to act on peripheral targets. The same neurons may also interact by synaptic or modulatory action in the CNS in different neuropil regions of the thoracic ganglion.

Using neuropharmacology to distinguish between excitatory and inhibitory movement detection mechanisms in the fly Calliphora erythrocephala

By combining neuropharmacology and electrophysiology, we tried to determine whether the main neuronal mechanism responsible for direction-selective motion detection in the fly is based on an excitatory or an inhibitory synaptic interaction. By blocking inhibitory interactions with picrotoxinin, an antagonist of the inhibitory neurotransmitter GABA, we could abolish most of the directional selectivity of a large-field movement-sensitive neuron (H1-cell) in the lobula plate of the blowfly Calliphora erythrocephala. These modifications are similar to changes observed in the optomotor response of the fruitfly Drosophila melanogaster after application of picrotoxinin (Bulthoff and Bulthoff 1987a, b). Assuming a simplified logical model, these results are compatible with inhibitory synaptic interactions at the level of the elementary movement detectors. The picrotoxinin-induced changes in direction selectivity are not due to modifications of the peripheral visual processing in the retina and lamina. This was shown by simultaneous recordings of the electroretinogram and the H1-cell. The latencies between drug injections into various parts of the brain and their first effects on the H1-cell suggest that the inhibitory mechanism for motion detection is located in the medulla rather than in the lobula plate.

Quantitative analysis of resting membrane electrogenesis in insect (diptera) skeletal muscle. II. Testing of a model involving contributions from potassium and sodium ions, and the anomalous effect of reducing extracellular sodium.

A model of resting membrane electrogenesis in skeletal muscles of prepupal Calliphora erythrocephala was formulated. From experiments in which reversible effects of changing extracellular K+ and Na+ activities on the membrane potential (EM) were measured, three different values of alpha (the ratio of the partial permeabilities of the membrane to Na+ and K+) were derived, each from a different range of extracellular Na+ and K+ activities. Two independent tests were carried out to determine the most realistic value of alpha. Intracellular K+ and Na+ activities and EM values were measured in a population of cells, and the EM values predicted using the Goldman-Hodgkin-Katz equation for different values of alpha. The best fit for the data was obtained for alpha = 0.036. In ionic substitution experiments, in which passive movements of Cl- were prevented or minimized, the changes in EM around the resting level could be explained with a high degree of accuracy by assuming again that alpha = 0.036. However, tests of the model by investigation of direct effects of reducing extracellular Na+ concentration over a wide range of EM values gave an anomalous result. In low-Na+ Ringer, EM values became more positive than the respective resting levels. The anomalous effect of low-Na+ Ringer on EM did not involve a change in the K+ equilibrium potential. Instead, it was probably due to a reduction in the K+ permeability of the membrane. Possible mechanisms underlying this effect are discussed.

Serotonin levels in the central nervous system of the blowfly Calliphora erythrocephala during postembryonic development

The levels of serotonin in the central nervous system (CNS) of the blowfly, Calliphora erythrocephala were studied by means of high performance liquid chromatography with fluorometric detection. The levels of serotonin were found to vary during development. Serotonin levels in the larval CNS begin to increase 2 days after hatching and reach a maximum at the onset of pupariation. At the start of adult development the serotonin levels drop first to a minimum and then increase, reaching a peak on the fifth day. Thereafter the levels decline for about 3 days and then increase exponentially. The quantitative data reported here are consistent with previous immunocytochemical studies on the development of serotonergic neurons in Calliphora erythrocephala.