Pigment-dispersing hormone-immunoreactive neurons and their relation to serotonergic neurons in the blowfly and cockroach visual system

Summary The pigment-dispersing hormone (PDH) family of neuropeptides comprises a series of closely related octadecapeptides, isolated from different species of crustaceans and insects, which can be demonstrated immunocytochemically in neurons in the central nervous system and optic lobes of some representatives of these groups (Rao and Riehm 1989). In this investigation we have extended these immunocytochemical studies to include the blowfly Phormia terraenovae and the cockroach Leucophaea maderae. In the former species tissue extracts were also tested in a bioassay: extracts of blowfly brains exhibited PDH-like biological activity, causing melanophore pigment dispersion in destalked (eyestalkless) specimens of the fiddler crab Uca pugilator. Using standard immunocytochemical techniques, we could demonstrate a small number of pigment-dispersing hormone-immunoreactive (PDH-IR) neurons innervating optic lobe neuropil in the blowfly and the cockroach. In the blowfly the cell bodies of these neurons are located at the anterior base of the medulla. At least eight PDH-IR cell bodies of two size classes can be distinguished: 4 larger and 4 smaller. Branching immunoreactive fibers invade three layers in the medulla neuropil, and one stratum distal and one proximal to the lamina synaptic layer. A few fibers can also be seen invading the basal lobula and the lobula plate. The fibers distal to the lamina appear to be derived from two of the large PDH-IR cell bodies which also send processes into the medulla. These neurons share many features in their laminamedulla morphology with the serotonin immunoreactive neurons LBO-5HT described earlier (see Nässel 1988). It could be demonstrated by immunocytochemical double labeling that the serotonin and PDH immunoreactivities are located in two separate sets of neurons. In the cockroach optic lobe PDH-IR processes were found to invade the lamina synaptic region and form a diffuse distribution in the medulla. The numerous cell bodies of the lamina-medulla cells in the cockroach are located basal to the lamina in two clusters. Additional PDH-IR cell bodies could be found at the anterior base of the medulla. The distribution and morphology of serotonin-immunoreactive neurons in the cockroach lamina was found to be very similar to the PDH-IR ones. It is hence tempting to speculate that in both species the PDH-and serotonin-immunoreactive neurons are functionally coupled with common follower neurons. These neurons may be candidates for regulating large numbers of units in the visual system. In the flies photoreceptor properties may be regulated by action of the two set of neurons at sites peripheral to the lamina synaptic layer, possibly by paracrine release of messengers.     

Postembryonic development of serotonin-immunoreactive neurons in the central nervous system of the blowfly

Summary Neurons immunoreactive with antibodies to serotonin (5-HT) were mapped in the thoracico-abdominal ganglia of the blowfly, Calliphora erythrocephala, during postembryonic development. Reconstructions from serial sections of tissue processed with a preincubation PAP-method permitted a detailed analysis of the morphological changes occurring in 5-HT-immunoreactive (5-HTi) neurons.All the 5-HTi cell bodies in the thoracico-abdominal ganglia of the 3rd instar larva, except two in the metathoracic ganglion, retain their immunochemical phenotype throughout pupal development. Hence, all the adult 5-HTi neurons in these ganglia differentiate during embryonic development. The finer processes of the larval 5-HTi neurons undergo a substantial regression during the first 24 h of pupal development, and thereafter new branches form on the primary processes of the same cell bodies. The slight change in relative position of 5-HTi cell bodies and the reorganization of the neuropil into an adult pattern occur during the first half of pupal development. The neuropil mass and extent of 5-HTi processes continue to increase during the following days and appear to be fully developed two days (80% of pupal development) before hatching.On the basis of the presented data, some of the basic processes are discussed that lead to the transformation of the larval nervous system into its adult form.     

Postembryonic development of Arg-Phe-amide-like and cholecystokinin-like immunoreactive neurons in the blowfly optic lobe

Summary The adult optic lobes of the blowfly Calliphora erythrocephala were found to be innervated by more than 2000 neurons immunoreactive to antisera raised against the neuropeptides FMRFamide, its fragment RFamide, and gastrin/cholecystokinin (CCK). All of the CCK-like immunoreactive (CCK-IR) neurons also reacted with antisera to RFamide, FMRFamide and pancreatic polypeptide. A few RFamide/FMRFamide-like immunoreactive (RF-IR) neurons did not react with CCK antisera; they reacted instead with antisera to Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷. The RF-IR neurons are, thus, heterogeneous with respect to their contents of immunoreactive peptides. Two of the RF-IR neuron types innervating the adult optic lobes could be traced in their entirety only after following their postembryonic development, because of the complexity of the trajectories of the immunoreactive neuronal process in the adult insect. The majority of the cell bodies of the RF-IR and CCK-IR neurons lie within the optic lobes and are derived from imaginal neuroblasts of the inner and outer optic anlagen. Six of the peptidergic neurons are, however, metamorphosing larval neurons with their cell bodies in the central part of the protocerebrum. The full extent of immunoreactivitiy is not attained in some of the neurons until the late pupal or early adult stage. The larval optic center was also found to be innervated by neurons immuno-reactive with both RFamide and CCK antisera. The cell bodies of these RF-IR/CCK-IR neurons are located near the developing lamina (one on each side). In the 24 h pupa, the cell bodies of these neurons are still immunoreactive, but thereafter they cannot be immunolabeled apparently due to cell death or a change in transmitter phenotype.     

Distribution of GABA-like immunoreactive neurons in the optic lobes of Periplaneta americana

Summary Specific antisera against protein-conjugated -aminobutyric acid (GABA) were used in immunocytochemical staining procedures to study the distribution of the putative GABA-like immunoreactive neurons in the optic lobes of Periplaneta. GABA-like immunoreactive structures are evident in all three optic neuropil regions. Six different populations of GABAergic neurons, whose perikarya are grouped around the medulla, are found within the optic lobe. The number of these immunoreactive cells varies greatly and corresponds to the number of ommatidia of the eye. In the proximal part of the lamina, a coarse network of GABA-positive fibres is recognizable. These are the processes of large field tangential cells whose fibres pass through the distal surface of the medulla. A second fibre population of the lamina is made up of the processes of the centrifugal columnar neurons whose perikarya lie proximally to the medulla. The medulla contains 9 layers with GABAergic elements of variable immunoreactivity. Layers 1, 3, 5, 7 and 9 exhibit strong labelling, as a result of partial overlapping of the processes of centrifugal and centripetal columnar neurons, tangential fibres and/or lateral processes of perpendicular fibres and (possibly) processes of amacrines. A strong immunoreactivity is found in the proximal and distal layers of the lobula.     

The developmental expression of serotonin-immunoreactivity in the brain of the pupal honeybee

The biogenic amine serotonin is a neurotransmitter and modulator in both vertebrates and invertebrates. In the CNS of insects, serotonin is expressed by identifiable subsets of neurons. In this paper, we characterize the onset of expression in the brain and suboesophageal ganglion of the honeybee during pupal development. Several identified serotonin-immunoreactive neurons are present in the three neuromeres of the suboesophageal ganglion the dorsal protocerebrum, and the deutocerebrum at pupal ecdysis. Further immunoreactive neurons are incorporated into the developing pupal brain in two characteristic developmental phases. During the first phase, 5 days after pupal ecdysis, serotonin immunoreactivity is formed in the protocerebral central body, the lamina and lobula, and the deutocerebral antennal lobe. During the second phase, 2 days later, immunoreactivity appears in neurons of the protocerebral noduli of the central complex, the medulla, and the pedunculi and lobes of the mushroom bodies. Three novel serotonin-immunoreactive neurons that innervate the central complex and the mushroom bodies can be individually identified.     

中华蜜蜂工蜂视叶胚后发育过程中的细胞凋亡

本研究通过形态解剖和原位末端标记法(TUNEL),对中华蜜蜂Apis cerana cerana视叶胚后发育过程中的细胞凋亡进行了研究,结果表明:视叶内的细胞程序性死亡开始出现在1龄幼虫末期,随后凋亡细胞数量逐渐增加;在视叶的胚后发育过程中,细胞凋亡经历了3个高峰期,即2龄幼虫、5龄幼虫和蛹发育的第2天;在视叶3个部分的发育中,视髓层中细胞凋亡的数量远远多于视小叶和视神经节层,而视神经节层最少,说明了细胞凋亡的数量和位置与各部分结构发育的时间以及神经投射有关。广泛的细胞凋亡是蜜蜂视叶发育过程中的一个显著特征。     

白斑迷蛱蝶视觉系统中GABA和5-HT能神经元的分布

采用树脂石蜡(Colophony-Paraffin,CP)组织包埋切片技术和链霉菌抗生物素蛋白一过氧化物酶(Streptavidin—peroxidase,SP)免疫组织化学方法,首次报道了GABA和5-HT两种神经递质在白斑迷蛱蝶视觉系统(复眼及视叶)中的分布。与以往所报道的昆虫不同,白斑迷蛱蝶复眼中部分光感细胞对GABA和5-HT抗血清产生免疫反应。每侧视叶中约有2600多个GABA能阳性神经元,它们共分为6群。其中3群位于外髓附近(M1-3),另外三群位于内髓复合体边缘(LC1-3)。GABA能神经元发出的轴突在整个视叶的3个神经纤维网中都有分布。相比之下,视叶对5-HT抗血清的反应较弱,视叶神经纤维网中不存在代表5-HT阳性反应的粗大静脉曲张状纤维,只有一些排列规则的细小纤维。每侧视叶只有位于外髓附近的25个神经元呈现阳性反应,它们的分布位置与部分M3群的GABA能样神经元相同。本文还探讨了5-HT和GABA在调节视觉信息时可能发挥的作用[动物学报50(5)：770—777,2004]。     

Pigment-dispersing hormone (PDH)-immunoreactive neurons form a direct coupling pathway between the bilaterally symmetric circadian pacemakers of the cockroach Leucophaea maderae

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier     

Spatio-temporal pattern of programmed cell death in the developing Drosophila optic lobe

A large number of cells die via programmed cell death during the normal development of the Drosophila optic lobe. In this study, we report the precise spatial and temporal pattern of cell death in this organ. Cell death in the developing optic lobe occurs in two distinct phases. The first phase extends from the start of metamorphosis to the mid-pupal stage. During this phase, a large number of cells die in the optic lobe as a whole, with a peak of cell death at an early pupal stage in the lamina and medulla cortices and the region of the T2/T3/C neurons, and a smaller number of dead cells observed in the lobula plate cortex. The second phase extends from the mid-pupal stage to eclosion. Throughout this period, a small number of dying cells can be observed, with a small peak at a late pupal stage. Most of the dying cells are neurons. During the first phase, dying cells are distributed in specific patterns in cortices. The lamina cortex contains two distinct clusters of dying cells; the medulla cortex, four clusters; the lobula plate cortex, one cluster; and the region of the T2/T3/C neurons, one cluster. Many of the clusters maintain their distinct positions in the optic lobe but others extend the region they cover during development. The presence of distinct clusters of dying cells at different phases suggests that distinct mechanisms control cell death during different stages of optic lobe development in Drosophila.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

Serotonin-like immunoreactivity in the optic lobes of three insect species

The cellular localization of 5-HT in the optic lobes of three insect species was assayed with the use of antibodies raised against 5-HT. In Schistocerca, Periplaneta, and Calliphora all neuropil regions of the optic lobe, the lamina, medulla and lobula, contain 5-HT-immunoreactive varicose fibres in different patterns, like columns and layers. Such fibres also connect the lobula to neuropil in the lateral protocerebrum. In Calliphora also 5-HT-positive fibres of the medulla and lobula plate have projections to the lateral protocerebrum, whereas the origin of the lamina fibres is not certain. In all species the processes displaying 5-HT-like immunoreactivity appear to be derived from a relatively small number of cell bodies, each neuron thus having processes over a large volume of the neuropil of the optic lobe in different layers.     

Allatostatin-like immunoreactivity in the optic lobe of the fly Sarcophaga bullata.

An antiserum against Diploptera allastostain 1 (Dip-AST1) was used to map the distribution of allatostain containing neurons in the optic lobes of the fly Saccrophaga bullata. Strongly immunoreacting neurons were found in two areas of the optic ganglia, namely, the medulla and the area between medulla and lobula. These cells were generally interneurons arborizing the base of the medulla. The positive reaction of specific populations of the optic lobe neurons against allatostain antiserum suggests some role for this neuropeptide in the visual physiology of the fly.     

A genetic analysis of visual system development in Drosophilia melanogaster.

The eyes and optic lobes of adult Drosophila melanogaster comprise a highly organized system of interconnected neurons. The eye and optic lobe primordia are physically separate during the embryonic and larval stages of development, and these tissues do not come into contact until the third larval instar, as a consequence of axons growing from the receptor cells of the developing eyes to the primordial optic lobes. After this contact, the axons of the eyes arrange themselves into their complex and orderly adult pattern. Simultaneously, the optic lobe cells begin elaborating axons which organize into their precise adult array. One question posed by this system is: Does cellular pattern formation in either the eyes or optic lobes depend on eye-brain interactions, or do the two tissues organize autonomously? To answer this question, mutations were found which cause abnormal ommatidial array in the eyes and which also perturb the normal adult axon array in the optic lobes. By means of X ray-induced somatic recombination and by genetically controlled mitotic chromosome loss (gynandromorph formation), flies mosaic for genotypically mutant and normal tissue were constructed. Analysis of the neuronal array in mosaic flies in which eye and optic lobe tissue differed genotypically showed that the axon array phenotype of the optic lobe depends on the genotype of the eye tissue innervating that lobe, while the eye phenotype does not depend on optic lobe genotype. Thus, the axonal organization of the D. melanogaster optic lobe has been shown to depend on the transmission of information from the eyes to the optic lobes.     

Immunocytochemical demonstration of S-antigen (arrestin) in the brain of the blowfly Calliphora vicina

S-Antigen (arrestin)-immunoreaction can be considered as a marker for retinal and extraretinal photoreceptors in both vertebrate and invertebrate species. The present immunocytochemical study with the blowfly Calliphora vicina revealed S-antigen immunoreaction in retinal photoreceptors and various groups of neurons bilaterally distributed in the optic lobes and in the proto-, deuto- and tritocerebrum. S-Antigen-immunoreactive processes and terminal formations were found in the lower division of the central body complex and in the neuropil of the mushroom body. Also neuropil regions of the optic lobe, the lamina, medulla and lobula displayed S-antigen-immunoreactive fibers which were arranged in different patterns. These immunocytochemical data suggest that extraocular photoreceptors may be located in various parts of the blowfly brain. They provide a structural basis for further experiments which are needed to identify definitely these elements as extraretinal photoreceptors.     

Optic lobe projections of marginal ommatidia in Calliphora erythrocephala specialized for detecting polarized light

Summary The structure of ommatidia at the dorsal eye margin of the fly, Calliphora erythrocephala is specialized for the detection of the e-vector of polarized light. Marginal zone ommatidia are distinguished by R7/R8 receptor cells with large-diameter, short, untwisted rhabdomeres and long axons to the medulla. The arrangement of the R7 microvillar directions along the marginal zone is fan-shaped. Ommatidia lining the dorsal and frontal edge of the eye lack primary screening pigments and have foreshortened crystalline cones. The marginal ommatidia from each eye view a strip that is 5 °–20 ° contralateral to the fly's longitudinal axis and that coincides with the outer boundaries of the binocular overlap.Cobalt injection into the retina demonstrates that photoreceptor axons arising from marginal ommatidia define a special area of marginal neuropil in the second visual neuropil, the medulla. Small-field neurons arising from the marginal medulla area define, in turn, a special area of marginal neuropil in the two deepest visual neuropils, the lobula and the lobula plate. From these arise local assemblies of columnar neurons that relay the marginal zones of one optic lobe to equivalent areas of the opposite lobe and to midbrain regions from which arise descending neurons destined for the the thoracic ganglia.Optically, the marginal zone of the retina represents the lateral edge of a larger area of ommatidia involved in dorsofrontal binocular overlap. This binocularity area is also represented by special arrangements of columnar neurons, which map the binocularity area of one eye into the lobula beneath the opposite eye. Another type of binocularity neuron terminates in the midbrain.These neuronal arrangements suggest two novel features of the insect optic lobes and brain: (1) Marginal neurons that directly connect the left and right optic lobes imply that each lobe receives a common input from areas of the left and right eye, specialized for detecting the pattern of polarized light. (2) Information about the e-vector pattern of sky-light polarization may be integrated with binocular and monocular pathways at the level of descending neurons leading to thoracic motor neuropil.     

The optic lobes of Lepidoptera

Variants of the Golgi-Colonnier (1964) selective silver procedure have been used to show up neurons in insect brains. Neural elements are particularly clearly impregnated in the optic lobes. Three classes of nerve cells can be distinguished; perpendicular (class I), tangential (class II) and amacrine cells (class III). There are many types of neurons in each class which together have a very wide variety of form. Their components are related to specific strata in the optic lobe regions. Short visual cells from the retina terminate in the lamina in discrete groups of endings (optic cartridges). Pairs of long visual fibres from ommatidia pass through the lamina and end in the medulla. Class I cells link these two regions in parallel with the long visual fibres and groups of these elements define columns in the medulla. These in turn give rise to small-field fibres that project to the lobula complex. Tangential processes intersect the parallel arrays of class I cells at characteristic levels. Some are complex in form and may invade up to three regions. Another type provides a direct link between the ipsi- and contralateral optic lobe. Amacrine cells are intrinsic to single lobe regions and have processes situated at the same levels as those of classes I and II cells. A fifth optic lobe region, the optic tubercle, is connected to the medulla and lobula and also receives a set of processes from the mid-brain. There are at least six separate types of small-field relays which could represent the retina mosaic arrangement in the lobula.     

Morphological changes of the optic lobe from late embryonic to adult stages in oval squids Sepioteuthis lessoniana

The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro‐CT scans were acquired to reconstruct the 3D‐structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.     

Cell degeneration in the developing optic lobes of the sine oculis and small-optic-lobes mutants of Drosophila melanogaster

In the small-optic-lobes (sol) and sine oculis (so) mutants of Drosophila melanogaster extensive cell death occurs in the optic lobes during the first half of pupal development. Gynandromorph flies show that the sol mutation acts primarily on cells of the medulla cortex. Degeneration of medullar ganglion cells occurs at an early stage of cellular differentiation, when their axons have not yet participated in the formation of the second optic chiasma. The so gene, on the other hand, acts on the eye anlagen. The analysis of chimeric flies demonstrates that degeneration in the optic lobes of so flies is a consequence of eye reduction. At the level of the second optic chiasma extensive axonal degeneration can be observed in the mutant. Neurons seem to die after their failure to establish a sufficient number of functional contacts. In sol;so double mutants, the mutational effects are cumulative causing complete degeneration of columnar cell types in pupae without any eye anlage. The tiny rudiments of the optic lobes in eyeless double mutants still contain tangential neurons of the medulla and of the lobula complex. The central brain is reduced in size due to the missing visual fibers, however, its overall appearance is surprisingly normal.     

Neuropeptide Y-immunoreactive neuronal system and colocalization with FMRFamide in the optic lobe and peduncle complex of the octopus (Octopus vulgaris)

The distribution of neuropeptide Y (NPY)-like immunoreactivity and its colocalization with FMRFamide were investigated in the optic lobe and peduncle complex of the octopus ( Octopus vulgaris) by using immunohistochemical techniques. In the optic lobe cortex, NPY-immunoreactive (NPY-IR) fibers were observed in the plexiform layer, although no NPY-IR somata were observed in the outer or inner granular cell layers. In the optic lobe medulla, NPY-IR somata were seen in the cell islands, and abundant NPY-IR varicose fibers were observed in the neuropil. Most of the NPY-IR structures in the medulla showed FMRFamide-like immunoreactivity. In the peduncle lobe, abundant NPY-IR and FMRFamide-IR (NPY/FMRF-IR) varicose fibers were seen in the basal zone neuropil of the peduncle lobe. In the olfactory lobe, NPY/FMRF-IR varicose fibers were also abundant in the neuropil of the three lobules. NPY/FMRF-IR somata, with processes running to various neuropils, were scattered in the median and posterior lobules. In the optic gland, many NPY/FMRF-IR varicose fibers formed a honeycomb pattern. These observations suggest that NPY/FMRF-IR neurons in the optic lobes participate in the modulation of visual information and that those in the optic gland are involved in the regulation of endocrine function.