The color-vision circuit in the medulla of Drosophila

BACKGROUND: Color vision requires comparison between photoreceptors that are sensitive to different wavelengths of light. In Drosophila, this is achieved by the inner photoreceptors (R7 and R8) that contain different rhodopsins. Two types of comparisons can occur in fly color vision: between the R7 (UV sensitive) and R8 (blue- or green sensitive) photoreceptor cells within one ommatidium (unit eye) or between different ommatidia that contain spectrally distinct inner photoreceptors. Photoreceptors project to the optic lobes: R1-R6, which are involved in motion detection, project to the lamina, whereas R7 and R8 reach deeper in the medulla. This paper analyzes the neural network underlying color vision into the medulla. RESULTS: We reconstruct the neural network in the medulla, focusing on neurons likely to be involved in processing color vision. We identify the full complement of neurons in the medulla, including second-order neurons that contact both R7 and R8 from a single ommatidium, or contact R7 and/or R8 from different ommatidia. We also examine third-order neurons and local neurons that likely modulate information from second-order neurons. Finally, we present highly specific tools that will allow us to functionally manipulate the network and test both activity and behavior. CONCLUSIONS: This precise characterization of the medulla circuitry will allow us to understand how color vision is processed in the optic lobe of Drosophila, providing a paradigm for more complex systems in vertebrates.     

Comparative anatomy of pigment-dispersing hormone-immunoreactive neurons in the brain of orthopteroid insects

Summary In a comparative study, the anatomy of neurons immunoreactive with an antiserum against the crustacean -pigment-dispersing hormone was investigated in the brain of several orthopteroid insects including locusts, crickets, a cockroach, and a phasmid. In all species studied, three groups of neurons with somata in the optic lobes show pigment-dispersing hormone-like immunoreactivity. Additionally, in most species, the tritocerebrum exhibits weak immunoreactive staining originating from ascending fibers, tritocerebral cells, or neurons in the inferior protocerebrum. Two of the three cell groups in the optic lobe have somata at the dorsal and ventral posterior edge of the lamina. These neurons have dense ramifications in the lamina with processes extending into the first optic chiasma and into distal layers of the medulla. Pigment-dispersing hormone-immunoreactive neurons of the third group have somata near the anterior proximal margin of the medulla. These neurons were reconstructed in Schistocerca gregaria, Locusta migratoria, Teleogryllus commodus, Periplaneta americana, and Extatosoma tiaratum. The neurons have wide and divergent arborizations in the medulla, in the lamina, and in several regions of the midbrain, including the superior and inferior lateral protocerebrum and areas between the pedunculi and -lobes of the mushroom bodies. Species-specific differences were found in this third cell group with regard to the number of immunoreactive cells, midbrain arborizations, and contralateral projections, which are especially prominent in the cockroach and virtually absent in crickets. The unusual branching patterns and the special neurochemical phenotype suggest a particular physiological role of these neurons. Their possible function as circadian pacemakers is discussed.     

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

Summary The postembryonic development of serotonin-immunoreactive (5-HTi) neurons was studied in the optic lobe of the blowfly. In the adult fly there are 24 5-HTi neurons invading each optic lobe. The perikarya of two of these neurons are situated in the dorso-caudal part of the protocerebrum (LBO-5HT neurons; large bilateral optic lobe 5-HTi neurons). The cell bodies of the remaining 22 neurons are located anteriorly at the medial base of the medulla (2 innervating the lobula, LO-5HT neurons; and 20 neurons innervating the medulla, ME-5HT neurons). The two central neurons (LBO-5HT neurons) are derived from metamorphosing larval neurons, while the ME- and LO-5HT neurons are imaginai optic lobe neurons differentiating during pupal development.The 5-HTi neurons of the optic lobe seem to have different ancestors. The LBO-5HT neurons are probably derived from segmental protocerebral neuroblasts, whereas the ME-and LO-5HT neurons are most likely derived from the inner optic anlage. The first 5-HTi fibers to reach the imaginal optic lobes are seen in the late third instar larva and are derived from the LBO-5HT neurons. The first ME- and LO-5HT neurons become immunoreactive at 24 h (10%) pupal development. At about 96 h (40%) of pupal development all the 5-HTi neurons of the optic lobes have differentiated and attained their basic adult morphology. The further development mainly entails increase in volume of arborizations and number of finer processes. The differentiation and outgrowth of 5-HTi processes follows that of, e.g., columnar neurons in the optic lobe neuropils. Hence, 5-HTi processes invade neuropil relatively late in the differentiation of the optic lobe.     

Immunocytochemical characterization of the accessory medulla in the cockroach Leucophaea maderae

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.     

The antidromic activation of tectal neurons by electrical stimuli applied to the caudal medulla oblongata in the toad,Bufo bufo L.

In order to specify the tectal projection to the bulbar/spinal regions, the antidromic responses of the physiologically identified tectal neurons as well as the gross antidromic field responses in the optic tectum to electrical stimuli applied to the caudal medulla were examined in the paralyzed common toad, Bufo bufo. The antidromic field potential was recorded in the optic tectum in response to electrical stimuli applied to the ventral paramedian portion of the contralateral caudal medulla (where the crossed tecto-spinal pathway of Rubinson (1968) and Lázár (1969) runs), but generally not when they were applied to various parts of the ipsilateral caudal medulla. The antidromic field potential was largest at the superficial part of Layer 6 or at the border between Layers 6 and 7 of the optic tectum, indicating that neurons in these layers project to the contralateral caudal medulla. Mapping experiments of the antidromic field potential over the optic tectum showed that the antidromic field potential was recorded mainly in the lateral part of it, indicating that this part of the optic tectum is the main source of projection neurons to the contralateral caudal medulla. Various classes of tectal neurons as well as retinal ganglion neurons were identified from the characteristics of the response properties to moving visual stimuli and the properties of the receptive fields. Of these, the Class T1, T2, T3, T4, T5(1), T5(2), T5(3), and T5(4) tectal neurons were activated antidromically by stimuli applied to the contralateral caudal medulla. Only a limited proportion of the Class T5(1) neurons was activated antidromically by stimuli applied to the ipsilateral caudal medulla. On the other hand, the Class T7 and T8 neurons, as well as the Class R2, R3, and R4 retinal neurons, were not activated antidromically by stimuli applied to the caudal medulla of either side. These results suggest a possibility that these tectal neurons which project to the medullary regions form the substrate of the sensorimotor interfacing and contribute to the initiation or coordination of the visually guided behavior, such as prey-catching.     

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]。     

Immunocytochemical Localization of Amines and GABA in the Optic Lobe of the Butterfly, Papilio xuthus

Butterflies have sophisticated color vision. While the spectral organization of the compound eye has been well characterized in the Japanese yellow swallowtail butterfly, Papilio xuthus, neural mechanisms underlying its color vision are largely unexplored. Towards a better understanding of signal processing in the visual system of P. xuthus, we used immunocytochemical techniques to analyze the distribution of transmitter candidates, namely, histamine, serotonin, tyramine and γ-aminobutyric acid (GABA). Photoreceptor terminals in the lamina and medulla exhibited histamine immunoreactivity as demonstrated in other insects. The anti-histamine antiserum also labeled a few large medulla neurons. Medulla intrinsic neurons and centrifugal neurons projecting to the lamina showed serotonin immunoreactivity. Tyramine immunostaining was detected in a subset of large monopolar cells (LMCs) in the lamina, transmedullary neurons projecting to the lobula plate, and cell bodies surrounding the first optic chiasma. An anti-GABA antiserum labeled a subset of LMCs and populations of columnar and tangential neurons surrounding the medulla. Each of the four antisera also labeled a few centrifugal neurons that innervate the lobula complex from the central brain, suggesting that they have neuromodulatory roles. A distinctive feature we found in this study is the possibility that tyramine and GABA act as transmitters in LMCs of P. xuthus, which has not been reported in any other insects so far.     

5-HT-like immunoreactivity in the central nervous system of the crayfish,Pacifastacus leniusculus

An immunocytochemical technique with the use of three different antibodies raised against serotonin was applied to localize the immunoreactive neurons in the central nervous system of the crayfish, Pacifastacus leniusculus. Immunoreactive neurons were found in three optic ganglia (medulla externa, interna and terminalis). They appeared in three layers of the medulla externa and interna. The medulla terminalis displayed three prominent groups of immunoreactive perikarya and mainly marginal immunoreactive fibres. Immunoreactive areas of the brain comprised the protocerebral bridge, central body, paracentral lobes and two loci in the anterior portion of the protocerebrum, i.e., the terminal areas for immunoreactive fibres from the optic centres. The olfactory lobes showed a specific immunoreactive pattern. In addition, diffusely and sparsely distributed immunoreactive fibres were found throughout the brain. The immunoreactive neurons are largely localized in the same areas of the central nervous system as the catecholaminergic neurons although some distinct differences occur.     

Localization of corticotropin-releasing factor-immunoreactive nervous tissue and colocalization with neuropeptide Y-like substance in the optic lobe and peduncle complex of the octopus (<Emphasis Type="Italic">Octopus vulgaris</Emphasis>)

The distribution of corticotropin-releasing factor (CRF)-like immunoreactivity and its colocalization with neuropeptide Y (NPY)-like substances were investigated in the optic lobe and peduncle complex of the octopus (Octopus vulgaris) using immunohistochemical techniques. In the optic lobe cortex, CRF-immunoreactive (CRF-IR) and NPY-immunonegative varicose fibers were observed in the plexiform layer. In the medulla, CRF-IR somata were seen in the cell islands, and CRF-IR varicose fibers were observed in the neuropil. About half of the CRF-IR structures in the medulla showed NPY-like immunoreactivity. In the peduncle lobe, no CRF-IR somata but abundant CRF-IR varicose fibers were observed, and about half of them showed NPY-like immunoreactivity. In the olfactory lobe, CRF-IR somata and abundant CRF-IR varicose fibers were observed. Almost all the CRF-IR somata located in the posterior olfactory lobule showed NPY-like immunoreactivity, whereas those seen in the median olfactory lobule were immunonegative for NPY. About half of the CRF-IR fibers in the anterior lobule neuropil were immunopositive for NPY, but those in the median and posterior lobule neuropils were immunonegative for NPY. In the optic gland, almost all the CRF-IR varicose fibers were immunoreactive for NPY. Western blot analysis of the optic lobe and peduncle complex indicated that anti-CRF antiserum labeled approximate 16.4- and 14.6-kDa bands and that anti-NPY antiserum labeled an approximate 16.2-kDa band. CRF-IR and NPY-immunoreactive neurons in the optic lobe may participate in the modulation of visual information and those in the optic gland may be involved in the regulation of endocrine function.     

Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 Lom TK-LI neurons were found with cell bodies distributed in the proto-, deutoand tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodics that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the Lom TK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the Lom TK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functinal relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.     

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.     

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach <Emphasis Type="Italic">Leucophaea maderae</Emphasis>

Lesion and transplantation studies in the cockroach, Leucophaea maderae, have located its bilaterally symmetric circadian pacemakers necessary for driving circadian locomotor activity rhythms to the accessory medulla of the optic lobes. The accessory medulla comprises a network of peptidergic neurons, including pigment-dispersing factor (PDF)-expressing presumptive circadian pacemaker cells. At least three of the PDF-expressing neurons directly connect the two accessory medullae, apparently as a circadian coupling pathway. Here, the PDF-expressing circadian coupling pathways were examined for peptide colocalization by tracer experiments and double-label immunohistochemistry with antisera against PDF, FMRFamide, and Asn¹³-orcokinin. A fourth group of contralaterally projecting medulla neurons was identified, additional to the three known groups. Group one of the contralaterally projecting medulla neurons contained up to four PDF-expressing cells. Of these, three medium-sized PDF-immunoreactive neurons coexpressed FMRFamide and Asn¹³-orcokinin immunoreactivity. However, the contralaterally projecting largest PDF neuron showed no further peptide colocalization, as was also the case for the other large PDF-expressing medulla cells, allowing the easy identification of this cell group. Although two-thirds of all PDF-expressing medulla neurons coexpressed FMRFamide and orcokinin immunoreactivity in their somata, colocalization of PDF and FMRFamide immunoreactivity was observed in only a few termination sites. Colocalization of PDF and orcokinin immunoreactivity was never observed in any of the terminals or optic commissures. We suggest that circadian pacemaker cells employ axonal peptide sorting to phase-control physiological processes at specific times of the day.     

Antibodies against the PER protein of Drosophila label neurons in the optic lobe,central brain,and thoracic ganglia of the crickets Teleogryllus commodus and Teleogryllus oceanicus

We describe labeling of neurons in the central nervous system of two cricket species, Teleogryllus commodus and T. oceanicus, with both mono- and polyclonal antibodies against the PER protein. Western blots reveal that the monoclonal antibodies recognize a single protein with a molecular weight of approximately 94 kDa, i.e., similar to that of the PER protein of the moth, Anterea pernii. Neurons and their processes are labeled both in the optic lobes and in the central brain. Processes occur in the accessory medulla, the medulla, and proximal lamina, in the central complex, in the non-glomerular neuropil, and in the retrocerebral complex, suggesting that PER-containing neurons form a widely distributed network. Neurons and processes were also labeled in the meso- and metathoracic ganglia. Four to six PER-immunoreactive (ir) neurons with processes in the accessory medulla were double labeled by an antibody against pigment-dispersion factor (PDF), a peptide that is implicated in circadian rhythmicity in Drosophila. In the central brain, projections of fibers labeled by the anti-PER and anti-PDF antibodies were mainly distinct, with overlap only in a few restricted regions. In most neurons, including those projecting into the accessory medulla, PER labeling was restricted to the cytoplasm and there was no indication of circadian variation in the intensity of staining.     

Morphological and physiological identification of medulla interneurons in the visual system of the tiger beetle larva

The morphology of visual interneurons in the tiger beetle larva was identified after recording their responses. Stained neurons were designated as either medulla or protocerebral neurons according to the location of their cell bodies. Medulla neurons were further subdivided into three groups. Afferent medulla neurons extended processes distally in the medulla neuropil and a single axon to the brain through the optic nerve. They received their main input from stemmata on the ipsilateral side. Two distance-sensitive neurons, near-by sensitive and far-sensitive neurons, were also identified. Atypical medulla neurons extended their neurites distally in the medulla and proximally to the brain, as afferent medulla neurons, but their input patterns and the shapes of their spikes differed from afferent neurons. Protocerebral neurons sent a single axon to the medulla neuropil. They spread collateral branches in the posterior region of the protocerebrum on its way to the medulla neuropil. They received main input from stemmata on the contralateral side. Medulla intrinsic neurons did not extend an axon to the brain, and received either bilateral or contralateral stemmata input only. The input patterns and discharge patterns of medulla neurons are discussed with reference to their morphology.     

Insect optic lobe neurons identifiable with monoclonal antibodies to GABA

Five monoclonal antibodies against GABA were tested on glutaraldehyde fixed sections of optic lobes of three insect species, blowflies, houseflies and worker bees. The specificity of these antibodies was analyzed in several tests and compared with commercially available anti-GABA antiserum. A very large number of GABA-like immunoreactive neurons innervate all the neuropil regions of these optic lobes. Immunoreactive processes are found in different layers of the neuropils. The immunoreactive neurons are amacrines and columnar or noncolumnar neurons connecting the optic lobe neuropils. In addition some large immunoreactive neurons connect the optic lobes with centers of the brain. Some neuron types could be matched with neurons previously identified with other methods. The connections of a few of these neuron types are partly known from electron microscopy or electrophysiology and a possible role of GABA in certain neural circuits can be discussed.     

Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1-4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.     

Neural cell types surviving congenital sensory deprivation in the optic lobes of Drosophila melanogaster

Golgi staining of neuronal cell types in the optic lobe rudiments of adult eyeless flies of the sine oculis (so) mutant of Drosophila melanogaster reveals partial independence of optic lobe's development from compound eye formation. (1) Differentiation and maintenance of many neuronal cell types of medulla and lobular complex do not require innervation of the medulla from the retina and the lamina. Neurons derived from the outer and inner optic anlage have been found in adult eyeless flies. (2) The rudiments of ipsilateral medulla, lobula, and lobular plate are isotopically connected with each other. (3) Stratification of the lobular complex is retained. (4) Equivalent parts of the dorsal lobulae are connected by heterolateral small field neurons. (5) The shapes of many tangential neurons of the medulla show sprouting and compensatory innervation of the lobular complex. The basic results reported here for eyeless flies have many parallels in what is known about anophthalmic mice.     

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