Histamine-immunoreactive neurons in the brain of the cockroach Leucophaea maderae.

Histamine is the neurotransmitter of insect photoreceptor cells but has also been found in a small number of interneurons in the insect brain. In order to investigate whether the accessory medulla (AMe), the putative circadian pacemaker of the cockroach Leucophaea maderae receives direct visual input from histaminergic photoreceptors, we analyzed the distribution of histamine-like immunoreactivity in the optic lobe and midbrain of the cockroach. Intense immunostaining was detected in photoreceptor cells of the compound eye, which terminated in the first optic neuropil, the lamina, and in a distal layer of the medulla, the second optic neuropil. Histamine immunostaining in parts of the AMe, however, originated from a centrifugal neuron of the midbrain. Within the midbrain 21-23 bilaterally symmetric pairs of cell bodies were stained. Most areas of the brain were innervated by one or more of these neurons, but the protocerebral bridge and the mushroom bodies were devoid of histamine immunoreactivity. The branching patterns of most histamine-immunoreactive neurons could be reconstructed individually. While the majority of identified neurons arborized in both brain hemispheres, five cells were local neurons of the antennal lobe. A comparison with other insect species shows striking similarities in the position of certain histamine-immunoreactive neurons, but considerable variations in the presence and branching patterns of others. The data suggest a role for histamine in a non-photic input to the circadian system of the cockroach.     

Myoinhibitory peptides in the brain of the cockroach Leucophaea maderae and colocalization with pigment-dispersing factor in circadian pacemaker cells

Myoinhibitory peptides (MIPs) are a family of insect W(X(6))Wamides with inhibitory effects on visceral muscles and juvenile hormone synthesis. Although MIPs are widely distributed within the nervous system, a detailed analysis of their distribution and function in insect brains is still missing. We analyzed the distribution of MIPs in the brain of the cockroach Leucophaea maderae. We focused on the accessory medulla (AMe), a small neuropil near the medulla that acts as the master circadian clock. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and Nano-LC electrospray ionization (ESI) mass spectrometry revealed five Lem-MIPs in preparations of the AMe and corpora cardiaca. The complete sequences of two of these peptides were identified. Immunocytochemistry revealed wide distribution of MIP-related peptides in the cockroach brain. The superior median protocerebrum, parts of the central complex, and the tritocerebrum showed particularly dense immunostaining. In contrast, only a few local interneurons were stained in the antennal lobe and a few extrinsic neurons in the mushroom body, including a giant neuron innervating the calyces. The noduli of the AMe showed dense immunostaining, and neurons in all AMe cell groups except the anterior neurons were labeled. Pigment-dispersing factor- (PDF) and MIP immunostaining was colocalized in two neurons of the AMe. No colocalization of MIP- and PDF immunostaining was detected in the anterior optic commissure, but two small PDF-immunoreactive commissural fibers near the posterior optic commissure showed colocalized MIP immunostaining. The results suggest that several MIPs participate in different functional circuits of the circadian system and are involved in multiple brain circuits of the Madeira cockroach.     

Distribution of histamine in the cockroach brain and visual system: an immunocytochemical and biochemical study

The distribution of histamine-immunoreactivity in the carbodiimide-fixed brain and visual system of the cockroach was revealed immunocytochemically with an antiserum against histamine (HA). Histamine levels were measured with high-pressure liquid chromatography. The results show a widespread distribution of histamine-containing somata and fibers in the brain, particularly in the visual system. The most intense immunolabeling was seen in the retinal photoreceptors and in the first optic ganglion, the lamina, where the short visual fibers make synaptic connections with the monopolar neurons, which also displayed immunofluorescence. Immunoreactive long visual fibers traversed the lamina and outer chiasma, terminating in the distal medulla. Tracts of histamine-immunopositive fibers appeared to link the optic ganglia to the protocerebrum. Prominent histamine-containing neurons were situated in the lateral protocerebrum. Immunolabeled pathways consisting of large-diameter fibers also were seen in the cockroach brain. The central parts of the brain, including the central body, were reached by thick immunoreactive fibers that gave rise to intensely fluorescent varicose processes there. In the mushroom bodies, immunoreactivity was limited to the calyces. The protocerebral bridge was nonreactive. Immunofluorescence was seen also in the antennal lobes, but not in the antennal nerves. The biochemical measurements correlated well with the immunocytochemical data. The retinas and optic lobes, measured together, contained remarkably large amounts of histamine. These results reinforce the hypothesis presented by Hardie ('87) and Elias and Evans ('83) that histamine functions as a neurotransmitter in the photoreceptors of some, if not all, insect species.     

Postembryonic differentiation of serotonin-immunoreactive neurons in fleshfly optic lobes developing in situ or cultured in vivo without eye discs

The differentiation of serotonin-immunoreactive (5-HTi) neurons in the optic lobes of fleshflies was studied during in situ development and in in vivo cultures. All 5-HTi neurons with cell bodies in the imaginal optic lobes differentiate during postembryonic (pupal) development. These are local anaxonal neurons. In addition there are two large 5-HTi bilateral neurons that connect all optic lobe neuropil regions on both sides of the brain and have their cell bodies in the midbrain proper. Deafferentation of optic lobes cultured in vivo leads to drastic reduction in optic lobe volume and increased cell death. All the 5-HTi neurons differentiate after deafferentation but their morphology changes. The neuropil receiving the photoreceptor inputs, the lamina, degenerates but a disorganized "pseudolamina" is formed by the processes of the two large 5-HTi neurons. The layering of the optic lobe neuropils cannot be distinguished and 5-HTi processes form novel projectional patterns. Hence, the 5-HTi neurons do not require afferent inputs from the retina for their differentiation and survival, but the effect on other optic lobe interneurons is reflected in the morphological plasticity of the 5-HTi neurons.     

Implementation of pigment-dispersing factor-immunoreactive neurons in a standardized atlas of the brain of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae is an established model in circadian rhythm research. Its circadian clock is located in the accessory medulla of the brain. Pigment-dispersing factor-immunoreactive (PDF-ir) neurons of the accessory medulla act as circadian pacemakers controlling locomotor activity rhythms. To characterize the neuronal network of the circadian system in L. maderae, the PDF-ir neurons were implemented into a standardized three-dimensional atlas of the cockroach brain. Serial confocal images from 20 wholemount brains were used for the construction of the atlas comprising 21 neuropils. Two different standardization protocols were employed: the iterative shape averaging (ISA) procedure using an affine transformation followed by iterative non-rigid registrations, and the virtual insect brain (VIB) protocol employing local non-rigid transformations after global and local rigid transformations. Quantitative analysis of the 20 brains revealed that volumes of the accessory medulla are directly correlated with the volumes of the medulla, the protocerebral bridge, and the upper division of the central body, suggesting functional connections among these neuropils. For a standardized reconstruction of the circadian pacemaker network, the ISA protocol was used to register PDF-ir neurons in the standard cockroach brain. The registration revealed that two PDF-ir arborization areas in the brain are highly interconnected with other PDF-ir projection sites and appear to be contacted both by fibers in the posterior and the anterior optic commissures. The distances between PDF-ir branching areas show specific numerical relationships that might be physiologically relevant for temporal encoding.     

Postembryonic development of γ-aminobutyric acid-like Immunoreactivity in the brain of the sphinx moth Manduca sexta

We have investigated the distribution of immunocytochemical staining for the neurotransmitter γ-aminobutyric acid (GABA) in the brain of the sphinx moth Manduca sexta during larval, pupal, and adult development. In the larval brain, about 300 neurons are GABA-immunoreactive. All neuropil areas except the mushroom bodies and central complex show intense immunostaining. Only minor changes in the pattern of immunoreactivity occur during larval development. During metamorphosis, changes in immunostaining occur in two phases. Beginning in wandering fifth-instar larvae (stage W2), immunoreactivity appears in numerous neurons of the central body and optic lobe and becomes more intense during early pupal stages. At the same time, GABA-like immunoreactivity disappears in most neuropil areas of the brain and becomes faint in many immunoreactive somata. Neurons with arborizations in the ventrolateral protocerebrum, however, continue to exhibit intense immunostaining during this period, and strongly immunolabeled fibers connect these areas with the ventral nerve cord. The second phase of transformation begins around pupal stage P5/P6, when faint immunostaining appears in many previously nonimmunoreactive somata and most neuropil areas of the brain. In subsequent stages (P8–P10), this immunoreactivity disappears again in most somata, but in certain cell groups, it becomes more intense and gradually develops to the adult pattern. Most larval GABA-immunoreactive neurons appear to survive through metamorphosis into the adult. Neurons in the midbrain that acquire GABA-like immunoreactivity during metamorphosis usually lie adjacent to larval immunostained neurons, suggesting common lineages. The onsets of the two developmental phases of GABA-like immunoreactivity correlate with sharp rises in hemolymph titers of ecdysteroid hormones, suggesting a role for ecdysteroids in the regulation of GABA synthesis. We hypothesize that the disappearance of GABA in many areas of the brain starting 2 days prior to pupation dramatically alters its functional circuitry and thus may account for profound changes in the behavior of the animal. © 1994 Wiley-Liss, Inc.     

Optic lobe commissures in a three-dimensional brain model of the cockroach Leucophaea maderae: a search for the circadian coupling pathways.

The circadian rhythm of locomotor activity in the cockroach Leucophaea maderae is controlled by bilaterally symmetric, apparently directly coupled, circadian pacemakers in the optic lobes. Strong evidence predicts that ventromedial to the medulla, the accessory medulla with associated pigment-dispersing hormone-immunoreactive neurons is this circadian clock. In search for direct coupling pathways between both clocks, we performed horseradish peroxidase backfills from one optic stalk as well as dextran and horseradish peroxidase injections into one accessory medulla. Seven commissures with projections in the contralateral optic lobe were identified and reconstructed. Three of these commissures connected both accessory medullae. Two of these resembled the arborization pattern of the pigment-dispersing hormone-immunoreactive neurons, which are circadian pacemaker candidates in insects. This finding suggests that some of these pacemaker candidates form a direct circadian coupling pathway. For better visualization of reconstructed commissures, we implemented the reconstructions into a three-dimensional model of the cockroach brain.     

Immunocytochemistry of dopamine in the brain of the locust Schistocerca gregaria.

Catecholamine-induced histofluorescence studies have suggested a rich innervation of the locust brain by dopamine-containing neurons. To provide a basis for future studies on dopamine action in this insect, the location and morphology of neurons reacting with antisera against dopamine were investigated in the supraoesophageal ganglion of the locust, Schistocerca gregaria. In each brain hemisphere, about 100 interneurons in the midbrain and approximately 3,000 cells in the optic lobe show dopamine-like immunoreactivity. All major areas of the brain except the calyces of the mushroom body, the antennal lobe, large parts of the lobula, and some areas in the inferior lateral protocerebrum contain immunoreactive neuronal processes. The arborization patterns of most dopamine-immunoreactive cell types could be identified through detailed reconstructions. The central body exhibits the most intense immunostaining. It is innervated by at least 40 pairs of dopamine-immunoreactive neurons belonging to three different cell types. Additional arborizations of these neurons are in the superior protocerebrum and in the lateral accessory lobes. A group of 4 immunoreactive neurons with ramifications in the antennal mechanosensory and motor center gives rise to a dense meshwork of varicose fibers in the pedunculus and parts of the alpha- and beta-lobes of the mushroom body. Other cell types innervate the ventrolateral protocerebrum, the inferior protocerebrum and the posterior optic tubercles. Three descending neurons originating in the tritocerebrum exhibit dopamine-like immunoreactivity. In the optic lobe, about 3,000 columnar intrinsic neurons of the medulla and a group of centrifugal tangential cells with arborizations in the medulla and lamina are dopamine-immunoreactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA‐ and serotonin‐expressing neurons take part in inhibitory as well as excitatory input pathways to the circadian clock of the Madeira cockroach Rhyparobia maderae

In the Madeira cockroach, pigment‐dispersing factor‐immunoreactive (PDF‐ir) neurons innervating the circadian clock, the accessory medulla (AME) in the brain′s optic lobes, control circadian behaviour. Circadian activity rhythms are entrained to daily light–dark cycles only by compound eye photoreceptors terminating in the lamina and medulla. Still, it is unknown which neurons connect the photoreceptors to the clock to allow for light entrainment. Here, we characterized by multiple‐label immunocytochemistry the serotonin (5‐HT)‐ir anterior fibre fan and GABA‐ir pathways connecting the AME‐ and optic lobe neuropils. Colocalization of 5‐HT with PDF was confirmed in PDF‐ir lamina neurons (PDFLAs). Double‐labelled fibres were traced to the AME originating from colabelled PDFLAs branching in accessory laminae and proximal lamina. The newly discovered GABA‐ir medial layer fibre tract connected the AME to the medulla′s medial layer fibre system, and the distal tract fibres connected the AME to the medulla. With Ca²⁺ imaging on primary cell cultures of the AME and with loose‐patch‐clamp recordings in vivo, we showed that both neurotransmitters either excite or inhibit AME clock neurons. Because we found no colocalization of GABA and 5‐HT in any optic lobe neuron, GABA‐ and 5‐HT neurons form separate clock input circuits. Among others, both pathways converged also on AME neurons that coexpressed mostly inhibitory GABA‐ and excitatory 5‐HT receptors. Our physiological and immunocytochemical studies demonstrate that GABA‐ and 5‐HT‐immunoreactive neurons constitute parallel excitatory or inhibitory pathways connecting the circadian clock either to the lamina or medulla where photic information from the compound eye is processed.     

Movement-sensitive,polarization-sensitive,and light-sensitive neurons of the medulla and accessory medulla of the locust,Schistocerca gregaria

Several lines of evidence suggest that the accessory medulla of orthopteroid insects is implicated in the control of circadian rhythms. To investigate the role of this brain area in more detail, anatomical and physiological properties of accessory-medulla neurons of the locust were studied by intracellular recordings combined with Lucifer dye injections. The responses of these neurons to visual stimuli were compared with visual responses of adjacent tangential neurons of the medulla. Principal neurons of the accessory medulla showed weak tonic excitations to stationary light stimuli, but they were not sensitive to movement stimuli or to different e-vector orientations of polarized light. These neurons connected the accessory medulla to the lamina, the anterior medulla, and to several areas in the midbrain including the superior protocerebrum and the posterior optic tubercle. A second class of neurons had tangential arborizations in the medulla, a few sidebranches in the accessory medulla, and projections to the lamina or to the contralateral optic lobe. Several of these neurons were sensitive to polarized light. Finally, a third class of neurons had tangential arborizations in the medulla and axonal projections to the lobula and to the lateral protocerebrum. These neurons showed phasic responses to light and nondirectional selective responses to motion stimuli. The results show that neurons of the accessory medulla receive photic input and support an involvement of this neuropil in circadian timekeeping functions. The possible role of the accessory medulla in polarization vision is discussed. J. Comp. Neurol. 386:329-346,1997. © 1997 Wiley-Liss, Inc.     

Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I.

Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.     

Serotonin-immunoreactive neurons in the brain of the honeybee

The distribution of serotonin-immunoreactive neurons in the brain of the worker honey bee Apis mellifera was studied by means of immunocytochemical staining by using a well-characterized antibody to serotonin (5-HT). About 75 immunoreactive perikarya are grouped into clusters in the optic lobe and in the median and dorsal protocerebrum. Immunoreactive fibers were resolved in all areas of the brain. The optic lobe shows restricted layers of 5-HT-immunoreactive fibers in the lamina and medulla organized perpendicular to the retinotopic elements. Immunoreactive fibers in the lobula represent invasions of protocerebral giant wide-field neurons. The nonglomerular neuropil of the brain exhibits a meshwork of immunoreactive fibres invading glomerular neuropil of the mushroom bodies, central body complex, and antennal lobes. Mushroom body stalks and lobes contain immunoreactive fibers arranged perpendicular to the Kenyon cell fibers and matching subcompartments of these corpora pedunculata areas. The calyces are devoid of immunofluorescence. Serotonin-positive fibres in the central body complex are arranged in its subcompartments. No 5-HT immunoreactivity was found in the pons. Antennal glomeruli contain immunoreactive fibers restricted around the margin of the glomeruli. The selective mapping of 5-HT-immunoreactive neurons complements studies on the distribution of monoamine-containing neurons in the bee brain. Serotonin- and catecholamine-containing neurons often occur together in the same brain areas and subcompartments. The immunohistochemical approach in chemoneuroanatomy gives new evidence for a more complicated architecture of the brain than could be deduced from the classical neuroanatomical studies.     

Patterns of PERIOD and pigment-dispersing hormone immunoreactivity in the brain of the European honeybee (Apis mellifera): age- and time-related plasticity

We explored the neural basis of age- and task-related plasticity in circadian patterns of activity in the honeybee. To identify putative circadian pacemakers in the bee brain, we used antibodies against Drosophila melanogaster and Antheraea pernyi PERIOD and an antiserum to crustacean pigment-dispersing hormone (PDH) known to cross-react with insect pigment-dispersing factors (PDFs). In contrast to previous results from Drosophila, PDH and PER immunoreactivity (-ir) were not colocalized in bee neurons. The most intense PER-ir was cytoplasmic, in two groups of large neurons in the protocerebrum. The number of protocerebral PER-ir neurons and PER-ir intensity within individual cells were highest in brains collected during subjective night and higher in old bees than in young bees. These results are consistent with previous analyses of brain per mRNA in honeybees. Nuclear PER-ir was found throughout the brain, including the optic and antennal lobes. A single group of PDH-ir neurons (approximately 20/optic lobe) was consistently and intensely labeled at the medial margin of the medulla, independent of age or time of day. The processes of these neurons extended to specific neuropils in the protocerebrum and the optic lobes but not to the deutocerebrum. The patterns displayed by PER- and PDH-ir do not completely match any patterns previously described. This suggests that, although clock proteins are conserved across insect groups, there is no universal pattern of coexpression that allows ready identification of pacemaker neurons within the insect brain.     

Synaptic connections between pigment-dispersing factor-immunoreactive neurons and neurons in the pars lateralis of the blow fly Protophormia terraenovae

In females of the blow fly Protophormia terraenovae, neurons with cell bodies in the pars lateralis (PL) projecting to the retrocerebral complex (designated as PL neurons) are necessary for the induction of reproductive diapause under short-day and low-temperature conditions. In the present study, neural connections between PL neurons and pigment-dispersing factor (PDF)-immunoreactive neurons were examined via immunolight microscopy and immunoelectron microscopy combined with backfills through the cardiac-recurrent nerve. Immunolight microscopy showed that fibers of PL neurons overlapped with PDF-immunoreactive fibers in the dorsolateral region of the superior protocerebral neuropil. Immunoelectron microscopy showed that PDF-immunoreactive fibers formed output synapses with fibers of PL neurons and unlabeled neurons in a region dorsoanteriorly located with respect to the calyx of the mushroom body. The distribution of synaptic connections between PDF-immunoreactive fibers and the fibers of PL neurons was sparse. According to the projection patterns, PDF-immunoreactive fibers with synaptic connections with PL neurons appeared to originate from PDF-immunoreactive neurons with cell bodies at the base of the medulla of the optic lobe (medulla PDF neurons), which are putative circadian clock neurons in P. terraenovae. PDF immunoreactivity was restrictively detected in dense-core vesicles but not in clear synaptic vesicles. The present results suggest that medulla PDF neurons convey time or photoperiodic information to PL neurons for diapause induction through direct synaptic connections.     

Novel insect orcokinins: characterization and neuronal distribution in the brains of selected dicondylian insects

Orcokinins are a family of myotropic neuropeptides identified in various decapod crustaceans and recently in a cockroach. Their presence in the crustacean nervous system and hemolymph suggests that they act as hormones and as locally acting neuromodulators. To provide further evidence for the existence of orcokinins in insects, we identified a novel orcokinin-related peptide in the locust Schistocerca gregaria and used an antiserum against Asn13-orcokinin for immunostaining in the brains of selected dicondylian insects, including a silverfish, three polyneopteran species (a cockroach and two locusts), and three endopterygote species (a moth, a bee, and a fly). As analyzed by MALDI-TOF spectrometry and nanoelectrospray Q-TOF, the locust orcokinin is a novel tetradecapeptide with striking sequence similarity to crustacean orcokinins. Orcokinin immunostaining was widespread and occurred in similar patterns in the brain of the silverfish and the polyneopteran species. Prominent immunostaining was detected in the optic lobe, especially in the medulla and in the accessory medulla, in local interneurons of the antennal lobe, and in extrinsic and intrinsic mushroom-body neurons. All parts of the central complex and many other areas of the brains were densely stained. In the silverfish, the cockroach, and the locusts, processes in the corpora cardiaca showed orcokinin immunoreactivity, suggesting that orcokinins also serve a hormonal role. In contrast to the case in polyneopteran species, immunostaining was completely lacking in the brains of the honeybee, fruitfly, and sphinx moth. This indicates that orcokinins either are modified considerably or may be completely absent in the brains of endopterygote insects.     

Organization and neural connections of the anterior optic tubercle in the brain of the locust,Schistocerca gregaria

The anterior optic tubercle is a small neuropil in the insect brain and a major target of visual interneurons from the optic lobe. The functional role of the tubercle is poorly understood, but recent evidence from locusts points to a possible involvement in polarization vision. The present study examines the organization of the anterior optic tubercle in the locust Schistocerca gregaria and its connections with other brain areas. The tubercle of the locust consists of an upper and a lower subunit. Both units are connected in parallel with the medulla and lobula of the optic lobe, with the contralateral tubercle, and with the lateral accessory lobe in the median protocerebrum. Wide-field transmedullary neurons provide input from the medulla. Neurons with processes in the dorsal rim of the medulla, a relay station in the polarization vision pathway, project exclusively to the lower unit of the tubercle. Visual input from the lobula to the upper and lower unit originates from topographically distinct strata. The most prominent output target of the tubercle is the lateral accessory lobe in the median protocerebrum. Neurons from the upper unit project widely in the lateral accessory lobe, whereas neurons from the lower unit have focused projections confined to the median olive and to the lateral triangle. The two subunits of the anterior optic tubercle are, therefore, processing stages in two parallel visual pathways from the optic lobe to the median protocerebrum. Pathways via the lower unit of the tubercle appear to be involved in polarization vision.     

Pigment-dispersing hormone-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and of several mutants with altered circadian rhythmicity

Antisera against the crustacean pigment-dispersing hormone (β-PDH) were used in immunocytochemical preparations to investigate the anatomy of PDH-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and in that of several brain mutants of this species, some of which express altered circadian rhythmicity. In the wild-type and in all rhythmic mutants (small optic lobes, sine oculis, small optic lobes;sine oculis), eight cell bodies at the anterior base of the medulla (PDFMe neurons) exhibit intense PDH-like immunoreactivity. Four of the eight somata are large and four are smaller. The four large PDFMe neurons have wide tangential arborizations in the medulla and send axons via the posterior optic tract to the contralateral medulla. Fibers from the four small PDFMe neurons ramify in the median protocerebrum dorsal to the calyces of the mushroom bodies. Their terminals are adjacent to other PDH-immunoreactive somata (PDFCa neurons) which send axons via the median bundle into the tritocerebrum. The results suggest a possible involvement of the PDFMe neurons in the circadian pacemaking system of Drosophila. The location and size of the PDFMe neurons are identical with those of neurons containing the period protein which is essential for circadian rhythmicity. Changes in the arborizations of the PDFMe neurons in small optic lobes; sine oculis mutants are suited to explain the splitting in the locomotor rhythm of these flies. In the arrhythmic mutant, disconnected, the PDFMe neurons are absent. The arrhythmic mutant per°, however, shows normal PDH immunoreactivity and therefore, does not prevent the expression of PDH-like peptides in these neurons.© 1993 Wiley-Liss, Inc.     

Histamine-immunoreactive neurons in the midbrain and suboesophageal ganglion of sphinx moth Manduca sexta

This paper describes the distribution of histamine-like immunoreactivity in the midbrain and suboesophageal ganglion of the sphinx moth Manduca sexta. Intense immunocytochemical staining was detected in ten bilateral pairs of neurons in the median protocerebrum and in one pair of neurons in the suboesophageal ganglion. Whereas most areas of the brain and suboesophageal ganglion are innervated by one or more of these neurons, typically no immunoreactive fibers were found in the mushroom bodies, the protocerebral bridge, and the lateral horn of the protocerebrum. The 11 histamine-immunoreactive neurons were reconstructed from serial sections. Ten neurons have bilateral arborizations, often with axonal projections in symmetric areas of both hemispheres. One neuron, whose soma resides in the lateral protocerebrum, has only unilateral projections. Of the 11 neurons, 6 occur in pairs with similar morphological features. In addition to these neurons, weak histamine-like immunoreactivity was detected in 7-13 interneurons that were not reconstructed individually. The central projections of the ocellar nerves from the intracranial ocelli also exhibit histamine-like immunoreactivity. The single-cell reconstructions reveal similarities between the organization of histamine- and serotonin-immunoreactive neurons in the brain and suboesophageal ganglion of this insect.     

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.