The innervation of the corpus cardiacum of Locusta migratoria: A neuroanatomical study with the use of Lucifer yellow

Summary Neural connections of the corpus cardiacum (CC) in the African locust, Locusta migratoria, were labelled with the fluorescent tracer Lucifer yellow. (1) Unilateral anterograde labelling of the nervus corporis cardiaci I revealed fluorescent fibres in the storage lobe of the CC (CCS). Some fluorescent fibres in the CCS closely approached the ipsilateral border of the glandular lobes of the CC (CCG). Fluorescent fibres also projected into the neuropile of the hypocerebral ganglion via the ipsilateral nervi cardiostomatogastrici I and II, and from there into the oesophageal nerves. (2) Unilateral anterograde labelling of the nervus corporis cardiaci II revealed fluorescent fibres in the CCS and in the ipsilateral CCG. Fluorescent fibres also projected via the ipsilateral nervus corporis allati I into the corpus allatum. (3) Unilateral retrograde labelling of the nervus corporis allati I revealed a distinct fluorescent nerve tract that runs through the CCS and into the nervus corporis cardiaci II. The tract arises from about eight cell bodies in the brain at the rostroventral side of the ipsilateral calyx of the mushroom body. (4) Labelling of the recurrent nerve revealed fluorescent fibres and some fluorescent cell bodies in the hypocerebral ganglion and, via the nervi cardiostomatogastrici I and II, also in the CCS. Fluorescent fibres were also present in the oesophageal nerves.     

Serotonin-immunoreactive neurones in the brain of Locusta migratoria innervating the corpus cardiacum

Summary The serotoninergic innervation of the corpus cardiacum (CC) of Locusta migratoria was investigated using two antisera against serotonin. A dense network of immunoreactive nerve fibres was present in the storage lobe of the CC. Immunopositive fibres only sporadically crossed the border between the storage lobe and the glandular lobe of the CC. Immunopositive fibres entered the storage lobe of the CC via the nervus corporis cardiaci I (NCCI); NCCII was immunonegative. Unilateral retrograde fillings of the NCCI with the fluorescent tracer Lucifer yellow, followed by antiserotonin immunocytochemistry, revealed about 20 double-labelled neurones in the anterior part of the pars intercerebralis. The double-labelled neurones were scattered between fluorescent non-immunoreactive neurones. Additionally, 5–7 neurones labelled only with Lucifer yellow were found at the ventrolateral side of the tritocerebrum. No immunopositive neurones were observed in the hypocerebral ganglion. Immunopositive fibres from neurones in the frontal ganglion ran via the recurrent nerve and the neuropile of the hypocerebral ganglion into the paired oesophageal nerve. At most, a few immunopositive nerve fibres occurred in the cardiostomatogastric nerves II, which connect the storage lobe of the CC with the paired oesophageal nerve at the caudal end of the hypocerebral ganglion.     

Co-localization of the adipokinetic hormones I and II in the same glandular cells and in the same secretory granules of corpus cardiacum of Locusta migratoria and Schistocerca gregaria

Summary The immunocytochemical reactivity of the glandular cells of the corpus cardiacum (CCG-cells) of Locusta migratoria and Schistocerca gregaria was investigated at the electron-microscopic level, using the protein A-gold method, with three antisera against fragments of the adipokinetic hormones AKH I and AKH II. This combination of antisera permitted discrimination between anti-AKH I and anti-AKH II immunoreactivity. Fixation in a mixture of 2% glutaraldehyde and 2% formaldehyde, in combination with low-temperature embedding in Lowicryl K₄M, produced the highest and most consistent selective immunogold labelling of the secretory and ergastoplasmic granules. All secretory granules in all CCG-cells investigated possessed a distinct anti-AKH I-immunopositive reaction, whereas most secretory granules showed a weaker anti-AKH II immunoreaction. Ergastoplasmic granules reacted similar to the secretory granules. The average immunolabelling of the secretory granules was higher in the processes than in the cell bodies of the CCG-cells. The results in Schistocerca gregaria were essentially similar to those in Locusta migratoria. It is concluded that (i) the individual CCG-cells synthesize AKH I as well as AKH II; (ii) these hormones coexist in the same ergastoplasmic and secretory granules; and (iii) these granules contain a higher content of AKH I than AKH II.     

Ageing adipokinetic cells in Locusta migratoria: an ultrastructural morphometric study

Summary A morphometric study was made of the ultrastructure of adipokinetic cells in resting adults of Locusta migratoria at 3, 23, and 43 days after imaginal ecdysis. The nucleus, rough endoplasmic reticulum, and Golgi apparatus enlarge with age, which indicates that the synthesis and packaging of secretory substances increases during ageing. The size of the storage compartment, consisting of secretory and ergastoplasmic granules, does not increase earlier than 23–43 days after imaginal ecdysis. The lysosomal compartment markedly enlarges between 3 and 23 days; later on, the growth of this compartment, especially of autophagosomes, is less prominent. This suggests that lysosomal destruction initially compensates for the production of new secretory granules, assuming that exocytosis of secretory granules by adipokinetic cells is insignificant in resting locusts. Afterwards, lysosomal destruction may no longer be sufficient to prevent over-production of secretory granules, as is suggested by the increase in the number of these granules between 23 and 43 days. This coincides with the appearance of a considerable number of large ergastoplasmic granules, which represent a spatially more efficient form of storage of secretory material than the much smaller secretory granules. The increase with age in the amount of secretory products indicates that the biosynthetic activity of the adipokinetic cells is not (finely) tuned to their releasing activity.     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

Morphology of neurones in the storage part of the corpus cardiacum of Locusta migratoria: no evidence for their involvement in the regulation of adipokinetic cell activity

The storage part of the corpus cardiacum of Locusta migratoria consists of two compartments: a neural part on the haemocoelic side containing neuronal cell bodies that are protected by a blood-brain barrier, and a neurohaemal part adjacent to the aorta. Intracellular filling of the neurones in the neural part with Lucifer yellow followed by confocal laser scanning microscopy has revealed that these neurones can be divided into several classes. None of the neurones has processes extending into the glandular part of the corpus cardiacum. They are, therefore, not directly involved in the regulation of adipokinetic cell activity.     

Effect of azadirachtin A on neuroendocrine activity in Locusta migratoria

Summary The neurosecretory activity of azadirachtin A-treated Locusta migratoria that failed to mature was compared with that of rapidly maturing control females by means of histological techniques. High resolution drymount autoradiography was used to localize [22,23-³H₂]dihydroazadirachtin A in the brain and corpus cardiacum (CC). The neurosecretory system of the locusts treated with this insecticide was accompanied by an unusually high accumulation of paraldehyde fuchsin (PAF)-stainable neurosecretory material (NSM) in the brain fibers and in the storage lobes of the CC. The control females never had such an intense accumulation of NSM. The accumulation of NSM, resulting from its poor release, may influence its synthesis which is controlled by feed-back regulation. [22,23-³H₂]Dihydroazadirachtin A is localized mainly at the peripheral blood-brain barrier and does not penetrate into the brain effectively, whereas it completely covers the CC. It is concluded that azadirachtin A affects the endocrine activity of the brain in an indirect way.     

Ultrastructural and immunocytochemical studies of neuromuscular junctions in oviduct of Locusta migratoria

The ultrastructure of nerve endings in the oviduct visceral muscles of Locusta migratoria was studied by electron microscopy and by immunogold labeling for two kinds of neuromodulators, the pentapeptide proctolin and FMRFamide-related peptides. Nerve endings contained electron-lucent round vesicles and two kinds of granules (round and avoid), and formed two types of synapses or release sites with the muscle. The morphologically distinct nerve endings were classified into three different categories based on the composition of synaptic vesicles and granules. Type-I nerve endings were dominated by electron-lucent round vesicles and contained only a few round electron-dense granules. Type-II nerve endings contained mostly electron-dense round granules and electron-lucent round vesicles. A few electron-dense ovoid granules were also present. Electron-dense ovoid granules dominated the type-III nerve endings, which usually contained less electron-lucent vesicles than either type-I or II nerve endings. Both proctolin and FMRFamide-like immunoreactivity was associated with electron-dense round granules. However, FMRFamide-like immunoreactivity was only found in the type-II nerve endings, while proctolin immunoreactivity was found within type-I nerve endings as well as in some type-II nerve endings. Immunological results therefore allow us to further divide type-II nerve endings into type-IIa (immunonegative for proctolin) and type-IIb (immunopositive for proctolin). Type-III nerve endings show no immunolabeling to either proctolin or FMRFamide.     

GABA-like immunoreactivity of identified interneurons in the flight system of the locust,Locusta migratoria

Summary The transmitter content of identified inhibitory interneurons in the flight system of the locust, Locusta migratoria, has been characterized using antibodies raised against protein-conjugated gamma aminobutyric acid. Identified flight neurons were filled with the fluorescent dye, Lucifer Yellow. Serial sections of dye-filled neurons were incubated with an antibody to gamma aminobutyric acid which was subsequently tagged with a fluorescent marker. Excitatory motoneurons to wing muscles and 13 flight interneurons (3 excitatory, 7 inhibitory, and 3 with unknown synaptic effect) were examined. Neither the moto-neurons nor any of the 3 excitatory interneurons contained immunoreactive material. Six of the 7 inhibitory interneurons did contain immunoreactive material. All the neurons which contained immunoreactive material and whose synaptic effect is known were inhibitory. We conclude that most of the inhibitory flight interneurons which have been described use gamma aminobutyric acid as their transmitter. Interestingly, at least 1 set of interneurons known to be inhibitory does not use gamma aminobutyric acid. We predict that the 2 interneurons which do contain immunoreactive material and whose synaptic effect is not yet known will be found to have inhibitory roles in the operation of the flight circuitry.     

Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria,Locusta migratoria and Neobellieria bullata

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active protion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.     

Identification of a secretomotor centre in the brain of Locusta migratoria,controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum

Summary After retrograde filling of axons terminating in the glandular lobe of the corpus cardiacum (CC) of Locusta migratoria with cobalt chloride, a paired group of about 15 cobalt containing cells was demonstrated in the lateral area of the protocerebrum. The axons of these cells run via the NCC II into the glandular lobe of the CC. These small neurons have the characteristics of secretory cells; they contain secretory granules of about 1000 Å in diameter. The axon terminals in the glandular lobe, making synaptic contacts with the glandular cells, contain secretory granules of the same size. It is therefore concluded that the cell groups in the protocerebrum control the activity of the glandular cells which produce an adipokinetic hormone. Arborizations of fibers of the lateral secretomotor cells are present in the dorsal neuropile of the protocerebrum, ventral of the mushroom bodies and along the tracts of the NCC I within the brain. It is proposed that these arborizations are sites of synaptic input. It is discussed that the axons of these cells might receive additional synaptic input in the storage lobe of the CC.The localization of cell bodies, the axons of which enter the storage part of the CC is described. The course of the axon tracts of the various cell groups in the protocerebrum and their connections with the NCC I and NCC II are demonstrated.Supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The electron microscopical investigations were performed at the EM-unit of the Faculty of Biology, State University of Utrecht (Director: Prof. Dr. J.C. van de Kamer)The author is greatly indebted to Dr. A.M.Th. Beenakkers and Dr. H.H. Boer for their active interest and helpful advise. Thanks are also due to Mr. H. van Kooten and his staff for making the macro- and microphotographs, to Mr. L.W. van Veenendaal for preparing the electron micrographs and final assistance in the preparation of the photo pages and to Mr. D. Smit, who made the drawings     

Neuronal connections between central and enteric nervous system in the locust, Locusta migratoria

The number and location of neurons, in the central nervous system, that project into the frontal connective was studied in the locust by using retrograde neurobiotin staining. Staining one frontal connective revealed some 70 neurons in the brain. Most of these were located within both tritocerebral lobes. Additional groups of neurons were located within the deutocerebrum and protocerebrum. Some 60 neurons were labelled in the suboesophageal ganglion. These formed nine discernable populations. In addition, two neurons were located in the prothoracic ganglion and two neurons in the first abdominal neuromere of the metathoracic ganglion. Thus, some 250 neurons located within the head ganglia, and even neurons in thoracic ganglia, project into the ganglia of the enteric nervous system. This indicates that the coordination between the central and enteric ganglia is much more complex than previously thought. With the exception of some previously described dorsal unpaired median neurons and a few motor neurons in the head ganglia, the identity and function of most of these neurons is as yet unknown. Possible functions of the neurons in the thoracic ganglia are discussed.     

Crustacean cardioactive peptide in the nervous system of the locust,Locusta migratoria: an immunocytochemical study on the ventral nerve cord and peripheral innervation

Summary Crustacean cardioactive peptide-immunoreactive neurons occur in the entire central nervous system of Locusta migratoria. The present paper focuses on mapping studies in the ventral nerve cord and on peripheral projection sites. Two types of contralaterally projecting neurons occur in all neuromers from the subesophageal to the seventh abdominal ganglia. One type forms terminals at the surface of the thoracic nerves 6 and 1, the distal perisympathetic organs, the lateral heart nerves, and on ventral and dorsal diaphragm muscles. Two large neurons in the anterior part and several neurons of a different type in the posterior part of the terminal ganglion project into the last tergal nerves. In the abdominal neuromers 1–7, two types of ipsilaterally projecting neurons occur, one of which gives rise to neurosecretory terminals in the distal perisympathetic organs, in peripheral areas of the transverse, stigmata and lateral heart nerves. Four subesophageal neurons have putative terminals in the neurilemma of the nervus corporis allati II, and in the corpora allata and cardiaca. In addition, several immunoreactive putative interneurons and other neurons were mapped in the ventral nerve cord. A new in situ whole-mount technique was essential for elucidation of the peripheral pathways and targets of the identified neurons, which suggest a role of the peptide in the control of heartbeat, abdominal ventilatory and visceral muscle activity.Abbreviations AG abdominal ganglia - AM alary muscle - AMN alary muscle nerve - CA corpus allatum - CC corpus cardiacum - dPSO distal perisympathetic organ - LHN lateral heart nerve - LT CCAP-immunoreactive lateral tract - NCA nervus corporis allati - NCC nervus corporis cardiaci - NM neuromer - PMN paramedian nerve - PSO perisympathetic organ - SOG subesophageal ganglion - VDM ventral diaphragm muscles - VNC ventral nerve cord     

Immunocytochemical localization of Bombyx-PTTH-like molecules in neurosecretory cells of the brain of the migratory locust,Locusta migratoria

Summary Using a monoclonal antibody directed against a synthetic pentadecapeptide corresponding to the N-terminus of the prothoracicotropic hormone (PTTH) of Bombyx mori, we report the presence of immunoreactive molecules in a large number of median neurosecretory cells of the pars intercerebralis of the migratory locust, Locusta migratoria. These cells correspond to the A₁ cell type which we show to contain also neuroparsins, a family of predominant neurohormones of the migratory locust. In contrast, PTTH-like molecules are absent from A₂ cells of the pars intercerebralis which contain Locusta insulin-related peptide (LIRP). Developmental studies show the presence of PTTH-related substances in neurosecretory cells of Locusta migratoria from late embryogenesis to adult development, including ageing vitellogenic female adults.     

Ultrastructural differentiations in the developing follicle cortex of Locusta migratoria,with special reference to vitelline membrane formation

Summary Electron microscopic studies on developing follicles of Locusta migratoria show the vitelline membrane to be composed of two ultrastructurally distinguishable components: The vitelline membrane bodies (VMBs) and, in addition, fine granular material, cementing the VMBs together. VMBs form first in the oocyte-near zone within the oocyte-follicle cell space. Subsequently, the second vitelline membrane substance is secreted between the VMBs through apical protrusions of the follicle cells. The possible origin of the VMBs is discussed.Yolk uptake in Locusta seems to occur predominantly by pinocytosis. During oocyte development the oocyte membrane is enlarged by numerous microvilli and folds. In addition pinocytotic vesicles are pinched off. It is supposed that the latter loose their coat and eventually transform into large proteid yolk spheres.This work was supported by the Volkswagenstiftung, HannoverI wish to thank Prof. Dr. H. Emmerich, Techn. Hochschule Darmstadt, for valuable discussions     

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the rans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horseradish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.     

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus

Summary The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160–350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160–350 nm in diameter. Synaptoid configurations with 20–50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.This work was done while JTB was supported by USPHS Training Grant HD-0266 from NICHDI wish to express my thanks to Dr. Richard A. Cloney for sharing his expertise in electron microscopy     

Localization of Lom-AG-myotropin I-like substances in the male reproductive and nervous tissue of the locust,Locusta migratoria

Summary Lom-AG myotropin I (Lom-AG-MTI) was the first peptide to be isolated from the male accessory reproductive glands of the locust, Locust migratoria. It shows no sequence similarity to any of the peptides identified from vertebrate or invertebrate tissues. A polyclonal antiserum was used to localize Lom-AG-MTI-like material in the male reproductive system and nervous system of the locust. Immunoreactivity was found in two of the hyaline gland tubules. In the brain, cell bodies were detected in the proto- and deuterocerebrum as well as the frontal ganglion. Nerve fibers were stained in the neuropils of the brain and throughout the labial nerves into the recurrent nerve. Thoracic and last abdominal ganglia contained neurons which could be stained with Lom-AG-MTI antiserum. The pronounced reactivity in the central nervous system suggests a possible neuroregulatory function of the peptide.