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.     

GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria

Summary Neurones in the suboesophageal ganglion of the locust Schistocerca gregaria were stained with an antiserum raised against gamma amino butyric acid (GABA). This ganglion consists of the fused mandibular, maxillary and labial neuromeres. Immunoreactive cell bodies of similar size and distribution occur in the lateral, ventral and middorsal regions of all three neuromeres. Approximately 200 cell bodies stain in both the mandibular and maxillary neuromeres and 270 in the labial neuromere. A few distinctly larger cells occur in the ventral groups and one large pair occurs in the lateral group of the maxillary neuromere. Dorsal commissures DCIV and DCV are composed mainly of stained fibres, while DCI–DCIII are largely unstained. A ventral commissure also stains in the maxillary neuromere. All longitudinal tracts contain both stained and unstained fibres. Many processes within the neuropil are also immunoreactive. A stained axon is found in the posterior tritocerebral commissure which enters the anterior dorsal region of the mandibular neuromere. The salivary branch of the 7th nerve contains one stained axon and two axons stain in nerve 8 which innervates neck muscles.     

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.     

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.     

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     

Crustacean cardioactive peptide-immunoreactive neurons innervating brain neuropils,retrocerebral complex and stomatogastric nervous system of the locust,Locusta migratoria

The distribution and morphology of crustacean cardioactive peptide-immunoreactive neurons in the brain of the locust Locusta migratoria has been determined. Of more than 500 immunoreactive neurons in total, about 380 are interneurons in the optic lobes. These neurons invade several layers of the medulla and distal parts of the lobula. In addition, a small group of neurons projects into the accessory medulla, the lamina, and to several areas in the median protocerebrum. In the midbrain, 12 groups or individual neurons have been reconstructed. Four groups innervate areas of the superior lateral and ventral lateral protocerebrum and the lateral horn. Two cell groups have bilateral arborizations anterior and posterior to the central body or in the superior median protocerebrum. Ramifications in subunits of the central body and in the lateral and the median accessory lobes arise from four additional cell groups. Two local interneurons innervate the antennal lobe. A tritocerebral cell projects contralaterally into the frontal ganglion and appears to give rise to fibers in the recurrent nerve, and in the hypocerebral and ingluvial ganglia. Varicose fibers in the nervi corporis cardiaci III and the corpora cardiaca, and terminals on pharyngeal dilator muscles arise from two subesophageal neurons. Some of the locust neurons closely resemble immunopositive neurons in a beetle and a moth. Our results suggest that the peptide may be (1) a modulatory substance produced by many brain interneurons, and (2) a neurohormone released from subesophageal neurosecretory cells.     

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.     

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.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

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.     

Precocene-induced necrosis and haemocyte-mediated breakdown of corpora allata in nymphs of the locust Locusta migratoria

Light and electron microscopic studies, including the use of the vital dyes trypan blue and acridine orange, indicate that the topical application of precocene II rapidly triggers degenerative processes in the corpora allata (CA) leading within a few days to the virtual disappearance of the parenchymal cells of these glands. The following sequence of events was observed: 1) Within 2 h, many of the cells in fixed nymphal specimens showed increased electron density. During the next few hours, they decreased considerably in volume (shrinkage necrosis). Intercellular spaces increased simultaneously. Although a variable number of cells remained electron lucent, they showed nuclear and cytoplasmic degeneration later on (coagulative necrosis). 2) Haemocytes arrived at the CA sheath and invaded the CA in large numbers after 12 h. 3) CA cells became increasingly necrotic, and cell fragments were budded off and were phagocytosed most noticeably after 1 to 3 days. Thereafter no CA parenchyma remained. 4) Haemocytes dispersed.     

Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusts migratoria) at rest and during flight

Summary Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoprotein epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.     

The development of GABA-like immunoreactivity in the thoracic ganglia of the locust Schistocerca gregaria

Summary The development of GABA-like immunoreactivity was investigated in embryonic and juvenile locusts using an antibody raised against GABA-protein conjugates. GABA-like immunoreactivity was first detectable in the neuropile of embryonic ganglia at 55% development, and in neuronal somata at 62% development. The total number of immunoreactive somata increased between 62% and 85% embryonic development, and followed an anterio-posterior pattern of expression. At 85% development, the number of immunoreactive somata reached adult levels and no change in number was then seen. In embryonic stages and first and second juvenile instars two dorsal and four ventral groups of somata were labeled in all three thoracic ganglia, whilst in later juvenile instars one of the dorsal groups was visible as a separate entity only in the metathoracic ganglion. These early patterns were modified by alterations in the positions of some of the groups during late embryogenesis and during juvenile development to produce the adult pattern. The results show that the development of GABA expression is similar to that of other neurotransmitters. The characteristics of the development of immunoreactivity indicate that some of these immunoreactive clusters may be derived from clonally related neurones. Finally, we demonstrate the presence of immunoreactive somata and processes in embryos, which correspond to those of identified local and intersegmental interneurones studied in the adult.Abbreviations Ab1–3 first-third abdominal ganglion - CON connective - CI _1–3 common inhibitors 1–3 - CTC tract - DC I–VII dorsal commissures I–VII - DIT dorsal intermediate tract - DMT dorsal median tract - LDT lateral dorsal tract - LF lateral fibres - o, iLVT outer and inner lateral ventral tract - MVT median ventral tract - N1–5 nerves 1–5 - aPT anterior perpendicular tract - PT perpendicular tract - aRT anterior ring tract - R1–5 nerve roots 1–5 - PVC posterior ventral commissure - SMC supra-median commissure - T3 metathoracic neuromere - TT T tract - aVAC anterior ventral association centre - VC I ventral commissure I - d,vVCII dorsal and ventral parts of ventral commissure II - VF ventral fibres - VIT ventral intermediate tract - VLT ventral lateral tract - VMT ventral median tract - (d,v)LAG (dorsal and ventral) lateral anterior group - LDG lateral dorsal group - LVG lateral ventral group - MDG medial dorsal group - MPG medial posterior group - MVG medial ventral group     

GABA and glutamate-like immunoreactivity at synapses received by dorsal unpaired median neurones in the abdominal nerve cord of the locust

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.     

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.     

Correlation of electrophysiological,histochemical, and mechanical properties in fibres of the coxa rotator muscle of the locust,Locusta migratoria

Summary The fibre composition of the anterior coxa rotator muscle of the locust middle leg (M92) was examined. The muscle is composed of 90–100 fibres. Muscle fibres were characterized with regard to innervation pattern, electrophysiological properties, and morphological parameters. Activity and isoenzyme composition of myofibrillar ATPase, succinic acid dehydrogenase (SDH) activity and glycogen content were examined employing histochemical techniques. Shortening velocity and the dependence of tension on intracellular Ca²⁺ were determined in skinned fibre experiments. A close match was observed between the innervation pattern of the muscle fibres and their histochemical and physiological properties. The combination of all parameters examined allowed an accurate classification of the muscle fibres into three types. Within a given type, broad variability of some properties was observed (SDH activity, Ca²⁺ sensitivity) while others assumed distinct values (innervation pattern, shortening velocity). The comprehensive characterization of muscle fibre properties permits a functional interpretation of fibre heterogeneity with regard to muscle performance. Fibres with the same innervation pattern may be recruited specifically, according to their electric properties and Ca²⁺ sensitivities. The resulting specific recruitment of fibres with different mechanical responses should allow a subtle control of muscular force, with regard to force amplitude, temporal characteristics of contraction, and metabolic cost.Abbreviations CI₁ common inhibitory neurone one - ejp ijp excitatory, inhibitory junctional potential - EGTA ethylene glycol-bis[-aminoethyl ether] N,N,N,N-tetraacetic acid - mATPase myofibrillar adenosinetriphosphatase - MOPSO 3-[N-morpholino]-2-hydroxypropanesulfonic acid - M92 anterior rotator muscle of the coxa - n Hill coefficient - pCa₅₀ pCa corresponding to half-maximal tension - P₀ maximal isometric tension - SDH succinic acid dehydrogenase - V _max maximal shortening velocity     

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.     

An identified dorsal unpaired median neurone and bilaterally projecting neurones exhibiting bovine pancreactic polypeptide-like/FMRFamide-like immunoreactivity in abdominal ganglia of the migratory locust

Summary Three antisera were used to study the distribution and anatomy of bovine pancreatic polypeptide (BPP)-like/FMRFamide-like immunoreactive neurones within the unfused abdominal ganglia of the migratory locust, Locusta migratoria. All the antisera used stained two or more clusters of perikarya, localized anteriorly and posteriorly near the midline within each unfused abdominal ganglion. Double labelling experiments with intracellular dye injection, or differential backfilling, combined with subsequent immunostaining were carried out to identify these neurones. Two of the antisera (antisera 1 and 2, both raised against FMRFamide) stained three groups of midline neurones, located anterior dorsal, anterior ventral and posterior dorsal within the ganglion. Neurones of the former of these two clusters projected via the anterior median nerve to a neurohaemal organ. The posterior cluster of midline cells comprised immunopositive perikarya all but one of which also projected via the anterior median nerve to innervate the neurohaemal organ. Double labelling with Lucifer yellow and antisera 1 and 2 showed that the remaining neurone was the previously identified doral unpaired median (DUM)heart1 neurone. The third antiserum (AK141), also raised against FMRFamide, stained neurones within an anterior dorsal cluster, and in a posterior cluster. Double labelling with differential Co²⁺/Ni²⁺-backfilling and the antiserum 3 (AK141) demonstrated that the large neurones of both clusters belonged to the population of bilaterally projecting neurones (BPNs), including the DUMheart1 neurone. Since the antisera cross-react with BPP and fail to label neurones when preadsorped with BPP or FMRFamide, we conclude that the labelled neurones contain polypeptides of the FMRFamide/BPP-family.     

Immunochemical analysis of the distribution of the new ovary maturating neurohormone during development of the African locust,Locusta migratoria

Summary Following our prior identification of a gonadotropic neurohormone isolated from the neurosecretory lobe of the corpora cardiaca of the African locust, we have raised a polyclonal antiserum against this new molecule. In the present paper, we characterize this antiserum using enzyme-linked immunosorbent assay and Western blotting. The latter procedure reveals that the immune serum specifically recognizes the neurohormone, which we have termed ovary maturating parsin. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blotting were used to analyze the distribution of this gonadotropic neurohormone throughout the central nervous system during development. It is produced only by the type-B neurosecretory cells of the pars intercerebralis-corpora cardiaca system and is present both in males and females throughout life from embryo to adult. This permanent expression suggests that the neurohormone may have functions other than its primary direct gonadotropic role in females.