Characterization of a new neurosecretory cell type containing gastrin-cholecystokinin-like peptide in the locust pars intercerebralis: ultrastructural and immunocytochemical studies,in situ and in vitro

A new neurosecretory cell type of the locust pars intercerebralis, immunolabelled with an antiserum against a vertebrate peptide related to gastrin-cholecystokinin (CCK-8(s)), was characterized both in situ and in primary cell cultures. Semithin sections of pars intercerebralis were first immunostained in order to identify neurosecretory cells containing CCK-like material and then examined by electron microscopy. The neurosecretory cells containing CCK-like material were paraldehyde fuchsin negative and were unequivocally identified in ultrathin sections adjacent to immunostained semithin sections. They exhibited neurosecretory vesicles of variable electron density, ranging in diameter from 150 to 250 nm. Immunogold labelled ultrathin sections adjacent to unlabelled ultrathin sections allowed for the unambiguous localization of CCK-like immunoreactive material over the neurosecretory vesicles of the cells containing CCK-like material. Immunoreactivity towards CCK-8(s)-like peptide could also be detected in pars intercerebralis neurosecretory neurons grown in vitro. The CCK-like positive neurons showed a multipolar morphology with fine processes radiating from the cell body. The positive cells had the same ultrastructural characteristics as the in situ CCK-like neurons. The pattern of neurite outgrowth on reactive CCK-like neurosecretory cells in vitro and the neuroanatomical pathway of the CCK-like immunoreactive neurosecretory cells in situ could be correlated. On the basis of their number, size and localization in the locust pars intercerebralis, it is possible that the CCK-like neurosecretory cells correspond to neurosecretory cell type C, which has not, to date, been identified at the ultrastructural level.     

Ovary maturing parsin and diuretic hormone are produced by the same neuroendocrine cells in the migratory locust, Locusta migratoria

In the migratory locust, the CRF-related diuretic hormone that stimulates fluid secretion by the Malpighian tubules, and the ovary maturing parsin, a neurohormone able to stimulate oogenesis, are produced by the same neuroendocrine cells of the pars intercerebralis in the brain.     

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.     

Restricted occurrence of Locusta migratoria ovary maturing parsin in the brain-corpora cardiaca complex of various insect species

Ovary maturing parsin (OMP) is a gonadotrophic molecule previously isolated from the neurosecretory lobes of the corpora cardiaca of Locusta migratoria (acridian Orthoptera). A polyclonal antiserum directed against the two biologically active domains of the L. migratoria (Lom) OMP was used to investigate the occurrence of Lom OMP-like substances in brain-corpora cardiaca complexes of other insect species. Using immunohistochemistry, specimens of 40 different insect species belonging to 13 insect orders were tested. The Lom OMP-like substance was strictly limited to specimens of insect species belonging to the Acridae. It occurred in non-basophilic cells of the pars intercerebralis that project to the corpora cardiaca, as in Locusta. Although the antiserum only detected Lom OMP-like material in the Acridae, it is possible that related molecules exist in other insects. The antiserum may be very specific for domains of the Lom OMP molecule that have not been highly conserved during evolution or possibly these domains are not accessible to the antiserum in other insects.     

A comparative study of leucokinin-immunoreactive neurons in insects

Antisera were raised against leucokinin IV, a member of the leucokinin peptide family. Immunohistochemical localization of leucokinin immunoreactivity in the brain of the cockroach Nauphoeta cinerea revealed neurosecretory cells in the pars intercerebralis and pars lateralis, several bilateral pairs of interneurons in the protocerebrum, and a group of interneurons in the optic lobe. Several immunoreactive interneurons were found in the thoracic ganglia, while the abdominal ganglia contained prominent immunoreactive neurosecretory cells, which projected to the lateral cardiac nerve. The presence of leucokinins in the abdominal nerve cord was confirmed by HPLC combined with ELISA. Leucokinin-immunoreactive neurosecretory cells were also found in the pars intercerebralis of the cricket Acheta domesticus and the mosquito Aedes aegypti, but not in the locust Schistocerca americana or the honey bee Apis mellifera. However, all these species have leucokinin-immunoreactive neurosecretory cells in the abdominal ganglia. The neurohemal organs innervated by abdominal leucokinin-immunoreactive cells were different in each species.     

Système endocrine et physiologie de la diapause imaginale chez le criquet égyptien, Anacridium aegyptium

The Egyptian locust, Anacridium aegyptium, has four protocerebral neurosecretory centres: the A to B neurosecretory cells of the pars intercerebralis (the A cells are rich in fuchsinophil material and the B cells are devoid of fuchsinophil neurosecretion), the voluminous C neurosecretory cells poor in neurosecretion, and the median sub-ocellar neurosecretory cells.From September to the beginning of January, imaginal diapause is characterized by an accumulation of the median neurosecretion in the pars intercerebralis-corpora cardiaca system, by small corpora allata, and, in the female, by a stop in oöcyte development although the male's sexual activity is still not altered. Allatectomy suppresses neither the male's sexual behaviour nor its fecundity. From January, the increase of the photoperiod causes a release of the median neurosecretion in both sexes, an increase of the volume of the corpora allata, and breaks ovarian diapause.In autumn, the implantation of the male's or female's corpora allata of Anacridium does not stimulate ovarian growth of diapausing females. On the contrary, the implantation of corpora allata or of pars intercerebralis or of corpora cardiaca of Locusta migratoria migratorioides (locust without diapause) causes ovarian development of the diapausing females of Anacridium. Thus, in the two sexes of the Egyptian locust, the corpora allata are inactive during the female ovarian diapause. The imaginal diapause of Anacridium affects both sexes (stocking of median neurosecretion, arrest of the corpora allata). If diapause does not seem to affect the male's development, it is because its sexual activity is free from the pars intercerebralis and corpora allata.The corpora allata of Anacridium show a sexual dimorphism in the active adult: they are smaller in the male and have more mitosis in the female. An explanation of this dimorphism is advanced.     

Development of blood-cerebrospinal fluid barrier to horseradish peroxidase in the avian choroidal epithelium

Summary Four neurons in the brain of the migratory locust were immunohistologically identified with an anti-met-enkephalin antiserum. The perikarya of two of these cells are located in the center of each of the two groups of lateral protocerebral neurosecretory cells. The fibres coming from these perikarya terminate in numerous immunoreactive ramifications visible at the periphery of both tractus I to the corpora cardiaca, through which pass the neurosecretory products of the pars intercerebralis. The other two cell bodies are located at the bases of the two optic lobes; their fibres enter the posterior part of the protocerebrum and ramify around the root of the nervus corporis cardiaci II, another area through which neurosecretory products pass. The topographic distribution of these met-enkephalin arborizations suggests that these four neurons may act as neuromodulators of the acitivity of the major neurosecretory cells in the brain of this insect.     

Neurons projecting from the brain to the corpora allata in orthopteroid insects: anatomy and physiology

Retrograde and orthograde labeling of neurons projecting to the corpus allatum was performed in locust, grasshopper, cricket, and cockroach species in order to identify brain neurons that may be involved in the regulation of juvenile hormone production. In the acridid grasshopper Gomphocerus rufus L., and the locusts Locusta migratoria (R.&F.) and Schistocerca gregaria Forskal, the corpora allata are innervated by two morphologically distinguishable types of brain neurons. One group of 9–13 neurons (depending on species) with somata in the pars lateralis extend axons via the nervus corporis cardiaci 2 and nervus corporis allati 1 to the ipsilateral corpus allatum, whereas two cells in each pars lateralis have bilateral projections and innervate both glands. No direct connection between the pars intercerebralis and corpus allatum has been found. In contrast, neurons with paired axons innervating both glands are not present in Periplaneta americana (L.) and Gryllus bimaculatus de Geer. Instead, two cells in each pars lateralis project only to the gland contralateral to their somata. Electrophysiological experiments on acridid grasshoppers have confirmed the existence of a direct conduction pathway between the two glands via the paired axons of four cells that have been identified by neuroanatomy. These cells are not spontaneously active under experimental conditions. Ongoing discharges in the left and right nerves are unrelated, suggesting that the corpora allata receive independent neuronal inputs from the brain.     

Ultrastructural Analysis of the Neuroendocrine Apparatus of Oncopeltus fasciatus (Heteroptera)

• 1 In Oncopeltus fasciatus, the A-cells of the pars intercerebralis and their tracts are stainable in situ with the performic acid-victoria blue (PAVB) method. The axons from these cells, after traversing the corpus cardiacum, terminate in the anterior part of the aorta which thus serves as the neurohemal organ.
• 2 Ultrastructurally, four types of secretory neurons are distinguishable in the pars intercerebralis region: pic-I with granules measuring 1000–3000 Å in diameter; pic-II with granules of irregular size and shape, the elongate ones showing mean dimensions of 2400 × 1400 Å; pic-III with less electron-dense granules measuring 1000–2700 Å in diameter; pic-IV, present not only in the pars intercerebralis but also in adjacent regions of the brain, with variable proportions of granules measuring 700–1800 A and dense-cored vesicles measuring 1000–2400 Å.
• 3 The nervi corporis cardiaci contain at least three types of neurosecretory axons, based on granule content, presumably representing pic-I, pic-II and pic-III neurons.
• 4 The wall of the aorta contains endings of at least three distinct types, again representing pic-I, pic-II and pic-III neurons, and thus provides the neurohemal site for these three types of protocerebral neurosecretory cells. Axons of pic-IV neurons appear to enter the cerebral neuropil.
• 5 The corpus cardiacum is composed of two types of parenchymal secretory cells, with electron-dense granules measuring 1300–3000 Å and 1000–2300 Å in diameter, respectively. The corpus cardiacum also contains interstitial cells and some axons of extrinsic origin, with and without granules.
• 6 The corpus allatum may be paired or median, and receives a small number of at least two types of axons. The corpora allata of some reproducing females show a large number of PAVB-stainable inclusions which appear to be modified cytoplasmic organelles, but are definitely not neurosecretory material.
• 7 The hypocerebral ganglion is composed of two types of secretory-appearing neurons and glial cells. The two neuronal types contain secretory granules, 1000–3000 Å and 900–2100 Å in diameter, respectively. Axons of the recurrent nerve also may contain occasional granules.
• 8 In this heteropteran insect, the two principal functions of the corpus cardiacum appear to be spatially separated: the neurohemal function is subserved by the aortic wall, which permits release of material into both the aortic lumen and the hemocoel, and the intrinsic endocrine function is possessed by the parenchymal cells.

     

Experimental evidence for a neuroendocrine control of ecdysone biosynthesis in adult females of Locusta migratoria

The considerable increase in ecdysteroid concentration which occurs in normal Locusta ovaries at the end of each cycle of oöcyte maturation is prevented if the median neurosecretory cells of the pars intercerebralis are cauterized, or if the corpora cardiaca are excised 24 hr before the onset of ecdysone synthesis in normal females. Implantation of additional brain-corpora cardiaca complexes into young vitellogenic females advances the time of ecdysone synthesis by some 12 hr. Oöcyte growth itself is not affected in these different types of experiments.It is inferred from the data of the present study that ecdysone synthesis in the follicle cell epithelium of maturing Locusta ovaries is stimulated by a neurohormone produced in the median neurosecretory cells of the pars intercerebralis and secreted into the blood via the corpora cardiaca.     

Axonal projections within the brain-retrocerebral complex of the cricket,Teleogryllus commodus

Summary The cerebral origins and axonal trajectories of neurons projecting to the retrocerebral complex of the cricket, Teleogryllus commodus, were examined in silver-intensified nickel preparations. Spatially separate groups of somata in the pars intercerebralis (PI) and in the pars lateralis (PL), commonly accepted as neurosecretory loci, were found to give rise to axons which terminate in the nervus corporis allati 2, the corpus allatum, or the corpus cardiacum. Additional findings demonstrated a distinct group of somata from the PI whose axons run in the esophageal nerve (stomatogastric nervous system), nine somata in the subesophageal ganglion with axons projecting into the nervus corporis allati 2, and also a small cluster of tritocerebral perikarya with axons terminating in the corpus cardiacum. Somata residing in the PI and PL were found to be compartmentally organized based upon the retrocerebral destinations of their axons. Possible functional consequences of these results with respect to the insect neurosecretory system are discussed.     

Morphology of identified neurosecretory and non-neurosecretory cells in the cockroach pars intercerebralis

Electrophysiologically identified cells of the cockroach pars intercerebralis (Periplaneta americana) were injected with the dye Lucifer Yellow for morphological examination and with horseradish peroxidase for ultrastructural marking. In addition to this, uninjected cells were also studied to elaborate the findings from the injected material. The two electrophysiologically distinct classes of cells (type I and type II) correspond to two distinct morphological and ultrastructural classes. Type I cells are the medial neurosecretory cells of the pars intercerebralis, which project their axons to the retrocerebral neuro-hemal complex. Their cell bodies have a mean diameter of 17 microns, and they contain neurosecretory granules 200 nm in diameter. Arborizations emanate from the axon in the anterior part of the protocerebral neuropil. The type II cell bodies are larger (38 microns in diameter). Their axons project into the contralateral circumesophageal connective. These cells were usually multipolar, having somatic arborizations in the anterior portocerebral neuropil. The cell bodies contain vesicles 40 nm in diameter, numerous trophospongia, and a multi-layered glial envelope.     

The influence of some chemosterilants and caffeine on the neuroendocrine system of adult male Ephestia kühniella

Injection of caffeine into adult male Ephestia kühniella induced a reduction in the amount of neurosecretory material in the resorcin fuchsin stainable (A-) cells of the pars intercerebralis. Both hemel (hexamethyl melamine) and tepa (tris (1-aziridinyl) phosphine oxide) treatments resulted in an increase in stainable neurosecretory products in the cytoplasm of the A-cells. The increase after hemel treatment was largely a result of increased synthesis of neurosecretory material as indicated by increased nuclear volume and nuclear-cytoplasmic ratio. No changes were induced by hemel in the corpora cardiaca and allata of the adult male E.kühniella. This disruption in neuroendocrine function is believed to be involved in the observed mating aberrations (permanent copulation and sterile matings) by adult males after hemel treatment.     

Rupture de la diapause ovarienne d'Anacridium aegyptium par stimulation électrique des cellules neurosécrétrices médianes de la pars intercerebralis

Oögenesis and the physiological activity of the corpora allata were studied in adult females of the Egyptian locust (Anacridium aegyptium), in ovarian diapause, after electrical stimulation in vivo of the pars intercerebralis. This stimulation provokes (1) a decrease in the quantity of fuchsinophilic material present in the median neurosecretory cell bodies and in the internal cardiac tract, (2) an increase in the physiological activity of the corpora allata (measured by its chromatropic effect on larvae of Locusta), and (3) rupture of the ovarian diapause (advance of maturation of the oöcytes and oviposition by 5 months, and initiation of the ovarian cycle).In the control animals, the same electrical stimulations of various regions of the central nervous system (tritocerebrum, first ganglion of the abdominal cord) have no effect on these phenomena.In allatectomized females, electrical stimulations of the pars intercerebralis are followed by a slight growth of oöcytes, without a deposit of yellow vitellus. The diapause is not broken. Section of the allatocardiac nerves or rupture of the allatocardiac and allato-suboesophageal nervous connexions do not change the physiological state of the corpora allata. In the case of females in which the corpora allata have been disconnected, electrical stimulations of the pars intercerebralis succeed in activating the corpora allata and breaking the ovarian diapause. The aggregate of these results confirms that in locusts the control of the brain over the physiological activity of the corpora allata is above all neuroendocrine.     

Physical characterization and sequence identification of the ovary maturating parsin. A new neurohormone purified from the nervous corpora cardiaca of the African locust (Locusta migratoria migratorioides.

A novel neurohormone, which anticipates ovarian maturation, was recently purified using liquid chromatography from the African locust nervous corpora cardiaca. Both its function and production by the pars intercerebralis of Locusta migratoria lead to its name, the ovary maturating parsin (Lom OMP). In this study, the Lom OMP was physically and chemically characterized. Its multiply charged ion spectrum was interpreted as two peaks of quite equal size having molecular masses of 6923.4 Da (major peak) and 6907.3 Da. The Lom OMP presented no periodic secondary structure according to the far ultraviolet circular dichroism spectrum obtained. It is composed of 65 amino acids and included a high concentration of alanine but is devoid of cysteine, isoleucine, methionine, lysine and threonine. The amino acid sequence indicated only one microheterogeneity, observed at position 26, consisted in the replacement of serine by alanine. The calculated Mr of the two acidic isoforms (calculated pHi = 4.87) were found to be in agreement with mass spectrometry measurements. When compared to the sequence libraries, the Lom OMP, the first insect gonadotropic neurohormone, was revealed as an unique protein.     

Tyrosine hydroxylase in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the nonglomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.     

Hypothalamo-hypophysial Neurosecretory System in Normal and Hypophysectomised Catfish,Clarias Batrachus L

Two neurosecretory centers, nucleus preopticus (NPO) and nucleus lateralis tuberis (NLT), are distinguished in the hypothalamus of Clarias batrachus L. The cell bodies of NPO are grouped into nucleus preopticus magnocellularis (NPM) and nucleus preopticus parvocellularis (NPP). The NLT is recognised into three divisions: pars rostralis, pars medialis and pars ventrolateralis. The neurons of both the NLT and NPO are aldehyde-fuchsin (AF) positive. The axons from NPO run into four or five bundles ventrolaterally and caudally for some distance before all of them from either side join ventromedially into a single thick cord of neurosecretory fibers which finally enters the pars intermedia to form the neurointermediate lobe. The fibers from NLT, which are separate from the neurosecretory fibers of the NPO, anastomose in the region of the pars distalis. The neurosecretory material (NSM) of NPO is transported by axonal route whereas that of NLT is by axonal as well as ependymovascular pathways. Hypophysectomy results in the increase of AF-positive material in the neurons of NPO shortly after the operation, but later on, the material begins to deplete in them. The AF-positive material at the cut end of neurosecretory fibers, however, accumulates. The AF-positive material in the cell bodies of NLT is also depleted, but the nuclei increase in size, after hypophysectomy.     

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.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

The stomatogastric nervous system of the house cricket Acheta domesticus L. II. Iontophoretic study of neuron anatomy

The anatomy of neurons of the stomatogastric nervous system of Ascheta domesticus was studied using heavy metal iontophoresis through cut nerve ends followed by silver intensification. Nineteen categories of neuron are described and compared with neurons known from the stomatogastric nervous system of other insects. Possible functions for the neurons are suggested. Motor neuron candidates are suggested for all parts of the gut served by the stomatogastric nervous system, and axons of sensory neurons of the anterior pharynx are located. There are four neuron types that cannot readily be assigned motor, sensory, or interneuron functions: large dorsal cells of the frontal ganglion; the two neurons of the nervus connectivus, and two categories of neurons in the median neurosecretory cell group of the pars intercerebralis, the axons of which are contained in the stomatogastric nerves.