Study on the GABA gated channels in the neurosecretory cells of MTXO in the eyestalks of Eriocheir sinensis

The responses to rapid application of gamma-aminobutyric acid (GABA) and the GABA receptor characteristics of MTXO neurosecretory cells in the eyestalks of Chinese mitten-handed crab (Eriocheir sinensis) were examined by whole-cell patch clamp. Under current clamp mode, the depolarization and hyperpolarization were evoked from the three types of neurosecretory cells in response to the GABA (0.1 mmol/L) depending on the Nernst Cl- potential. Under voltage clamp mode, the inward Cl- channel currents (I-GABA) were resolved from all three types of neurosecretory cells in response to GABA (0.01similar to5 mmol/L). The GABA currents were activated within 1 200 ms and peaked within 800 ms. No obviously desensitization was observed during GABA application. The dose-response curve showed usual S-shape, with a just-discernible effect at 0.01 mmol/L and near-saturation at 0.5 mmol/L. The GABA currents had reversal potentials that followed Nernst Cl- potentials when [Cl-] was varied. The pharmacological results revealed that the GABA receptor of the crab neurosecretory cells was sensitive to the Cl- channel blockers picrotoxin and niflumic acid (0.5 mmol/L), insensitive to GABA(A) receptor antagonist bicuculline and GABA(C) receptor agonist cis-4-aminocrotonic acid (CACA 1 mmol/L) and trans-4-aminocrotonic (TACA 1 mmol/L).

Antizyme Inhibitor 2 : A Novel Regulator of Cellular Polyamine Homeostasis

Polyamines are organic polycations that participate in various physiological functions, including cell proliferation, differentiation and apoptosis. Cellular polyamines originate from endogenous biosynthesis and exogenous sources. Their subcellular pool is under strict control, achieved by regulating their uptake and metabolism. Polyamine-induced proteins called antizymes (AZ) act as key regulators of intracellular polyamine concentration. They regulate both the transport of polyamines and the activity and degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. AZs themselves are negatively regulated by antizyme inhibitor (AZIN). AZIN functions as a positive regulator of cellular polyamine homeostasis, which by binding to AZs reactivates ODC and induces the uptake of polyamines. In various pathological conditions, including cancer, polyamine levels are misregulated. Polyamine homeostasis has therefore become an attractive target for therapeutic interventions and it is thus crucial to characterize the molecular basis underlying the homeostatic regulation. A novel human AZIN-resembling protein was previously identified in our group. The purpose of this study was to elucidate the function and distribution of this protein, termed as an antizyme inhibitor 2 (AZIN2). According to my results, AZIN2 functions as a novel regulator of polyamine homeostasis. It shows no enzymatic activity, but instead it binds AZs and negates their activity, which subsequently leads to reactivation of ODC and inhibition of its degradation. Expression of AZIN2 is restricted to terminally differentiated cells, such as mast cells (MC) and neurosecretory cells. In these actively secreting cell types, AZIN2 localizes to subcellular vesicles or granules where its function is important for the vesicle-mediated secretion. In MCs, AZIN2 localizes to the serotonin-containing subset of MC granules, and its expression is coupled to MC activation. The functional role of polyamines as potential mediators of MC activity was also investigated, and it was observed that the secretion of serotonin is selectively dependent on activation of ODC. In neurosecretory cells, AZIN2-positive vesicles localize mainly to the trans-Golgi network (TGN). Depletion of AZIN2 or cellular polyamines causes selective fragmentation of the TGN and retards secretion of proteins. Since addition of exogenous polyamines reverses these effects, the data indicate that AZIN2 and its downstream effectors, polyamines, are functionally implicated in the regulation of secretory vesicle transport. My studies therefore reveal a novel function for polyamines as modulators of both constitutive and regulated secretion. Based on the results, I propose that the role of AZIN2 is to act as a local in situ activator of polyamine biosynthesis.

Characterisation of neurons in the pedal ganglia of the green-lipped mussel, Perna canaliculus, using antibodies raised against neuropeptides and neurotransmitters involved in gastropod egg-laying behaviour and bivalve reproduction and spawning

To characterise central neurons in the pedal ganglia of both male and female green lipped mussel, Perna canaliculus immunohistochemical techniques were used. Mollusc antibodies were used against neuropeptides and neurotansmitters known to control reproduction and spawning. Anti-ELH and anti-APGWamide showed very strong immunoreactivity in small type of neurons. Anti-5-HT and anti-DA immunoreactivity was mostly in large type of neurons. The labelled neurons are consistent with descriptions of neurosecretory cells implicated in the control of reproduction and spawning on the basis of earlier histological staining techniques used in this species. The use of selective immunological markers for peptides and amines appears to be a, promising tool for further characterisation of neurosecretory cells, and to isolate an'tl characterise neuropeptides and other biologically active materials involved in the control of reproduction in Perna canaliculus.

Neurons characterization in the cerebral ganglia of the green-lipped mussel, Perna canaliculus, using antibodies raised against neuropeptides and neurotransmitters involved in gastropod egg-laying behaviour and bivalve reproduction and spawning

Immunohistochemical techniques were used to characterise central neurons in the cerebral ganglia of both male and female Perna canaliculus. We used mollusc antibodies raised against neuropeptides and neurotransmitters known to control reproduction and spawning. Anti-ELH and anti-APGWamide showed very strong immunoreactivity in small type of neurons. Anti-5-HT and anti-DA immunoreactivity was mostly in large type of neurons. The labelled neurons are consistent with descriptions of neurosecretory cells implicated in the control of reproduction and spawning on the basis of earlier histological staining techniques used in this species. The use of selective immunological markers for peptides and amines appears to be a promising tool for further characterisation of neurosecretory cells, and to isolate and characterise neuropeptides and other biologically active materials involved in the control of reproduction in Perna canaliculus.

Urotensin II in the central nervous system of the frog Rana ridibunda: Immunohistochemical localization and biochemical characterization

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians. (C) 1996 Wiley-Liss, Inc.

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Photoperiod-dependent plasticity of circadian pacemaker center in the brain of the Madeira cockroach Rhyparobia maderae

To various degrees, insects in nature adapt to and live with two fundamental environmental rhythms around them: (1) the daily rhythm of light and dark, and (2) the yearly seasonal rhythm of the changing photoperiod (length of light per day). It is hypothesized that two biological clocks evolved in organisms on earth which allow them to harmonize successfully with the two environmental rhythms: (1) the circadian clock, which orchestrates circadian rhythms in physiology and behavior, and (2) the photoperiodic clock, which allows for physiological adaptations to changes in photoperiod during the course of the year (insect photoperiodism). The circadian rhythm is endogenous and continues in constant conditions, while photoperiodism requires specific light inputs of a minimal duration. Output pathways from both clocks control neurosecretory cells which regulate growth and reproduction. This dissertation focuses on the question whether different photoperiods change the network and physiology of the circadian clock of an originally equatorial cockroach species. It is assumed that photoperiod-dependent plasticity of the cockroach circadian clock allows for adaptations in physiology and behavior without the need for a separate photoperiodic clock circuit. The Madeira cockroach Rhyparobia maderae is a well established circadian clock model system. Lesion and transplantation studies identified the accessory medulla (aMe), a small neuropil with about 250 neurons, as the cockroach circadian pacemaker. Among them, the pigment-dispersing factor immunoreactive (PDF-ir) neurons anterior to the aMe (aPDFMes) play a key role as inputs to and outputs of the circadian clock system. The aim of my doctoral thesis was to examine whether and how different photoperiods modify the circadian clock system. With immunocytochemical studies, three-dimensional (3D) reconstruction, standardization and Ca2+-imaging technique, my studies revealed that raising cockroaches in different photoperiods changed the neuronal network of the circadian clock (Wei and Stengl, 2011). In addition, different photoperiods affected the physiology of single, isolated circadian pacemaker neurons. This thesis provides new evidence for the involvement of the circadian clock in insect photoperiodism. The data suggest that the circadian pacemaker system of the Madeira cockroach has the plasticity and potential to allow for physiological adaptations to different photoperiods. Therefore, it may express also properties of a photoperiodic clock.

Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.

Comparative distribution of a putative egg-laying hormone in neural and reproductive tissues of four Decapoda crustaceans

Evidence for the presence of a putative egg-laying (ELH) hormone has been previously described in the black tiger shrimp, Penaeus monodon, so a further investigation was carried out to detect its presence in a range of Decapoda crustaceans prior to a full molecular analysis. The crustaceans were represented by the Australian fresh water yabbie, Cherax destructor, the Australian southern rock lobster, Jasus edwardsii, the snow crab, Chionoecetes opilio, and the blue swimmer crab, Portunus pelagicus. Female cerebral ganglia, ventral nerve cords and gonads were investigated in a comparative study of the distribution of the immunoreactive hormone using immunoenzyme and immunofluorescence techniques. Immunoreactivity was detected in all tissues of interest, and the distribution patterns showed similarity within the four species, as well as that of P. monodon reported in the earlier study. There were minor variations. These data indicate that a putative ELH-like neuropeptide is widespread in crustaceans, and supports its previous identification in a range of molluscs and other invertebrates. Elucidation of the molecular structure of the peptide hormone and its encoding gene, as well as its involvement in spawning behaviour of crustaceans, is now fully under investigation.

The existence of gonadotropin-releasing hormone-like peptides in the neural ganglia and ovary of the abalone, Haliotis asinina L.

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the presence and distribution of two isoforms of GnRH-like peptides in neural ganglia and ovary of reproductively mature female abalone, Haliotis asinina, using immunohistochemistry. We found significant immunoreactivities (ir) of anti-lamprey(I) GnRH-III and anti-tunicate(t) GnRH, but with variation of labeling intensity by each anti-GnRH type. IGnRH-III-ir was detected in numerous type1 neurosecretory cells (NS1) throughout the cerebral and pleuropedal ganglia, whereas tGnRH-I-ir was detected in only a few NS1 cells in the dorsal region of cerebral and pleuropedal ganglia. In addition, a small number of type2 neurosecretory cells (NS2) in cerebral ganglion showed lGnRH-III-ir. Long nerve fibers in the neuropil of ventral regions of the cerebral and pleuropedal ganglia showed strong tGnRH-I-ir. In the ovary, lGnRH-III-ir was found primarily in oogonia and stage I oocytes, whereas tGnRH-ir was observed in stage I oocytes and some stage II oocytes. These results indicate that GnRH produced in neural ganglia may act in neural signaling. Alternatively, GnRH may also be synthesized locally in the ovary where it could induce oocytes development.

Ultrastructure of the urethra of the Mongolian gerbil

Objective: the urethra is the main port of entry of sexually transmitted pathogens. However, papers on the morphology of the urethra are scarce. The Mongolian gerbil is a rodent native of the Mongolia and China and has been utilized as a laboratory animal since the 1960s. This work describes the ultrastructure of the urethra of the Mongolian gerbil to provide data for future experimental studies. Methods: the urethra of ten adult male gerbils was studied by transmission electron microscopy. Results: the epithelium of the pelvic urethra possesses two cell types: I and II, without the formation of cellular layers, while the penile urethra possesses cellular layers: basal, intermediate and superficial. The urethra presents neurosecretory cells belonging to the amine precursor uptake and decarboxylation system. Conclusions: the urethral epithelium of the gerbil is a neurosecretory epithelium, part of the amine precursor uptake and decarboxylation system.

Enhanced Expression of Heme Oxygenase-1 and Carbon Monoxide Excitatory Effects in Oxytocin and Vasopressin Neurones During Water Deprivation

A growing body of evidence indiates that carbon monoxide (CO) acts as a gas neurotransmitter within the central nervous system. Although CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying the actions of CO within the magnocellular neurosecretory system remain largely unknown. In the present study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as the actions of CO on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs), in euhydrated (EU) and 48-h water-deprived rats (48WD). Our results show the expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalisation with OT and VP neurones within the SON and PVN, was significantly enhanced in 48WD rats. Inhibition of HO activity with chromium mesoporphyrin IX chloride (CrMP; 20 mu m) resulted in a slight membrane hyperpolarisation in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarisation, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found that the expression and actions of CO were enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.

Leiomyomatoid angiomatous neuroendocrine tumor (LANT) of the pituitary: a distinctive biphasic neoplasm with primitive secretory phenotype and smooth muscle-rich stroma

We describe a hitherto undocumented variant of dimorphic pituitary neoplasm composed of an admixture of neurosecretory cells and profuse leiomyomatous stroma around intratumoral vessels. Radiologically perceived as a macroadenoma of 3.8 cm in diameter, this pituitary mass developed in an otherwise healthy 43-year-old female. At the term of a yearlong history of amenorrhea and progressive bitemporal visual loss, subtotal resection was performed via transsphenoidal microsurgery. Discounting mild hyperprolactinemia, there was no evidence of excess hormone production. Histologically, solid sheets, nests and cords of epithelial-looking, yet cytokeratin-negative cells were seen growing in a richly vascularized stroma of spindle cells. While strong immunoreactivity for NCAM, Synaptophysin and Chromogranin-A was detected in the former, the latter showed both morphological and immunophenotypic hallmarks of smooth muscle, being positive for vimentin, muscle actin and smooth muscle actin. Architectural patterns varied from monomorphous stroma-dominant zones through biphasic neuroendocrine-leiomyomatous areas, to pseudopapillary fronds along vascular cores. Only endothelia were labeled with CD34. Staining for S100 protein and GFAP, characteristics of sustentacular cells, as well as bcl-2 and c-kit was absent. Except for alpha-subunit, anterior pituitary hormones tested negative in tumor cells, as did a panel of peripheral endocrine markers, including serotonin, somatostatin, calcitonin, parathormone and vasoactive intestinal polypeptide. Mitotic activity was absent and the MIB-1 labeling index low (1-2%). While assignment of this lesion to any established neoplastic entity is not forthcoming, we propose it is being considered as a low-grade neuroendocrine tumor possibly related to null cell adenoma.

Neurosecretory habituation in PC12 cells: modulation during parallel habituation.

PC12 cells habituate during repetitive stimulation with acetylcholine, bradykinin, or high potassium. Interspersing these stimulants did not affect the rate of habituation of the others, but it could modulate the amplitude of the norepinephrine secretion each could achieve. Stimulation with acetylcholine inhibited norepinephrine secretion caused by high potassium and bradykinin stimulation, while high potassium had no effect on acetylcholine or bradykinin, and bradykinin increased secretion caused by acetylcholine. Changes in norepinephrine secretion resulting from any of these stimulants correlated with changes in internal calcium levels. Cyclic AMP-, protein kinase C-, and calmodulin-dependent second messenger pathways all modulated norepinephrine secretion caused by acetylcholine and high potassium and showed a distinct hierarchy in their effectiveness. These data demonstrate that different receptor pathways can change the norepinephrine response of one another while not changing the levels of the molecules responsible for habituation.

Identification and characterization of the egg-laying hormone from the neurosecretary bag cells of Aplysia

This investigation has resulted in the chemical identification and isolation of the egg-laying hormone from Aplysia californica, Aplysia vaccaria, and Aplysia dactylomela. The hormone, which was originally identified as the Bag Cell-Specific protein (BCS protein) on polyacrylamide gels, is a polypeptide of molecular weight ≈ 6000, which is localized in the neurosecretory bag cells of the parietovisceral ganglion and the surrounding connective tissue sheath which contains the bag cell axons. All three species produce a hormone of similar molecular weight, but varying electrophoretic mobility as determined on polyacrylamide gels. As tested, the hormone is completely cross-reactive among the three species.

Although the bag cells of sexually immature animals contain the active hormone, sexual maturation of the animal results in a 10-fold increase in the BCS protein content of these neurons.

A seasonal variation in the BCS protein content was also observed, with 150 times more hormone contained in the bag cells of Aplysia californica in August than in January. This correlates well with the variation in the animals' ability to lay eggs throughout the year (Strumwasser et al., 1969). There are some indications that the receptivity of the animal to the available hormone also fluctuates during the year, being lower in winter than in swmner. The seasonal rhythm of the other species, Aplysia vaccaria and Aplysia dactylomela, has not been investigated.

A polyacrylamide gel electrophoresis analysis of water-soluble proteins in Aplysia californica revealed several other nerve-specific proteins. One of these is also located in the bag cell somas and stains turquoise with Amido Schwarz. The function of this protein has not been investigated.