α1 and ß3 adrenergic receptor-mediated excitatory effects of adrenaline on the caudal neurosecretory system (CNSS) in olive flounder, Paralichthys olivaceus.

Adrenaline is one of the most important neurotransmitters in the central nervous system and is produced during stress. In this study, we investigated the modulatory role of adrenaline and adrenergic receptors on the neuroendocrine Dahlgren cells in the caudal neurosecretory system (CNSS) of olive flounder. Ex vivo electrophysiological recordings revealed that adrenaline significantly increased the firing frequency and altered the firing pattern of Dahlgren cells. Moreover, treatment with adrenaline led to a significant upregulation of ion channels and major hormone secretion genes in CNSS at the mRNA levels. Additionally, treatment with adrenaline resulted in a significantly elevation in the expression levels of α1- and ß3-adrenergic receptors. Furthermore, the ß3-adrenergic receptor antagonist exerts a significant inhibitory effect on adrenaline-induced enhancement firing activities of Dahlgren cells, whereas the α1-adrenergic receptor antagonist displays a comparatively weaker inhibitory effect. Additionally, the enhanced firing activity induced by adrenaline could be effectively suppressed by both α1- and ß3-adrenergic receptor antagonists. Taken together, these findings provide strong evidence in favor of the excitatory effects of adrenaline through α1 and ß3 adrenergic receptors in CNSS to stimulate the secretion of stress-related hormones, ß3-adrenergic receptor plays a more dominant role in the modulation of firing activities of Dahlgren cells by adrenaline and thereby regulates the stress response in olive flounder.

Effects of chlorpyrifos toxicity on brain, pseudobranchial neurosecretory system and swimming performance of a catfish, Heteropneustes fossilis.

In the present study, it was aimed to evaluate the adverse effects of CPF on the histopathology of the optic tectum and cerebellum, pseudobranchial neurosecretory system (PNS), biochemical assays of brain tissue, and locomotory behavior in catfish, Heteropneustes fossilis. The fishes were exposed to an environmentally relevant concentration of 0.09 and 0.192 mg/L of CPF for 7, 15, and 30 d. The CPF toxicity induced degenerative changes with significantly decreased cell size, number, and nucleo-cytoplasmic (N/C) ratio of the PNS; and altered neuro-architectural pattern of optic tectum with degenerative changes in mononuclear and granular cells and necrotic variation in granular and Purkinje cells of the cerebellum. The Acetylcholinesterase (AChE) and Catalase (CAT) activity in the CPF-exposed brain was significantly decreased, whereas Superoxide dismutase (SOD) and Malondialdehyde (MDA) level was significantly increased in comparison with control. In CPF-exposed fishes, the respiratory movements and locomotory behavioral pattern like swimming speed, total distance traveled, time mobile, absolute turn angle, head: distance traveled, maximum speed were significantly decreased, whereas time immobile and time freezing episodes were significantly increased as compared to control fishes. The present study concludes that environmentally relevant concentration of CPF may induce histopathological, biochemical, physiological, and behavioral disturbances in a non-target organism, H. fossilis.

Evaluation of salivary α-amylase and immunoglobulin A responses after endurance exercise in adolescent males and females with similar aerobic fitness.

The objective of this study was to clarify whether there are sex-specific differences in salivary α-amylase and immunoglobulin A responses following acute endurance exercise in adolescent males and females with equivalent cardiorespiratory fitness levels. Twenty-six aerobically trained adolescent males and females with similar training status were enrolled in this study. Each individual executed a 1-h prolonged cycling exercise corresponding to a constant power output at 65% of peak oxygen uptake. Unstimulated whole salivary samples were taken with the passive drooling method at the 10-min period before and after exercise for the determination of salivary responses [α-amylase, immunoglobulin A, total protein and flow rate]. Salivary α-amylase activity, immunoglobulin A concentration and total protein concentration were significantly augmented immediately after acute endurance exercise. Regarding sex differences, only the salivary flow rate was significantly lower in females than in males. The findings of the present study imply that adolescent males and females appear to have similar salivary responses after acute endurance exercise, as represented by salivary α-amylase activity and immunoglobulin A concentration in connection with total protein concentration and salivary flow rate, when they are matched for peak oxygen uptake relative to fat-free body mass.

The excitatory effect of 5-HT1A and 5-HT2B receptors on the caudal neurosecretory system Dahlgren cells in olive flounder, Paralichthys olivaceus.

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an essential role in the regulation of neural activity via multiple receptors. Here, we investigated the functional role of serotoninergic input on the Dahlgren cell population in the caudal neurosecretory system (CNSS) of olive flounder. In this study, the effect of 5-HT on the firing activity of Dahlgren cells was explored in terms of changes in firing frequency and firing pattern using multicellular recording electrophysiology ex vivo, and the role of several 5-HT receptor subtypes in the regulation was determined. The results revealed that 5-HT increased the firing frequency in a concentration-dependent manner and altered the firing pattern of Dahlgren cells. The effect of 5-HT on the firing activity of Dahlgren cells was mediated through the 5-HT1A and 5-HT2B receptors, selective agonists of both receptors effectively increased the firing frequency of Dahlgren cells, and selective receptor antagonists could also effectively inhibit the increase in firing frequency caused by 5-HT. In addition, the mRNA levels of major signaling pathway-related genes, ion channels, and major secretion hormone genes were significantly upregulated in CNSS after treatment with 5-HT. These findings demonstrate that 5-HT acts as an excitatory neuromodulator on Dahlgren cells and enhances neuroendocrine activity in CNSS.

Acute toxicity of broflanilide on neurosecretory system and locomotory behavior of zebrafish (Danio rerio).

Broflanilide, a novel meta-diamide insecticide, possesses moderate acute toxicity to zebrafish, with a 96-h median lethal concentration (96-LC50) of 0.76 mg/L. However, its effect on fish behavior and the underlying mechanisms are still unclear. The present study evaluated the effects of broflanilide on the zebrafish brain over a 96-h exposure by comparing the histopathological changes and relative expression of targeted genes with the behavioral metrics. The results of the toxicity test showed that broflanilide could cause deformities, such as deformation of the operculum and spinal curvature, at 0.6, 0.82 and 1.15 mg/L. Results also showed tissue damage and apoptosis in the cerebellum under 0.27 and 0.6 mg/L exposure. Additionally, broflanilide affected the neurotransmitters, metabolites and transcripts of genes associated with dopamine, gamma-aminobutyric acid expression. and the signaling pathways in zebrafish brains at 0.60 mg/L after 1 h and 96 h of exposure, while the levels of glutamate, glutamate decarboxylase, GABA transaminase, nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) were also inhibited at 0.27 mg/L after 96 h of exposure. The accumulated swimming distance was significantly longer and the average speed was significantly faster than the control at 0.27 and 0.6 mg/L after 1-h of exposure, while these metrics were lowered at 0.6 mg/L after 96 h of exposure. The study results demonstrates that broflanilide affects the zebrafish brain, neurotransmitters and associated fish behaviors. This study also provides deeper insight into the mechanistic understanding of the effects of broflanilide on the zebrafish brain.

Acute toxic effects of chlorpyrifos on pseudobranchial neurosecretory system, brain regions and locomotory behavior of an air-breathing catfish, Heteropneustes fossilis (Bloch 1794).

In the late twentieth century, tremendous use of second-class organophosphate insecticides especially chlorpyrifos (CPF) resulting into heavy accumulation in different non-targeted aquatic species including fishes leads to apparent structural and biochemical changes in different organs and related abnormal behavioral responses. The present study has been undertaken as a pioneer attempt to assess the toxic effects of CPF on histopathological changes in pseudobranchial neurosecretory cells (PNSCs) of a neuroendocrine system of gill region, optic tectum (OT) and cerebellum, biochemical changes (acetylcholinesterase (AChE) activity and antioxidant markers) in the brain and associated locomotory behavioral alterations in air-breathing catfish, Heteropneustes fossilis. The fishes were exposed to CPF concentration of 1.92 mg/l for four days and their locomotor activities were recorded by ANY-MAZE software (Stoelting, Kiel, WI), an automated behavior tracking device. The acute exposure of CPF induced pathological changes in PNSCs, subtle changes in granular cells of the cerebellum and neuroarchitectural pattern of different layers of OT as compared to control. In the CPF exposed brain, AChE activity was significantly decreased while antioxidant enzymatic activity such as SOD activity was increased but CAT activity was substantially decreased. The CPF exposed fishes displayed significantly reduced locomotory activities with symptoms of motionless, loss of equilibrium and erratic movements. This study concludes that acute exposure to CPF for short duration may induce dys-regulation of neurosecretory activity of PNSCs, altered biochemical activity of brain and reduced locomotory/swimming performances in fishes.

[The caudal neurosecretory system, the other "neurohypophysial" system in fish]. / Le système neurosécréteur caudal, l'autre système « neurohypophysaire ¼ des poissons.

The caudal neurosecretory system (CNSS) is a neuroendocrine complex whose existence is specific to fishes. Structurally, it has many similarities with the hypothalamic-neurohypophyseal complex of other vertebrates. However, it differs regarding its position at the caudal end of the spinal cord and the nature of the hormones it secretes, the most important being urotensins. The CNSS was first described more than 60 years ago, but its embryological origin is totally unknown and its role is still poorly understood. Paradoxically, it is almost no longer studied today. Recent developments in imaging and genome editing could make it possible to resume investigations on CNSS in order to solve the mysteries that still surround it.

Title: Le système neurosécréteur caudal, l'autre système « neurohypophysaire ¼ des poissons. Abstract: Le système neurosécréteur caudal (SNSC) est un complexe neuroendocrinien propre aux poissons. Sur le plan structural, il présente de nombreuses similitudes avec le complexe hypothalamo-neurohypophysaire d'autres vertébrés. Il s'en distingue toutefois par sa position, à l'extrémité caudale de la moelle épinière, et par la nature des hormones qu'il sécrète, les plus importantes étant les urotensines. Le SNSC a été décrit pour la première fois il y a plus de 60 ans, mais son origine embryologique est totalement inconnue et son rôle reste mal compris. Paradoxalement, il n'est presque plus étudié aujourd'hui. Les développements récents en imagerie et en génie génétique pourraient justifier la reprise d'investigations sur le SNSC afin de lever les mystères qui continuent de l'entourer.

Neuromodulatory effects of GnRH on the caudal neurosecretory Dahlgren cells in female olive flounder.

Gonadotropin-releasing hormone (GnRH) is considered a key player in reproduction. The caudal neurosecretory system (CNSS) is a unique neurosecretory structure of fish that may be involved in osmoregulation, nutrition, reproduction, and stress-related responses. However, a direct effect of GnRH on Dahlgren cells remains underexplored. Here, we examined the electrophysiological response of Dahlgren cell population of the CNSS to GnRH analog LHRH-A2 and the transcription of related key genes of CNSS. We found that GnRH increased overall firing frequency and may be changed the firing pattern from silent to burst or phasic firing in a subpopulation of Dahlgren cells. The effect of GnRH on a subpopulation of Dahlgren cells firing activity was blocked by the GnRH receptor (GnRH-R) antagonist cetrorelix. A positive correlation was observed between the UII and GnRH-R mRNA levels in CNSS or gonadosomatic index (GSI) during the breeding season. These findings are the first demonstration of the ability of GnRH acts as a modulator within the CNSS and add to our understanding of the physiological role of the CNSS in reproduction and seasonal adaptation.

GABAA receptor-mediated inhibition of Dahlgren cells electrical activity in the olive flounder, Paralichthys olivaceus.

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. We investigated its potential role as a neurotransmitter in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the flounder Paralichthys olivaceus. The application of GABA in vitro resulted in a decrease in electrical activity of Dahlgren cells, followed by an increase of the number of silent cells, together with a decreased firing frequency of all three activity patterns (tonic, phasic, bursting). GABAA receptor agonist etomidate decreased Dahlgren cell firing activity, in a similar way to GABA. The response to GABA was blocked by the GABAA receptor antagonist bicuculline. GABAA receptor gamma2 subunit (Gabrg2) and chloride channel (Clcn2) mRNA expression were significantly upregulated in the CNSS after GABA superfusion. These data suggest that GABA may modulate CNSS activity in vivo mediated by GABAA receptors.

Inhibitory role of taurine in the caudal neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus.

Taurine plays role in neural development and physiological functions such as endocrine regulation in the central nervous system (CNS), and it is one of the most abundant free amino acid there. We investigated its potential effect as a neurotransmitter in the group of neuroendocrine Dahlgren cells at flounder Paralichthys olivaceus caudal neurosecretory system (CNSS). The application of taurine in vitro led to a reduction in electrical activity of Dahlgren cells, followed by a rise in the number of silent cells, at the same time the frequency of all three activity patterns (tonic, phasic, bursting) in Dahlgren cells was reduced. Both strychnine (a glycine receptor antagonist) and bicuculline (a GABAA receptor antagonist) can block the response to taurine separately. Transcriptome sequencing analysis showed the existence of glycine receptor (GlyR) and GABAA receptor (GABAAR) in the flounder CNSS, and the GlyR, GABAAR, and Cl- channel mRNA expression were significantly raised after taurine superfusion according to quantitative RT-PCR results. These data indicate that taurine may mediate Dahlgren cell population of CNSS activity in vivo through GlyR and GABAAR, thereby, regulating stress-response.

The caudal neurosecretory system: A novel thermosensitive tissue and its signal pathway in olive flounder (Paralichthys olivaceus).

Ectotherm animals, such as fish, are vulnerable when facing an extreme temperature fluctuation as a result of their inability to maintain body temperature. The caudal neurosecretory system (CNSS) is unique to fish and has been shown to maintain homeostasis in response to seasonal changes. However, its temperature sensitivity is unknown. Here, we used in vitro electrophysiological and anatomical approaches to investigate a thermosensory pathway in the CNSS. We showed that the CNSS responds directly to local hypothermal challenge via the TRP channel, and transmits this signal using the neurotransmitter, GABA, to the neurosecretory Dahlgren cells of the CNSS. These findings are the first demonstration of the thermal perception of the CNSS and add to our understanding of the physiological role of the CNSS in thermoregulation and seasonal adaptation.

Paraneuronal pseudobranchial neurosecretory system in tank goby Glossogobius giuris with special reference to novel neurohaemal contact complex.

Various putative oxygen chemosensory cells are reported to be present throughout the vertebrate body performing pivotal roles in respiration by initiating responses during acute hypoxia. Since air-breathing fishes often are exposed to the oxygen-deficient milieu, in such conditions various chemosensory cells operate in an orchestrated fashion. The Pseudobranchial neurosecretory system (PSNS) a newly discovered system, is one of these. It has been placed in the category of "Diffuse NE systems (DNES)". It is found in all the catfish species and in some other non-catfish group of teleosts. In catfishes, it is present in close association with the carotid labyrinth- a chemosensory structure, known in fish and amphibians. The presence of this system in Glossogobius giuris, in association with the pseudobranch, a structure considered to be precursor of carotid labyrinth, is a significant finding. In an attempt to study the structure and organization of the pseudobranchial neurosecretory system in a non-catfish species of teleost, the present investigation was undertaken on a goby G. giuris. The histological observations, using a neurosecretion-specific stain, revealed the presence of this system in G. giuris. The findings are discussed in the light of the association of PSNS with pseudobranch and the type of "neurohaemal contact complex" formed between this neurosecretory system and the elements of the circulatory system.

Age-dependent increase in Thy-1 protein in the rat supraoptic nucleus.

Mature mammalian CNS neurons often do not recover successfully following injury. To this point, unilateral lesion of the hypothalamo-neurohypophysial tract results in collateral sprouting from uninjured axons of the supraoptic nucleus (SON) in 35-day-old but not in 125-day-old rats. Thus, it appears that there are age-related changes within the SON that preclude the older rat from recovering following axotomy. We hypothesize that the intrinsic capacity for axon reorganization may depend, in part, on age-related alterations in cell adhesion molecules that allow normal astrocyte-neuron interactions in the SON. In support of our hypothesis, numerous reports have shown that Thy-1 is increased in neurons at the cessation of axon outgrowth. Therefore, we compared protein levels of Thy-1 and the Thy-1 interacting integrin subunits, alpha-v (αv), beta-3 (ß3), and beta-5 (ß5), in 35- and 125-day-old SON using western blot analysis. Our results demonstrated that there was significantly more Thy-1 protein in the 125-day-old SON compared to 35-day-old SON, but no change in the protein levels of the integrin subunits. Furthermore, we localized Thy-1-, αv integrin-, ß3 integrin-, and ß5 integrin-immunoreactivity to both neurons and astrocytes in the SON. Altogether, our results suggest that the observed increase in Thy-1 protein levels in the SON with age may contribute to an environment that prevents collateral axonal sprouting in the SON of the 125-day-old rat.

The paraneuronal gill neuroendocrine system in ocellated puffer fish Leiodon cutcutia.

Pseudobranchial neurosecretory system (PSNS) is the third Neuroendocrine (NE) system found in the gill region of fishes in close association with pseudobranch/carotid labyrinth/carotid gland and can suitably be placed under the category of "Diffused NE system (DNES)." The cells belonging to this system fall under the category of "Paraneurons," a concept proposed by Fujita and coworkers. It is found uniformly in all the catfish species and some other noncatfish group of teleosts as Atheriniformes, Channiformes, Perciformes, and Clupeiformes. The fishes, in which the PSNS is present, belong to different breathing habits. Most of these have the capacity to tolerate low O2 conditions. Leiodon cutcutia although not an air-breathing fish, is known to retain air in its stomach for varied periods when threatened. In an attempt to verify the veracity of this system in a fish of another peculiar breathing habit, ocellated puffer fish L. cutcutia (order Tetradontiformes) was investigated. The histological observations undertaken on L. cutcutia revealed the presence of a well-developed extrabranchial NE system. The findings are discussed in the light of the association of PSNS with chemosensory system and its evolution in fishes, especially in the view of the transition from aquatic to terrestrial life.

Caracterización de pacientes con tumores neuroendocrinos en un hospital de referencia de alta complejidad / Characterization of patients with neuroendocrine tumors in a referral hospital

Introducción. Los tumores neuroendocrinos comprenden una amplia familia tumoral, siendo de mayor frecuencia aquellos localizados en el tracto gastrointestinal, los pulmones, el timo y el páncreas. Ocurren con poca frecuencia y cursan con un comportamiento biológico variable. El objetivo del presente estudio fue describir las características demográficas y clínicas de los pacientes con esta entidad en un hospital de alta complejidad. Métodos. Estudio descriptivo con revisión de historias clínicas correspondientes a pacientes con tumor neuroendocrino, cuya muestra histológica fue analizada en el Departamento de Patología del Hospital Pablo Tobón Uribe, Medellín, Colombia, entre 2010 y 2015. Se describen frecuencias de localización, grado de diferenciación y perfil de inmunohistoquímica. Resultados. Se revisaron 111 historias, de las que el 51,5 % correspondió a pacientes masculinos, con una mediana de edad al momento del diagnóstico de 48 años. Las comorbilidades más frecuentes fueron: hipertensión arterial (38,6 %), hipotiroidismo (15,8 %) y diabetes mellitus (11,9 %). El antecedente de neoplasia estuvo presente en el 13,9 % de los casos. Un 59,4 % de las muestras correspondieron a tumores primarios, el 34,8 % estaban localizados en el páncreas. El hígado fue el órgano más comprometido por metástasis (73,3 %). De los tumores neuroendocrinos, el 27 % correspondió a tumores grado 1 del tracto gastrointestinal o del páncreas, el 36 % a grado 2 y el 27,9 % a grado 3. El 3,6 % correspondió a tumores pulmonares de grado bajo o intermedio y el 5,4 % a tumores pulmonares de alto grado. Los marcadores tumorales con mayor positividad fueron sinaptofisina (97,2 %), citoqueratinas AE1/AE3 (95 %), CD56 (91,3 %) y cromogranina (87,8 %). Discusión. Los tumores neuroendocrinos tienen características clínicas e histopatológicas diversas. Conocer las características de los pacientes afectados permite una aproximación de la epidemiología local aplicable a futuras investigaciones

Introduction. Neuroendocrine tumors comprise a broad family of tumors, the most common being those located in the gastrointestinal tract, lungs, thymus, and pancreas. They occur infrequently and have a variable biological behavior. The objective of this study was to describe the demographic and clinical characteristics of patients with this entity in a highly complex hospital.Methods. Descriptive study with review of medical records corresponding to patients with neuroendocrine tu-mors, whose histological sample was analyzed in the Pathology Department of the Pablo Tobón Uribe Hospital in Medellín between 2010 and 2015. Frequencies of location, degree of differentiation and immune-histochemistry profile are described.Results. One hundred eleven histories were reviewed, of which 51.5% corresponded to male patients, with a median age at diagnosis of 48 years. The most frequent comorbidities were arterial hypertension (38.6%), hypothyroidism (15.8%) and diabetes mellitus (11.9%). Medical history of neoplasia was present in 13.9% of the cases. 59.4% of the samples corresponded to primary tumors, 34.8% were located in the pancreas. The liver was the organ most compromised by metastasis (73.3%). Of the neuroendocrine tumors, 27% corresponded to grade 1 tumors of the gastrointestinal tract or pancreas, 36% to grade 2 and 27.9% to grade 3; 3.6% corresponded to low or intermediate grade lung tumors and 5.4% to high-grade lung tumors. The tumor markers with the highest positivity were synaptophysin (97.2%), cytokeratins AE1/AE3 (95%), CD56 (91.3%) and chromogranin (87.8%).Discusion. Neuroendocrine tumors have diverse clinical and histopathologic features. Knowing the characteristics of affected patients allows an approximation of local epidemiology applicable to future research

Nitric Oxide and the Neuroendocrine Control of the Osmotic Stress Response in Teleosts.

The involvement of nitric oxide (NO) in the modulation of teleost osmoresponsive circuits is suggested by the facts that NO synthase enzymes are expressed in the neurosecretory systems and may be regulated by osmotic stimuli. The present paper is an overview on the research suggesting a role for NO in the central modulation of hormone release in the hypothalamo-neurohypophysial and the caudal neurosecretory systems of teleosts during the osmotic stress response. Active NOS enzymes are constitutively expressed by the magnocellular and parvocellular hypophysiotropic neurons and the caudal neurosecretory neurons of teleosts. Moreover, their expression may be regulated in response to the osmotic challenge. Available data suggests that the regulatory role of NO appeared early during vertebrate phylogeny and the neuroendocrine modulation by NO is conservative. Nonetheless, NO seems to have opposite effects in fish compared to mammals. Indeed, NO exerts excitatory effects on the electrical activity of the caudal neurosecretory neurons, influencing the amount of peptides released from the urophysis, while it inhibits hormone release from the magnocellular neurons in mammals.

Gene expression and hormone secretion profile of urotensin I associated with osmotic challenge in caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

The caudal neurosecretory system (CNSS) is a part of stress response system, a neuroendocrine structure unique to fish. To gain a better understanding of the physiological roles of CNSS in fluid homeostasis, we characterized the tissue distribution of urotensin I (UI) expression in European flounder (Platichthys flesus), analyzed the effect chronic exposure to seawater (SW) or freshwater (FW), transfer from SW to FW, and reverse transfer on mRNA levels of UI, L-type Ca2+ channels and Ca-activated K+ channels transcripts in CNSS. The tissue distribution demonstrated that the CNSS is dominant sites of UI expression, and UI mRNA level in fore brain appeared greater than other non-CNSS tissues. There were no consistent differences in CNSS UI expression or urophysis UI content between SW- and FW-adapted fish in July and September. After transfer from SW to FW, at 8 h CNSS UI expression was significantly increased, but urophysis UI content was no significantly changes. At 24 h transfer from SW to FW, expression of CNSS UI was no apparent change and urophysis UI content was reduced. At 8 h and 24 h after transfer from FW to SW UI expression and urophysis UI content was no significantly effect. The expression of bursting dependent L-type Ca2+ channels and Ca-activated K+ channels in SW-adapted fish significantly decreased compared to those in FW-adapted. However, there were no differences in transfer from SW to FW or from FW to SW at 8 h and 24 h. Thus, these results suggest CNSS UI acts as a modulator in response to osmotic stress and plays important roles in the body fluid homeostasis.

Dynamic Responses of the Caudal Neurosecretory System (CNSS) Under Thermal Stress in Olive Flounder (Paralichthys olivaceus).

Temperature is a critical environmental factor that affect most biological and physiological processes in fish. The caudal neurosecretory system (CNSS) is unique to fish and is proved to maintain homeostasis during seasonal alterations. However, the dynamic expression and secretion pattern of its major hormones, corticotrophin-releasing hormone (CRH), urotensin I (UI), and urotensin II (UII), and their response to thermal stress has not been studied. CRH, UII and cortisol in plasma, gene expression levels of CRH, UI, and UII in the CNSS of olive flounder (Paralichthys olivaceus) were therefore characterized. UI- and UII-positive Dahlgren cells, as well as cell proliferation in the CNSS, were also quantified. The results showed that plasma cortisol and CRH were increased in both low temperature (LT) and high temperature (HT) groups. However, there was no difference in plasma UI and UII during thermal stress. In CNSS, CRH, UI, and UII mRNA levels were all significantly elevated in response to acute hypothermal stress and recovered back to the control (normal) level after 8 days of adaptation. During hyperthermal challenge, gene expression of CRH and UI only significantly increased after 8-days of transfer but no change in UII was observed. We also demonstrated an increasing percent of UI-positive Dahlgren cells in the CNSS of 8-days hyperthermal stressed fish. However, no BrdU-labeled Dahlgren cells were found among the three treatment groups. Collectively, our results demonstrate that the CNSS is subjected to dynamic responses under thermal stress and expands upon the role of the CNSS in thermoregulation. The dynamic responses of hormone levels and the gene expression of CRH, UI and UII in CNSS are all involved in the process of hyper- or hypo-thermal stress and adaptation.

Modulatory effect of dopamine receptor 5 on the neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus.

A neuromodulatory role for dopamine has been reported for magnocellular neuroendocrine cells in the mammalian hypothalamus. We examined its potential role as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder Paralichthys olivaceus. In vitro application of dopamine (DA) caused an increase in electrical activity (firing frequency, recorded extracellularly) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The dopamine precursor, levodopa (L-DOPA), also increased firing frequency, cell recruitment and enhanced bursting and tonic activity. The effect of dopamine was blocked by the D1, D5 receptor antagonist SCH23390, but not by the D2, D3, D4 receptor antagonist amisulpride. Transcriptome sequencing revealed that all DA receptors (D1, D2, D3, D4, and D5) were present in the flounder CNSS. However, quantitative RT-PCR revealed that D5 receptor mRNA expression was significantly increased in the CNSS following dopamine superfusion. These findings suggest that dopamine may modulate CNSS activity in vivo, and therefore neurosecretory output, through D5 receptors.

Modulatory effect of glutamate GluR2 receptor on the caudal neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus.

A neuromodulatory role for glutamate has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined the potential role of glutamate as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) in the euryhaline flounder Paralichthys olivaceus. In pharmacological experiments in vitro, glutamate (Glu) caused an increase in electrical activity of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The glutamate substrate, glutamine (Gln), led to increased firing frequency, cell recruitment and enhanced bursting activity. The glutamate effect was not blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, or the GluR1/GluR3 (AMPA) receptor antagonist IEm1795-2HBr, but was blocked by the broad-spectrum α-amino-3-hydroxy- 5- methyl-4-isoxazo-lepropionic acid (AMPA) receptor antagonist ZK200775. Our transcriptome sequencing study revealed three AMPA receptor (GluR1, GluR2 and GluR3) in the olive flounder CNSS. Quantitative RT-PCR revealed that GluR2 receptor mRNA expression was significant increased following dose-dependent superfusion with glutamate in the CNSS. GluR1 and GluR3 receptor mRNA expression were decreased following superfusion with glutamate. L-type Ca2+ channel mRNA expression had a significant dose-dependent decrease following superfusion with glutamate, compared to the control. In the salinity challenge experiment, acute transfer from SW to FW, GluR2 receptor mRNA expression was significantly higher than the control at 2 h. These findings suggest that GluR2 is one of the mechanisms which can medicate glutamate action within the CNSS, enhancing electrical activity and hence secretory output.