Metacognitive therapy for generalized anxiety disorders in group: A case study.

OBJECTIVES: In Metacognitive therapy (MCT), homework is used, for example, to increase awareness of thoughts and thought processes, to challenge metacognitive beliefs in real-life situations, and to practice new ways of processing thoughts, feelings, and symptoms. All MCT treatment manuals include homework assignments to be given between each session. METHOD: The following study provides a detailed description of the implementation of homework in a group-based MCT treatment for generalized anxiety disorder (GAD) at an outpatient clinic in Norway. The treatment described in this case consisted of 10 weekly group sessions (7 patients) lasting two hours. RESULTS: This case study demonstrates that group-based MCT can be used to treat GAD and describes how the use of homework can facilitate therapeutic change. CONCLUSION: Overall, the effectiveness of MCT was found to be high. Homework gives patients the opportunity to take charge of their therapy and develop a sense of responsibility for their own progress, both during and after treatment.

Pituitary multi-hormone cells in mammals and fish: history, origin, and roles.

The vertebrate pituitary is a dynamic organ, capable of adapting its hormone secretion to different physiological demands. In this context, endocrinologists have debated for the past 40 years if endocrine cells are mono- or multi-hormonal. Since its establishment, the dominant "one cell, one hormone" model has been continuously challenged. In mammals, the use of advanced multi-staining approaches, sensitive gene expression techniques, and the analysis of tumor tissues have helped to quickly demonstrate the existence of pituitary multi-hormone cells. In fishes however, only recent advances in imaging and transcriptomics have enabled the identification of such cells. In this review, we first describe the history of the discovery of cells producing multiple hormones in mammals and fishes. We discuss the technical limitations that have led to uncertainties and debates. Then, we present the current knowledge and hypotheses regarding their origin and biological role, which provides a comprehensive review of pituitary plasticity.

A computational model for gonadotropin releasing cells in the teleost fish medaka.

Pituitary endocrine cells fire action potentials (APs) to regulate their cytosolic Ca2+ concentration and hormone secretion rate. Depending on animal species, cell type, and biological conditions, pituitary APs are generated either by TTX-sensitive Na+ currents (INa), high-voltage activated Ca2+ currents (ICa), or by a combination of the two. Previous computational models of pituitary cells have mainly been based on data from rats, where INa is largely inactivated at the resting potential, and spontaneous APs are predominantly mediated by ICa. Unlike in rats, spontaneous INa-mediated APs are consistently seen in pituitary cells of several other animal species, including several species of fish. In the current work we develop a computational model of gonadotropin releasing cells in the teleost fish medaka (Oryzias latipes). The model stands out from previous modeling efforts by being (1) the first model of a pituitary cell in teleosts, (2) the first pituitary cell model that fires sponateous APs that are predominantly mediated by INa, and (3) the first pituitary cell model where the kinetics of the depolarizing currents, INa and ICa, are directly fitted to voltage-clamp data. We explore the firing properties of the model, and compare it to the properties of previous models that fire ICa-based APs. We put a particular focus on how the big conductance K+ current (IBK) modulates the AP shape. Interestingly, we find that IBK can prolong AP duration in models that fire ICa-based APs, while it consistently shortens the duration of the predominantly INa-mediated APs in the medaka gonadotroph model. Although the model is constrained to experimental data from gonadotroph cells in medaka, it may likely provide insights also into other pituitary cell types that fire INa-mediated APs.

Gonadotrope plasticity at cellular, population and structural levels: A comparison between fishes and mammals.

Often referred to as "the master gland", the pituitary is a key organ controlling growth, maturation, and homeostasis in vertebrates. The anterior pituitary, which contains several hormone-producing cell types, is highly plastic and thereby able to adjust the production of the hormones governing these key physiological processes according to the changing needs over the life of the animal. Hypothalamic neuroendocrine control and feedback from peripheral tissues modulate pituitary cell activity, adjusting levels of hormone production and release according to different functional or environmental requirements. However, in some physiological processes (e.g. growth, puberty, or metamorphosis), changes in cell activity may be not sufficient to meet the needs and a general reorganization of cell composition and pituitary structure may occur. Focusing on gonadotropes, this review examines plasticity at the cellular level, which allows precise and rapid control of hormone production and secretion, as well as plasticity at the population and structural levels, which allows more substantial changes in hormone production. Further, we compare current knowledge of the anterior pituitary plasticity in fishes and mammals in order to assess what has been conserved or not throughout evolution, and highlight important remaining questions.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman.

The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

Sex steroids differentially regulate fshb, lhb and gnrhr expression in Atlantic cod (Gadus morhua).

Depending on the stage of gonad maturation, as well as other factors, gonadal steroids can exert either a positive or negative feedback at the brain and pituitary level. While this has been demonstrated in many teleost species, little is known about the nature of steroid feedback in Gadiform fish. Using an optimized in vitro model system of the Atlantic cod pituitary, the present study investigated the potential effects of two physiologically relevant doses of estradiol, testosterone (TS) or dihydrotestosterone (DHTS) on cell viability and gene expression of gonadotropin subunits (fshb/lhb) and two suggested reproduction-relevant gonadotropin-releasing hormone receptors (gnrhr1b/gnrhr2a) during three stages of sexual maturity. In general, all steroids stimulated cell viability in terms of metabolic activity and membrane integrity. Furthermore, all steroids affected fshb expression, with the effect depending on both the specific steroid, dose and maturity status. Conversely, only DHTS exposure affected lhb levels, and this occurred only during the spawning season. Using single-cell qPCR, co-transcription of gnrhr1b and gnrhr2a was confirmed to both fshb- and lhb- expressing gonadotropes, with gnrhr2a being the most prominently expressed isoform. While steroid exposure had no effect on gnrhr1b expression, all steroids affected gnrhr2a transcript levels in at least one maturity stage. These and previous results from our group point to Gnrhr2a as the main modulator of gonadotropin regulation in cod and that regulation of its gene expression level might function as a direct mechanism for steroid feedback at the pituitary level.

Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1).

NCX1 (Na(+)/Ca(2+) exchanger 1) is an important regulator of intracellular Ca(2+) and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman), a substrate for protein kinases A and C, has been suggested to regulate NCX1 activity. However, although several studies have demonstrated that binding of phosphorylated PLM (pSer(68)-PLM) leads to NCX1 inhibition, other studies have failed to demonstrate a functional interaction of these proteins. In the present study, we aimed to analyse the biological function of the pSer(68)-PLM-NCX1 interaction by developing high-affinity blocking peptides. PLM was observed to co-fractionate and co-immunoprecipitate with NCX1 in rat left ventricle, and in co-transfected HEK (human embryonic kidney)-293 cells. For the first time, the NCX1-PLM interaction was also demonstrated in the brain. PLM binding sites on NCX1 were mapped to two regions by peptide array assays, containing the previously reported PASKT and QKHPD motifs. Conversely, the two NCX1 regions bound identical sequences in the cytoplasmic domain of PLM, suggesting that NCX1-PASKT and NCX1-QKHPD might bind to each PLM monomer. Using two-dimensional peptide arrays of the native NCX1 sequence KHPDKEIEQLIELANYQVLS revealed that double substitution of tyrosine for positions 1 and 4 (K1Y and D4Y) enhanced pSer(68)-PLM binding 8-fold. The optimized peptide blocked binding of NCX1-PASKT and NCX1-QKHPD to PLM and reversed PLM(S68D) inhibition of NCX1 activity (both forward and reverse mode) in HEK-293 cells. Altogether our data indicate that PLM interacts directly with NCX1 and inhibits NCX1 activity when phosphorylated at Ser(68).

Molecular basis of calpain cleavage and inactivation of the sodium-calcium exchanger 1 in heart failure.

Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

Single-Cell Isolation and Gene Analysis: Pitfalls and Possibilities.

During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research.

Day length regulates gonadotrope proliferation and reproduction via an intra-pituitary pathway in the model vertebrate Oryzias latipes.

In seasonally breeding mammals and birds, the production of the hormones that regulate reproduction (gonadotropins) is controlled by a complex pituitary-brain-pituitary pathway. Indeed, the pituitary thyroid-stimulating hormone (TSH) regulates gonadotropin expression in pituitary gonadotropes, via dio2-expressing tanycytes, hypothalamic Kisspeptin, RFamide-related peptide, and gonadotropin-releasing hormone neurons. However, in fish, how seasonal environmental signals influence gonadotropins remains unclear. In addition, the seasonal regulation of gonadotrope (gonadotropin-producing cell) proliferation in the pituitary is, to the best of our knowledge, not elucidated in any vertebrate group. Here, we show that in the vertebrate model Japanese medaka (Oryzias latipes), a long day seasonally breeding fish, photoperiod (daylength) not only regulates hormone production by the gonadotropes but also their proliferation. We also reveal an intra-pituitary pathway that regulates gonadotrope cell number and hormone production. In this pathway, Tsh regulates gonadotropes via folliculostellate cells within the pituitary. This study suggests the existence of an alternative regulatory mechanism of seasonal gonadotropin production in fish.

Developmental tracing of luteinizing hormone ß-subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function.

BACKGROUND: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH-producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue-specific gene expression was examined. RESULTS: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH-producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb-GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. CONCLUSIONS: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function.

Optimized conditions for primary culture of pituitary cells from the Atlantic cod (Gadus morhua). The importance of osmolality, pCO2, and pH.

Protocols for primary cultures of teleost cells are commonly only moderately adjusted from similar protocols for mammalian cells, the main adjustment often being of temperature. Because aquatic habitats are in general colder than mammalian body temperatures and teleosts have gills in direct contact with water, pH and buffer capacity of blood and extracellular fluid are different in fish and mammals. Plasma osmolality is generally higher in marine teleosts than in mammals. Using Atlantic cod (Gadus morhua) as a model, we have optimized these physiological parameters to maintain primary pituitary cells in culture for an extended period without loosing key properties. L-15 medium with adjusted osmolality, adapted to low pCO(2) (3.8mm Hg) and temperature (12°C), and with pH 7.85, maintained the cells in a physiologically sounder state than traditional culture medium, significantly improving cell viability compared to the initial protocol. In the optimized culture medium, resting membrane potential and response to releasing hormone were stable for at least two weeks, and the proportion of cells firing action potentials during spawning season was about seven times higher than in the original culture medium. The cells were moderately more viable when the modified medium was supplemented with newborn calf serum or artificial serum substitute. Compared to serum-free L-15 medium, expression of key genes (lhb, fshb, and gnrhr2a) was better maintained in medium containing SSR, whereas NCS tended to decrease the expression level. Although serum-free medium is adequate for many applications, serum supplement may be preferable for experiments dependent on membrane integrity.

Long extensions with varicosity-like structures in gonadotrope Lh cells facilitate clustering in medaka pituitary culture.

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhß protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.

Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol.

BACKGROUND: The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. RESULTS: Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with ß-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. CONCLUSION: Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

Plasticity in medaka gonadotropes via cell proliferation and phenotypic conversion.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) produced by the gonadotropes play a major role in control of reproduction. Contrary to mammals and birds, Lh and Fsh are mostly produced by two separate cell types in teleost. Here, we investigated gonadotrope plasticity, using transgenic lines of medaka (Oryzias latipes) where DsRed2 and hrGfpII are under the control of the fshb and lhb promotors respectively. We found that Fsh cells appear in the pituitary at 8 dpf, while Lh cells were previously shown to appear at 14 dpf. Similar to Lh cells, Fsh cells show hyperplasia from juvenile to adult stages. Hyperplasia is stimulated by estradiol. Both Fsh and Lh cells show hypertrophy during puberty with similar morphology. They also share similar behavior, using their cellular extensions to make networks. We observed bi-hormonal gonadotropes in juveniles and adults but not in larvae where only mono-hormonal cells are observed, suggesting the existence of phenotypic conversion between Fsh and Lh in later stages. This is demonstrated in cell culture, where some Fsh cells start to produce Lhß, a phenomenon enhanced by gonadotropin-releasing hormone (Gnrh) stimulation. We have previously shown that medaka Fsh cells lack Gnrh receptors, but here we show that with time in culture, some Fsh cells start responding to Gnrh, while fshb mRNA levels are significantly reduced, both suggestive of phenotypic change. All together, these results reveal high plasticity of gonadotropes due to both estradiol-sensitive proliferation and Gnrh promoted phenotypic conversion, and moreover, show that gonadotropes lose part of their identity when kept in cell culture.

Plasticity of Lh cells caused by cell proliferation and recruitment of existing cells.

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control reproduction in vertebrates. Using a transgenic line of medaka, in which green fluorescent protein expression is controlled by the endogenous lhb promotor, we studied development and plasticity of Lh cells, comparing juveniles and adults of both genders. Confocal imaging and 3D reconstruction revealed hypertrophy and hyperplasia of Lh cells in both genders from juvenile to adult stages. We show that Lh cell hyperplasia may be caused by recruitment of existing pituitary cells that start to produce lhb, as evidenced by time lapse recordings of primary pituitary cell cultures, and/or through Lh cell proliferation, demonstrated through a combination of 5-bromo-2'-deoxyuridine incubation experiments and proliferating cell nuclear antigen staining. Proliferating Lh cells do not belong to the classical type of multipotent stem cells, as they do not stain with anti-sox2. Estradiol exposure in vivo increased pituitary cell proliferation, particularly Lh cells, whereas pituitary lhb and gpa expression levels decreased. RNA-seq and in situ hybridization showed that Lh cells express two estrogen receptors, esr1 and esr2b, and the aromatase gene cyp19a1b, suggesting a direct effect of estradiol, and possibly androgens, on Lh cell proliferation. In conclusion, our study reveals a high degree of plasticity in the medaka Lh cell population, resulting from a combination of recruitment and cell proliferation.

Gnrh1-Induced Responses Are Indirect in Female Medaka Fsh Cells, Generated Through Cellular Networks.

Reproductive function in vertebrates is stimulated by GnRH that controls the synthesis and release of the two pituitary gonadotropins, FSH and LH. FSH and LH, which regulate different stages of gonadal development, are produced by two different cell types in the fish pituitary. This is in contrast to the situation in mammals and birds, and it enables investigation of their differential regulation. In the present study, we used fluorescence in situ hybridization to show that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This result was confirmed by patch-clamp recordings and by cytosolic Ca2+ measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, responded to Gnrh1 by biphasic alteration in action-potential frequencies and cytosolic Ca2+ levels. In contrast, both Fsh and Lh cells are able to respond to Gnrh1 in brain-pituitary tissue slices both electrically and by elevating the cytosolic Ca2+ levels. Using Ca2+ uncaging in combination with patch-clamp recordings and cytosolic Ca2+ measurements, we show that Fsh and Lh cells form homotypic and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka are indirect and probably mediated via Lh cells.

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish.

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 °C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries.

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question. The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Electrophysiological properties of pituitary cells in primary culture from Atlantic cod (Gadus morhua).

The aim of the present study was to explore the electrophysiological properties of pituitary cells from Atlantic cod (Gadus morhua), as a basis for future studies of the signaling pathways involved in the control of pituitary secretion in this species. Primary cultures of pituitary cells from maturing Atlantic cod were prepared by trypsin treatment and mechanical dispersion. Electrophysiological recordings were performed using the perforated patch clamp method. A subpopulation of large cells were selected for recordings. Spontaneous action potentials were observed in about 30% of the cells. The action potentials displayed a fast initial spike followed by a prolonged plateau. Correspondingly, the inward current elicited by depolarizing steps consisted of both a transient, tetrodotoxin-sensitive Na(+) component and a nifedipine-sensitive Ca(2+) component that was sustained when Ba(2+) replaced Ca(2+) as current carrier. The outward current was partially blocked both by 5 mM tetraethylammonium and 10 mM 4-aminopyridine. The voltage-activated ion channels present in these cells largely correspond to the ion channels of pituitary cells in other teleosts (goldfish, Carassius auratus, and tilapia, Oreochromis mossambicus) and mammals, although differences exist regarding the shape and duration of action potentials.