Name: healthy brain-pituitary slices for electrophysiological investigations of pituitary cells in teleost fish
Uploaded: 2018-08-16T12:30:00
Description: Watch this Scientific Journal Video about Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish at JoVE.com

We thank Ms. LourdesCarreon G Tan for her help maintaining the medaka facility and Anthony Peltier for the illustrative figures. This work was funded by NMBU and by the Research Council of Norway, grant numbers 244461 (Aquaculture program) and 248828 (Digital Life Norway program).

All animal handling was performed according to the recommendations for the care and welfare of research animals at the Norwegian University of Life Sciences, and under the supervision of authorized investigators.
1. Preparation of Instruments and Solutions
NOTE: All solutions should be sterile. Careful attention should be given to the pH and the osmolality (osmol/kg water) of all solutions, which should be carefully adapted to the extracellular environment of the stud...

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Electrophysiological recordings using the patch-clamp technique on brain-pituitary slices require careful optimization. Well-optimized protocols for conducting live-cell investigations specifically in teleosts are limited, with the majority of publications using protocols based on mammalian systems. In this regard, it is important to be aware of the fact that several physiological parameters like pH and osmolality are not only species dependent, but also very much dependent on whether the organism in question lives on la...

The pituitary is a key endocrine organ in vertebrates located below the hypothalamus and posterior to the optic chiasm. It produces and secretes six to eight hormones from the specific cell types. Pituitary hormones constitute an intermediate between the brain and peripheral organs and drive a wide range of essential physiological processes including growth, reproduction, and regulation of homeostasis. Similar to neurons, endocrine cells of the pituitary are electrically excitable with the ability to fire action potentials spontaneously 1. The role of these action potentials is cell dependent. In several cell types of the mammalian pituitary, action potentials can elevate the intracellular Ca2+ sufficiently for a sustained release of hormone 2. In addition, the pituitary receives both stimulatory and inhibitory information from the brain that affects the membrane potential of the cells 3,4,5,6. Typically, stimulatory input increases the excitability and often involves the release of Ca2+ from intracellular stores as well as increased firing frequency&#160;7. Understanding how the cell utilizes the ion channel composition and adapts to these input signals from the brain is key to understanding hormone synthesis and release.
The patch-clamp technique was developed in the late 1970s by Sakmann and Neher 8,9,10 and further improved by Hamill 11, and allows detailed investigations of electrophysiological properties of cells down to single ion channels. Moreover, the technique can be used for studying both current and voltage. Today, patch-clamping is the gold standard for measuring electrophysiological properties of the cell. Four major configurations of the tight seal patch-clamp technique have been developed 11; the cell-attached, the inside-out, the outside-out, and the whole-cell patch. The three first configurations are typically used for single ion channel investigations. For the fourth, following the cell-attached configuration, a hole in the cell membrane is made using sub-atmospheric pressure. This configuration also allows investigations of the ion channel composition of the whole cell 12. However, one limitation of this technique is that cytoplasmic molecules are diluted by the patch pipette solution 13 (Figure 1A), thus affecting the electrical and physiological responses of the studied cells. Indeed, some of those molecules may play important roles in the transduction of the signal or in the regulation of different ion channels. To avoid this, Lindau and Fernandez 14 developed a method where a pore-forming compound is added to the patch pipette. Following the cell-attached configuration, the compound will incorporate into the plasma membrane under the patch and slowly perforate the membrane creating electrical contact with the cytosol (Figure 1B). Several different antifungals such as nystatin 15 and amphotericin B 16, or surfactants such as the saponin beta-escin 17,18 can be used. These compounds create pores large enough to allow monovalent cation and Cl- diffusion between the cytosol and the patch pipette while preserving the cytosolic levels of macromolecules and larger ions like Ca2+ 15,16.
The challenge of using perforated patch is the potentially high series resistance. Series resistance (Rs) or access resistance is the combined resistance over the patch pipette relative to the ground. During patch-clamp recordings, the Rs will be in parallel with membrane resistance (Rm). Rm and Rs in parallel work as a voltage divider. With the high Rs, the voltage will fall over the Rs giving errors in the recordings. The error will become larger with larger currents recorded. In addition, the voltage divider is also frequency dependent creating a low-pass filter, thus affecting the temporal resolution. In effect, the perforated patch may not always allow recordings of large and fast currents like the voltage gated Na+ currents (for detailed readings see reference&#160;19). Also, Rs may vary during patch-clamp recordings, again leading to changes in the recorded current. Thus, false positives may occur in situations where Rs changes during drug application.
The electrophysiology on the sliced tissue was first introduced by the Andersen lab to study electrophysiological characteristics of the neurons in the brain 20. The technique paved the way for detailed investigations of single cells as well as cell-cell communications and cell circuits in a more intact environment. A similar technique for making pituitary slices was introduced in 1998 by Gu&#233;rineau et al.&#160;21. However, it was not before 2005, that brain-pituitary slice preparation was used successfully for patch-clamp studies in teleost 22. In this study, the authors also reported the use of perforated patch-clamp recordings. However, by far, most of the electrophysiological investigations of pituitary cells have been conducted in mammals, and only a handful of other vertebrates, including teleost fish 1,2,22,23. In teleosts, almost all studies were performed on primary dissociated cells 24,25,26,27,28,29,30.
In the present paper, we outline an optimized protocol for preparation of healthy brain-pituitary slices from the model fish medaka. The approach represents several advantages compared to primary dissociated cell cultures. First, the cells are recorded in a relatively preserved environment compared to dissociated cell culture conditions. Second, slice preparations allow us to study indirect pathways mediated by cell-cell communication 22, which is not possible in dissociated cell culture conditions. Furthermore, we demonstrate how to conduct electrophysiological recordings on the obtained tissue slices using the perforated whole-cell patch-clamp technique with amphotericin B as the pore-forming agent.
Medaka is a small freshwater fish native to Asia, primarily found in Japan. The physiology, embryology, and genetics of medaka have been extensively studied for over 100 years 31, and it is a commonly used research model in many laboratories. Of particular importance to this paper is the distinct morphological organization of the hypothalamus-pituitary complex in teleost fish: Whereas in mammals and birds the hypothalamic neurons release their neuro-hormones regulating pituitary endocrine cells into the portal system of the median eminence, there is a direct nervous projection of hypothalamic neurons onto the endocrine cells of the pituitary in teleost fish 32. Thus, carefully conducted brain-pituitary slicing is of particular importance in fish, allowing us to investigate electrophysiological characteristics of the pituitary cells in a well-preserved brain-pituitary network, and in particular how pituitary cells control their excitability and thereby Ca2+ homeostasis.

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	</tbody>
</table>

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 &#176;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.

This protocol demonstrates a step by step protocol of how to achieve reliable electrophysiological recordings from pituitary (gonadotrope) cells, using a medaka transgenic line [Tg(lhb-hrGfpII)] where the target cells (Lh-producing gonadotropes) are labeled with green fluorescent protein (GFP).
Initially, the electrophysiological investigations were conducted using whole-cell configuration. However, spontaneous action p...

Watch this Scientific Journal Video about Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish at JoVE.com

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

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

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