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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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 ...

This method can help answering key questions in the field of neuroendocrinology. Such as how do pituitary cells communicate, respond to releasing factor from the brain? The main advantage of this technique is that compared to this first primary cell culture this procedure allows the investigation of cells in a more intact environment.Although this method can provide insight into the electrical properties of luteinizing hormone producing cells in Medaka. With only minor adjustments, it can also be used for other cell types or for other fish species. While holding a euthanized Teleost fish with ethanol sterilized strong forceps under a dissecting microscope, sever the two optic nerves behind the eyes with sharp forceps and break the skull bones from the posterior to the anterior side.Use the forceps to peel off the skull and to sever the spinal cord. Gently grasping the spinal cord with the forceps flip the brain from the posterior to the anterior orientation, and place the organ in a dish filled with ice cold, calcium free, extra cellular medium. Next fill a one point five to two cubed centimeter metal hold with liquid agarose and place the mold on ice.Just before the agarose solidifies, use the forceps to gently pick up the brain by the spinal cord, dry the forceps with a piece of lab paper, and place the brain into the agarose. Then quickly mix the agarose to dilute any traces of extra cellular medium left on the tissue and adjust the orientation of the brain within the mold as necessary. When the agarose has solidified, use a scalpel blade to trim a square block of agarose around the tissue and use surgical glue to attach the sample onto a vibratome specimen holder.Fill the cuvette of the vibratome holder with ice cold, calcium free extra cellular medium and place the holder into the vibratome. Using a high frequency and a low speed, cut the agarose block to make 150 micrometer parasagittal sections, placing the selected section into the patch-clamp recording chambers containing three milliliters of ice cold, calcium free extra cellular solution. It is important to move quickly once the brain has been added to the agarose, as the tissue will dry out if left in the molded agarose for too long and has limited access to air before it is sectioned.When all of the sections have been obtained, place a grid harp onto the tissue section and replace the calcium free extra cellular medium with calcium containing extra cellular medium supplemented with bovine serum albumen. Then let the tissue rest for 10 minutes. To set up the patch-clamp, place the recording chamber with the brain pituitary slice onto the patch-clamp recording microscope stage, and use the 10X objective to locate the pituitary gland.Place the grounding electrode through the 2%agar bridge into the extra cellular medium bath, and use the 40X objective to locate a healthy cell. Set the amplifier to voltage clamp mode and open the seal test window. Select the bath configuration and use a five millivolt pulse to monitor the pipette resistance.Backfill the tip of the patch pipette with antifungal free intra cellular medium before using a microfiller syringe to add intra cellular solution supplemented with Amphotericin B to the posterior section of the patch pipette. Use a one milliliter syringe to add a slightly positive air pressure into the patch pipette to avoid contamination of the pipette tip and assess the patch pipette resistance. If no particles are attached to the tip of the pipette, use the micromanipulators to guide the patch pipette down to the cell.Readjusting the pipette when it is a few microns above the cell so that the tip of the pipette will touch the cell a third of the distance from the middle of the cell. To touch the cell, release the pressure and apply gentle suction to make a seal. It's essential to apply the gigaseal quickly as positive pressure within the patch pipette will result in antifungal solution leakage after about one minute damaging the cells.When the seal is in place, immediately switch to the patch window in the patch-clamp software and adjust the holding potential to between negative 50 and negative 60 millivolts. Zero out the fast capacitance made up by the glass pipette and switch to the cell window to begin monitoring the access resistance, zeroing out the membrane capacitance in the amplifier software after sufficient access. Switch to the current clamp and adjust the correct value of the fast capacitive currents of the pipette in the I-Clamp window, and check the resulting membrane potential.Then with the cell firmly attached to a tissue with a membrane potential below negative 40 millivolts, begin the experimental recordings according to the manufacturer's protocol. Action potentials can be triggered in a subset of pituitary cells using a five to nine picoampere current injections from a resting potential between negative 50 and negative 60 millivolts. These action potentials have a fast rundown and after about four to six minutes, triggered action potentials are no longer achievable.Switching to the perforated patch configuration using Amphotericin B, spontaneous action potentials can be observed in about 50%of the recorded cells and the action potentials can be triggered in 95%of these cells with no observable rundown, even after up to one hour of recordings. Gonadotropin releasing hormone one, puff ejected onto cells in the perforated patch configuration reveals a biphasic response, during which the cells are hyper-polarized in the first phase of the response resulting in calcium release from internal stores followed by de-polarization of the cells and increased action potential firing frequency from one to two hertz to three hertz in the second phase. While attempting this procedure, it is important to remember that all of the solutions should be adapted to the physiology of the experimental organism that you're using, in this case, Medaka, and you need to have particular attention to pH as this value varies across species living at different temperatures.

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