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

Summary

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.

Abstract

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.

Introduction

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 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 Ca^{2+ 15,16}.

The challenge of using perforated patch is the potentially high series resistance. Series resistance (R_s) or access resistance is the combined resistance over the patch pipette relative to the ground. During patch-clamp recordings, the R_s will be in parallel with membrane resistance (R_m). R_m and R_s in parallel work as a voltage divider. With the high R_s, the voltage will fall over the R_s 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 ¹⁹). Also, R_s may vary during patch-clamp recordings, again leading to changes in the recorded current. Thus, false positives may occur in situations where R_s 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 Guérineau et al. 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.

Protocol

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 studied species. pH and osmolality should be adjusted with precise electronic equipment such as pH meter and freezing point osmometer respectively.

1. Make 500 mL of extra-cellular solution without Ca²⁺ (Ca²⁺ free EC): 150 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 4 mM glucose, 10 mM Hepes.
   1. Dissolve 4.38 g of NaCl, 186.37 mg of KCl, 61.89 mg of MgCl₂, 360.32 mg of Glucose and 1.19 g of HEPES in 450 mL of ultrapure (0.055 uS/cm at 25 °C, the resistivity of 18.2 MOhm) H₂O in a glass beaker using a magnetic agitator.
   2. Adjust the pH to 7.75 with NaOH.
   3. Transfer the solution into a 500 mL volumetric flask. Fill the volumetric flask with ultrapure H₂O until 500 mL.
   4. Mix the solution well before measuring the osmolality. Adjust to 290–300 mOsm with mannitol. Filter sterilize the solution using water vacuum system and 0.2 µm filter.
2. Make 500 mL of extra-cellular solution (EC): 150 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1.3 mM MgCl₂, 4 mM glucose, 10 mM Hepes.
   1. Dissolve 4.38 g of NaCl, 186.37 mg of KCl, 147.01 mg of CaCl₂, 61.89 mg of MgCl₂, 360.32 mg of Glucose and 1.19 g of HEPES in 450 mL of ultrapure H₂O in a glass beaker using a magnetic agitator.
   2. Adjust the pH to 7.75 with NaOH.
   3. Transfer the solution into a 500 mL volumetric flask. Fill the volumetric flask with ultrapure H₂O until 500 mL.
   4. Mix the solution well before measuring the osmolality. Adjust to 290–300 mOsm with mannitol. Filter sterilize the solution using water vacuum system and 0.2 µm filter.
3. Make 500 mL of extra-cellular solution with 0.1% bovine serum albumin (EC with BSA): 150 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1.3 mM MgCl₂, 4 mM glucose, 10 mM Hepes.
   1. Dissolve 4.38 g of NaCl, 186.37 mg of KCl, 147.01 mg of CaCl₂, 61.89 mg of MgCl₂, 360.32 mg of Glucose and 1.19 g of HEPES in 450 mL of ultrapure H₂O in a glass beaker using a magnetic agitator.
   2. Adjust the pH to 7.75 with NaOH.
   3. Transfer the solution into a 500 mL volumetric flask. Fill the volumetric flask with ultrapure H₂O until 500 mL.
   4. Mix the solution well before measuring the osmolality. Adjust to 290–300 mOsm with mannitol. Filter sterilize the solution using water vacuum system and 0.2 µm filter. Add 50 mg BSA.
4. Make 250 mL of intracellular solution (IC): 110 mM KOH, 20 mM KCl, 10 mM Hepes, 20 mM sucrose.
   1. Dissolve 1.54 g KOH, 327.75 mg of KCl, 595.75 mg of HEPES and 1.712 g of sucrose in 450 mL of ultrapure H₂O in a glass beaker using a magnetic agitator.
   2. Adjust the pH to 7.2 with (N-morpholino) ethane sulfonic (MES) acid.
   3. Transfer the solution into a 250 mL volumetric flask. Fill the volumetric flask with ultrapure H₂O until 250 mL.
   4. Mix the solution well before measuring the osmolality. Adjust to 280–290 mOsm (10 mOsm lower than EC) with sucrose. Filter sterilize the solution using water vacuum system and 0.2 µm filter.
5. Make 100 mL of 2% low melting agarose solution in Ca²⁺ and BSA free EC (2 g of low melting agarose in 100 mL of Ca²⁺ free EC). Dissolve using a microwave and place the melted agarose in a water bath at 40 °C. Let it cool down until it reaches 40 °C before use on the tissues.
   NOTE: EC, Ca²⁺ free EC may be stored at 4 °C for several weeks if sterile and IC solutions at -20 °C, for several months. Agarose can be stored for a week at 4 °C and can be reused 3–5 times by briefly microwaving. Excessive reuse will lead to evaporation and alteration of agarose and salt concentration and quality.
6. Make agar bridge:
   1. Heat 7.5 mm long borosilicate glass capillaries with outside diameter (O.D.) 2 mm and inside diameter (I.D.) 1.16 mm using a Bunsen burner at a focal point about 1/3 from one end until the glass starts to bend. Remove the glass from the flame and allow the glass to bend with an angle of about 120 degrees (Figure 2A).
   2. Melt 2% agar in normal EC without glucose using a microwave, and while the agarose is liquid fill the pre-made glass capillary using capillarity forces by putting one of the ends of the glass into the liquid and waiting for few minutes. Store bridges until use at 4 °C in EC without glucose.
7. Prepare borosilicate glass with filament patch pipettes using a suitable pipette puller. Refer to the pipette puller manual to obtain the desired electrode resistance.
   ​NOTE: The taper of the pipette should be as short as possible. Short pipette taper is achieved by using 4–6 pulling steps. The electrode resistance should be between 2 and 6 MΩ depending on cell size.
8. To avoid movement of tissue during the patch-clamp experiments coat the surface of the recording chamber with 0.1% of polyethylenimine (PEI) in borate buffer.
   1. Prepare 500 mL of borate buffer (25 mM):
      1. Dissolve 4.768 g of Na₂B₄O₇/10H₂O in 450 mL of ultrapure H₂O in a glass beaker using a magnetic agitator.
      2. Adjust pH to 8.4 with HCl.
      3. Transfer the solution into a 500 mL volumetric flask. Fill the volumetric flask with ultrapure H₂O until 500 mL and 0.2 µm filter sterilize the solution using water vacuum system.
   2. Prepare a 1% stock solution of PEI (1 mL 50% PEI to 49 mL 25 mM borate buffer) and a 0.1% PEI dilution by diluting 10 µL of 1% stock solution in 100 µL of borate buffer for final use.
   3. Add 2 mL of 0.1% PEI on the glass of the slice holder and let the coating incubate for 1 minute. Then briefly wash the glass twice with 5 mL of ultrapure H₂O and let air dry until use.
9. Prepare amphotericin B solutions.
   1. Make 60 mg/mL stock solution by dissolving 3 mg of amphotericin B powder in 50 µL dimethyl sulfoxide (DMSO), in a 1 mL tube protected from light. Vortex at the maximum speed for 30 s and sonicate for 15 min to homogenize. Store aliquots into 3–5 tubes at -20 °C and use one aliquot per day.
      NOTE: If the amphotericin B stock solution is not clear and yellow color but milky (still yellow) after 15–20 min sonication make a new stock solution.
   2. Make IC solution with amphotericin B by pipetting 2 mL of the IC into a clean glass beaker with more than 10 mL capacity. Add 8 µL of amphotericin B stock solution into the pipette solution and mix by pipetting. Wrap the beaker with aluminum foil and place on ice until use and renew it every 3 h.

2. Dissection and Slicing with Vibratome

1. Prepare the dissection tools before dissecting, including one sharp and one strong forceps with scissors. Clean the tools (tissue holder, brush, forceps) with ethanol and make sure that they are used only for dissection purposes and never in contact with fixed tissues. Keep them in a separate box to avoid contamination.
2. Euthanize the fish in cold ice water for 1 min.
3. Follow the subsequent steps to quickly dissect the medaka brain and pituitary with sharp forceps (use no more than 3 min).
   1. While holding the fish with the strong forceps severe the two optic nerves behind the eyes with scissors or sharp forceps.
   2. Open the dorsal part of the skull from the posterior to the anterior side by breaking the skull bones step by step with strong forceps.
   3. Peel off the skull on one side with strong forceps.
   4. Severe the spinal cord with scissors or sharp forceps.
   5. Gently, grab the spinal cord with the forceps, flip the brain from posterior to anterior and place it into a dish with the ice-cold Ca²⁺ free EC.
4. Fill a 1.5–2 cm³ metal mold with liquid agarose and place it on the ice.
5. Just before the agarose solidifies (at around 25–30 °C), gently grab the brain by the spinal cord with the forceps, dry the forceps with a piece of fine paper and put the tissue into the agarose. Quickly mix the agarose to dilute the traces of EC left around the tissue and orientate the tissue and let the mold on ice for a few seconds until the agarose hardens.
   NOTE: It is essential for this step to be fast. The agarose solidifies quickly as the metal mold is cooled down by the ice.
6. Trim a square block of agarose containing the tissue with a scalpel blade and glue it on the vibratome specimen holder using surgery (non-toxic) glue.
7. Fill the cuvette of the vibratome receiving the specimen (brain-pituitary) holder with ice-cold Ca²⁺ free EC.
8. Place the specimen holder into the vibratome and cut the agarose block to make parasagittal sections of 150 µm, using high frequency and the low speed for the sectioning. Collect the selected sections and place them into the patch-clamp recording chamber with 3 mL of ice-cold Ca²⁺ free EC.
9. Place the grid harp (Figure 2B) on the tissue section.
   NOTE: The harp will together with the coating stabilize the tissue and prevent it from moving during the patch-clamp recordings.
10. After the harp is in place, change the Ca²⁺ free EC medium to the normal EC with Ca²⁺ and BSA. Let the tissue rest for 10 min.

3. Perforated Patch-clamp and Electrophysiological Recordings

1. Before starting the experiment, chloride the silver wire electrodes by the following steps: First, use a fine sandpaper (p180) to clean the silver wire electrodes. Second, rinse with 2 mL, 70% ethanol. Third, dip the wires in 2 mL, 2–5% chlorine for 10 min. Finally, rinse with 2 mL, ultrapure H₂O.
2. Place the recording chamber with the brain-pituitary slice in the microscope stage and locate the target area (pituitary) using 10X objective and inspect the cells using 40X objective.
   NOTE: If the preparation is successful, very few round cells should be visible. These round cells are usually damaged cells that are detaching from the tissue.
3. Place the grounding electrode through the 2% agar bridge into the EC bath.
4. Locate a healthy cell using the 40X objective.
   NOTE: A healthy cell should be firmly attached to the tissue slice and not rounded up and detached from the slice.
5. Set the amplifier in voltage-clamp (VC; Figure 4A point 1) mode. Open the seal test window and press the bath configuration (Figure 4B point 1 and 2. Use a 5-mV pulse to monitor pipette resistance (Figure 4B point 3).
6. Backfill the tip of the patch pipette with the antifungal free IC solution before adding the IC solution with amphotericin B using a micro-filler syringe (see Figure 3).
7. Add a slightly positive air pressure into the patch pipette using 1 mL syringe.
   NOTE: This makes a small liquid flow from the patch pipette that avoid contamination of the tip.
8. Once in the bath, assess the patch pipette resistance and make sure no particles are attached to the tip of the pipette (Figure 4C).
   NOTE: A small piece of tape is attached onto the monitor displaying the video recording form the field of view. This makes the positioning of the patch pipette relative to cell easier when the objective is not focusing on the cell.
9. Guide the patch pipette down to the cell using micromanipulators.
   NOTE: Make sure that the pipette is clean by looking for small particles that may have attached to the tip of the pipette. Sudden changes in resistance may also be a result of particles stuck in the pipette tip.
10. Readjust the pipette when a few microns above the cell so that the tip of the pipette will touch the cell 1/3 from the middle of the cell, as shown in Figure 5. When touching the cell, release the pressure and apply the gentle suction to make a seal.
    NOTE: The time from the patch pipette enters the bath to a successful seal should be kept as short as possible. Not more than 1–1.5 min in order to avoid amphotericin B reaching the tip of the pipette and leak into the bath.
11. Immediately switch to the patch window in the patch-clamp software (Figure 4D point 1) and have a holding potential between -50 and -60 mV (Figure 4D point 2). Zero out the fast capacitance made up by the glass pipette (Figure 4D point 3).
12. Switch to the cell window in patch-clamp software (Figure 4E point 1 and 2) and start monitoring access resistance, Ra, displayed on the window. Zero out the membrane capacitance in the amplifier software (Figure 4E point 3) after sufficient access.
    NOTE: Sufficient access might take 10 to 30 min (Figure 4E point 2). A good access for current clamp recordings should be below 20 M .
13. Switch to current clamp, IC, in amplifier software window (Figure 4F point 1). Adjust the fast-capacitive currents in the I-clamp window of the amplifier software (Figure 4F point 2 and 3) to the same value as in 3.12. Check membrane potential (Figure 4F point 1).
    NOTE: Importantly, if the membrane potential is shallow (typical above -35 mV) the cell might be damaged. Cancel the recording and find a new cell.
14. After finding a healthy cell (firmly attached to the tissue with membrane potential below -40 mV), start the experimental recordings as detailed in the manufacturer's protocol ^33,34.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vibratome	Leica	VT1000 S
Chirurgical glue	WPI	VETBOND	3M Vetbond Tissue Adhesive
Stainless steel blades	Campden Instruments	752-1-SS
metal molds	SAKURA	4122
steel harp	Warner instruments	64-1417
PBS	SIGMA	D8537
Ultrapure LMP agarose	invitrogen	166520-100
patch pipettes	Sutter Instrument	BF150-110-10HP	Borosilicate with filament O.D.:1.5 mm, I.D.:1.10 mm
Microscope Slicescope	Scientifica	pro6000
P-Clamp10	Molecular Devices	#1-2500-0180	sofware
Digitizer Digidata 1550A1	Molecular Devices	DD1550
Amplifier Multiclap 700B Headstage CV-7B	Molecular Devices	1-CV-7B
GnRH	Bachem	4108604	H-Glu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-OH trifluoroacetate salt
pipette puller	Sutter Instrument	P-1000
amphotericin B	SIGMA	A9528	pore-forming antibiotic
polyethylenimine	SIGMA	P3143	50% PEI solution
microfiler syringe	WPI	MF28/g67-5
glass for the agar bridge	Sutter Instrument	BF200-116-15	Borosilicate with filament O.D.:2.0 mm, I.D.:1.16 mm Fire polished
Micro-Manager software			Open Source Microscopy Software
optiMOS sCMOS camera	Qimaging	01-OPTIMOS-R-M-16-C
sonicator	Elma	D-7700 singen
NaCl	SiGMA	S3014
KCl	SiGMA	P9541
MgCl2	SiGMA	M8266
D-Glucose	SiGMA	G5400
Hepes	SiGMA	H4034
CaCl2	SiGMA	C8106
Sucrose	SiGMA	84097
D-mannitol	SiGMA	63565
MES-acid	SIGMA	M0895
BSA	SIGMA	A2153