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, a....

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.

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