Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo β-Cell Function

Summary

Here, the study presents a protocol for calcium imaging and glucose stimulation of the pancreatic β-cells of the zebrafish in vivo.

Abstract

The pancreatic β-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in β-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, β-cells experience an influx of Ca²⁺. Thus, imaging the glucose-stimulated Ca²⁺ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying β-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca²⁺ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how β-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate β-cell activity in vivo. Here, a detailed protocol for Ca²⁺ imaging and glucose stimulation to investigate β-cell function in vivo is presented. This technique allows to monitor the coordinated Ca²⁺ dynamics in β-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect β-cell function.

Introduction

The pancreatic β-cells exhibit the unique capability for insulin secretion in response to glucose. After a carbohydrate-rich meal, the blood sugar increases and enters the β-cells, where it is quickly metabolized to produce ATP. The increase in the intracellular ratio of ATP/ADP leads to the closure of the ATP-dependent K⁺ channels, depolarizing the cell membrane and activating the voltage-dependent Ca²⁺ channels. The rapid increase in intracellular Ca²⁺ stimulates insulin-granule secretion by the β-cells.

Imaging of the islet cells within the intact pancreas in mice is demanding and requires ....

Protocol

The previously established transgenic lines used in this study were Tg(ins:GCaMP6s;cryaa:mCherry)⁶, Tg(ins:cdt1-mCherry;cryaa:CFP)¹⁴. All experiments were carried out in compliance with European Union and German laws (Tierschutzgesetz) and with the approval of the TU Dresden and the Landesdirektion Sachsen Ethics Committees (approval numbers: AZ 24D-9168,11-1/2013-14, TV38/2015, T12/2016, and T13/2016, TVV50/2017, TVV 45/2018, and TVV33-2019). In this study, all live imaging in vivo and glucose injections, as well as experimental procedures were performed with zebrafish larvae that did not exce....

Results

Using this protocol, the glucose response of individual β-cells in their native environment was characterized. For this purpose, the zebrafish larva is mounted on a glass-bottom Petri dish. Using a 3D manipulator, a glass capillary was inserted into the circulation, targeting the SV (Figure 1 and Figure 2). This permits the injection of specific volumes of solutions with a defined concentration. Simultaneously, the glucose-induced influx of Ca²⁺ for a.......

Discussion

In this protocol, the Ca²⁺ dynamics of β-cells in their native microenvironment with single-cell resolution was explored. This is possible by stimulating the zebrafish β-cells with a glucose injection in the circulation while recording their Ca²⁺ dynamics using GCaMP6s.

The protocol provides three main advantages. First, researchers have demonstrated that zebrafish β-cells show a coordinated response to glucose stimulation in vivo^.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm diameter glass-bottom dishes	Mattek	P35G-1.5-14-C	We use this glass-bottom dish to mount the zebrafish larvae and perform confocal microscopy
Blue-Green filter cube	ZEISS	489038-9901-000	Filter Set 38 HE
Confocal Microscope	ZEISS	LSM 980
D-(+)-Glucose	Sigma-Aldrich	G7528
Excel (2016)			https://www.office.com/
Femtojet	Eppendorf	5252000013	This equipment is a pneumatic micropump, which allows precise volume delivery and is accompanied by a capillary holder. We use the micropump Femtojet (injection pressure between 500-1000 hPa; compensation pressure = 0 hPa; and delivery time = 1 second).
Femtotips, glass capillaries ready to be used.	Eppendorf	5242952008	This are ready to use glass capillaries that can substitute the pulled-capillaries.
FIJI, using ImageJ Version: 1.51c			https://fiji.sc/
Fluorescence lamp	ZEISS	423013-9010-000	Illuminator HXP 120 V
Glass capillaries 3.5"	Drummonds Scientific Company	3-000-203-G/X	We use these glass capillaries to prepare the injection capillaries by pulling them with a capillary -puller
Injectman	Eppendorf	5192000019	This equipment allows for 3D manipulation of the capillary holder
Low melting agarose	Biozym	Art. -Nr.: 840101	We use the agarose to mount the zebrafish onto the glass-bottom dish
Microloader tip for glass microcapillaries 0.5 – 20 µL, 100 mm	Eppendorf	5242956003	Long tips for loading the glass capillaries with the solutions
Micro-tweezers	Dumont Swiss made	0102-4-PO	We use the micro-tweezers to move the zebrafish larvae during the mounting and to cut off the tip of the glass capillary (eg. Dumont, size 4).
Mineral oil	Sigma-Aldrich	M5904	We use the mineral oil to calibrate the drop size to inject
P-1000 Next Generation Pipette Puller	Science Products	P-1000	We use this capillary puller to prepare the glass capillary using the Drumond capillaries. We use the P-1000 capillary puller with the following parameters: Heat: 650, Pull: 20, Vel: 160, Time: 200, Pressure: 500.
PTU (1-phenyl-2-thiourea )	Sigma-Aldrich	P7629	We use this compound to inhibit pigmentation during zebrafish development
Red Filter Cube	ZEISS	000000-1114-462	Filter set 45 HQ TexasRed
Stereo microscope	ZEISS	495015-0001-000	SteREO Discovery.V8
Syringe filters, sterile. Pore size 0.2 µm	Pall Corporation	4612	We use these to filter all the solutions and prevent the capillary needle clothing
Transgenic Zebrafish line:Tg(ins:cdt1-mCherry;cryaa:CFP); Tg(ins:GCaMP6s;cryaa:mCherry)
tricaine methanesulfonate (MS222)	Sigma-Aldrich	E10521	We use this compound to anesthetize the fish larvae