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Developmental Biology

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

Published: September 21st, 2021

DOI:

10.3791/62347

1Centre for Regenerative Therapies TU Dresden, 2German Center for Diabetes Research (DZD e.V.), Paul Langerhans Institute Dresden of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus of TU Dresden

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

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.

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 ....

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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 stud.......

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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+ .......

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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.......

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Nikolay Ninov received funding from the Center for Regenerative Therapies Dresden at TU Dresden and the German Center for Diabetes Research (DZD), as well as research grants from the German Research Foundation (DFG) and the International Research Training Group (IRTG 2251), Immunological and Cellular Strategies in Metabolic Disease. We are grateful to the Light Microscopy Facility at the CRTD for the support in all the imaging techniques. We thank the Fish Facility at the CRTD for all the fish technical assistance and support.

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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

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