Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Podsumowanie

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

Streszczenie

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Wprowadzenie

Circadian clocks help organisms to adapt to the periodic environmental changes generated by the 24 h rotation of the Earth. The interlocked transcriptional-translational feedback loops commonly underlie the molecular machinery of circadian clocks across species¹. The circadian pacemaker circuit composed of clock-containing neurons integrates time-of-day information conveyed by the environmental cues, such as light/dark (LD) and temperature cycles, to orchestrate the rhythms of a plethora of daily physiological and behavioral processes^2,3. The coordination of molecular rhythms with neuronal inputs and outputs is critically important for the operation of the circadian circuit but remains only partially understood.

In Drosophila, at the core of the molecular clock, the CLOCK/CYCLE (CLK/CYC) heterodimer activates the transcription of period (per) and timeless (tim). PER and TIM form a complex and enter the nucleus, where they inhibit transcriptional activity of CLK/CYC and consequently their own transcription. Post-transcriptional and post-translational regulations cause delays between CLK/CYC-mediated transcription and repression by PER/TIM, ensuring the generation of circa 24-h molecular oscillations^1,3,4. About 150 neurons containing these molecular clocks form a circuit to control circadian behavior of adult flies⁵. A much simpler yet fully functional circadian circuit consisting of 3 groups of clock neurons - 5 ventral Lateral Neurons (LNvs; 4 PDF-positive LNvs and one PDF-negative LNv, see below), 2 Dorsal Neuron 1s (DN1s) and 2 Dorsal Neuron 2s (DN2s) - is present in the larval brain^6,7.

The simple larval circadian circuit offers an excellent model to study the interactions between inter-neuronal communication and the molecular clockwork. Using our newly developed fluorescent reporter PER-TDT, which mimics the levels of PER protein and its subcellular location, we sought to characterize the dynamics of the molecular clockwork in different clock neuron subgroups in the larval circadian circuit⁸. Furthermore, knowing the key role of the neuropeptide pigment-dispersing factor (PDF) produced by 4 LNvs in regulating circadian rhythms at the neuronal level^9,10,11, we wanted to examine the direct effect of PDF on the molecular clocks. To this end, we developed a method to monitor circadian gene expression rhythms in larval brain explant over multiple days by time-lapse confocal microscopy. The protocol was also adapted for pharmacological assays to test the effect of PDF or other compounds on the level of PER-TDT. Thus, the adaptation consists of making the brain explant culture accessible to drug application, increasing temporal resolution and imaging for a shorter duration.

Ex vivo culture of Drosophila brains of different developmental stages have been previously established^{12,13,14,15,16,17,18}. Whereas these protocols have been used for imaging various biological phenomena, some of them are not compatible with imaging at single-cell resolution or do not support the culture for more than several hours. Alternative methods to perform long-term live imaging of circadian neurons in Drosophila include bioluminescence imaging of molecular rhythms^19,20,21 and fluorescence imaging of calcium indicator with light sheet microscopy^22,23. Although bioluminescence imaging can achieve higher temporal resolution and light sheet microscopy can be adaptable for in vivo imaging, they are limited on the spatial resolution and require specialized microscope systems.

The method described here is tailored to visualize fluorescent signals in the whole brain culture at single-cell resolution over multiple days. This facile and versatile method could be adapted to image cultured adult fly brains and for pharmacological experiments to study many different problems in Drosophila neurobiology.

Protokół

1. Preparation of Stock Solutions Under a Culture Hood

1. Prepare 400 mL of 1x Schneider Active Medium (SAM) optimized for ex vivo culture of larval brains (modified from reference^24,25) (Table 1). Aliquot to 5 mL and flash freeze in liquid nitrogen (LN₂), store at - 80 °C.
2. Prepare 1x Dissecting Saline Solution (DSS) by diluting 10x stock solution (modified from reference²⁶) (Table 2).
3. Aliquot fibrinogen to 10 mg and store at 4 °C for a single use.
   Caution: Weigh fibrinogen under laminar flow, wearing gloves, as it is harmful.
4. Prepare thrombin stock solution in SAM (1500 NIH unit/mg) and aliquot to 10 µL, flash freeze in LN₂ and keep at -80 °C.
   Caution: Wear gloves when handling thrombin as it is harmful.
5. Aliquot stock solution of 100x Penicillin-Streptomycin. Keep at -20 °C.
6. Cut circles of polytetrafluoroethylene (PTFE) membrane (see Materials Table) to the size that fits the inside of a glass bottom dish. Rinse them in 70% EtOH and then in autoclaved dH₂O. Sterilize and dry under laminar flow with UV. Keep in a sterile Petri dish. Single use.
   NOTE: PTFE is a porous flexible membrane that allows gas exchange and prevents medium from evaporating during long-term recording.

2. Time-lapse Microscopy Setup

NOTE: The following setup is for a tandem scanner inverted confocal microscope (see Materials Table) equipped with a resonant scanner (8,000 Hz). The system includes a highly sensitive photo-multiplier detector, which together with the resonant scanner prevents phototoxicity and photobleaching. Temperature and humidity are controlled within a microscope environmental chamber (see Materials Table) that covers most of the microscope.

1. Place a stage-top humidity chamber.
2. Turn on the temperature and humidity controllers to equilibrate the microscope chamber to 25 °C and 80% humidity.
3. To perform experiments in constant darkness (DD), cover the microscope environmental chamber with an opaque cloth (unless the microscope chamber is made of an opaque material).
4. If using a water-immersion objective, position a Water Immersion Micro Dispenser on top of the objective and program a protocol with the Autoimmersion Objective Controller software to dispense water at a constant rate during time-lapse imaging.
   NOTE: Water-immersion objectives together with a water dispenser or dry objective lens are recommended for long-term live imaging, as other type of immersion medium would dry out.
5. Place a Petri dish holder on the stage, inside of the humidity chamber.
6. Launch the microscope software and select the resonant scanner mode from the first dialogue box. Select OK to initialize the stage when prompted. Go to Configuration tab and to Hardware configuration to switch on the relevant laser lines.
7. Go to Acquire tab and to XY panel to select bidirectional mode and set image acquisition parameters.
   NOTE: The described imaging setup presented here (Figure 2, Figure 3 and Movie 1) is optimized to observe circadian expression of specific fluorescent reporters. The following parameters were chosen to best fit our specifications: 40X water immersion objective, 8X line average with 512 × 512 image resolution. For multicolor imaging, sequential image acquisition is required when the emission wavelength of one fluorophore and the excitation wavelength of another overlap (here, wavelengths of Yellow Fluorescent Protein (YFP) emission and Tomato excitation overlap). Choose the sequential mode "in between stack" as it is the fastest and the movement of clock neurons is negligible during the acquisition of a Z-stack. Microscopy settings should be adapted to suit the aim of each experiment.

3. Setup of Larval Brain Explant Culture

NOTE: The whole procedure from dissection to embedding of brain explants takes ~30 min.

1. Preparation of reagents under a culture hood.
   1. Thaw an aliquot of thrombin and keep at room temperature until the embedding step (step 3.3). Single use.
   2. Thaw quickly an aliquot of SAM at 37 °C and keep on ice for a single use.
   3. Add SAM to a 10 mg aliquot of fibrinogen to 10 mg/mL, flick gently and incubate at 37 °C for at least 30 min and during the embedding step (step 3.3). Single use.
      NOTE: Do not incubate the fibrinogen solution more than 2 h as it starts to polymerize.
   4. Thaw an aliquot of 100x stock Penicillin-Streptomycin. Prepare 2.5 mL of SAM containing 1x Penicillin-Streptomycin (SAM/antibiotics). Keep at room temperature.
      NOTE: Store the remaining 100x stock Penicillin-Streptomycin at 4 °C up to 1 month.
   5. Prepare an aliquot of 500 µL from the remaining SAM. Keep on ice.
   6. For long-term imaging, apply autoclaved vacuum grease to the plastic part of the bottom of the glass bottom dish (Figure 1A) and sterilize under laminar flow with UV.
2. Dissection of the central nervous system and ventral nerve cord.
   1. Clean the forceps and 2 x three-well glass dissection dishes with 70% EtOH. Pour 400 µL of DSS into 4 wells and 400 µL of SAM in one well. Keep on ice.
   2. Scoop out fly medium containing larvae and pick non-wandering 3rd instar larvae (L3) with forceps. Rinse them successively in 2 DSS-filled wells. Keep them in the 3rd DSS-filled well on ice that anesthetizes the larvae.
      NOTE: L3 lasts approximately 2 days at 25 °C. To observe the brain in physiologically relevant timeframe we chose to use non-wandering L3 larvae. Other subgroups of clock neurons, such as l-LNvs and DN3s, start to be formed at wandering L3 stage but they are not necessarily cycling yet (data not shown). Therefore, although it is possible to image cycling clock neurons at this stage (data not shown), it is important to carefully distinguish the already functional larval clock neurons from developing ones for data analysis. Non-wandering L3 appear approximately 4 to 4.5 days after egg laying at 25 °C. To use this protocol for studying circadian rhythms, synchronize (entrain) the developing larvae in 12 h/12 h LD cycles at 25 °C for 3 days before collecting L3 larvae.
   3. Dissect larval brains with fine forceps in the 4th DSS-filled well, which has not been used for rinsing larvae, using the inside-out method²⁷.
      1. Briefly, cut the larva in two, gently pinch the mouth hooks with a pair of forceps and roll up inside-out the body wall using the other pair of forceps, thus exposing the brain. Free the brain by cutting out all the trachea and nerves that attach it to the rest of the body.
      2. Aspirate the brain in about 3 µL of DSS with a 20 µL pipette (pre-rinsed with DSS) and dispense in the SAM-filled well. Repeat the procedure for up to 7 brains.
         NOTE: Dissection should be performed on a cold surface to keep the larvae anesthetized and the brain chilled. For example, place the dissection dish on a cooling block connected to a pump to circulate ice-chilled water. The dissection procedure takes ~30 s per brain. It is important to keep the eye/antennal imaginal discs attached to the brains and the ventral nerve cord intact. Tearing off these parts will damage the brain and reduce the quality of the culture. Also, avoid exposing the brains to the air. Before aspirating the first brain, pipette up and down the DSS containing parts of the dissected larvae, as it prevents the brains from sticking to the wall of the tip.
3. Embedding of brain explants in a 3D matrix and mounting (~20 min).
   1. Submerge a dissected brain in fibrinogen working solution (fibrinogen final concentration ~3.3 mg/mL, 10.5 µL per brain) as follows:
   2. Aspirate 3.5 µL of SAM/antibiotics (with the same tip used in section 3.2.3). Aspirate a dissected brain from the dissection well into the same pipette tip without dispensing the SAM/antibiotics medium in the tip.
   3. Dispense the brain and the medium on the lid of a sterile Petri dish. Add another 3.5 µL of SAM/antibiotics and 3.5 µL of warm fibrinogen/SAM 10 mg/mL solution on the drop containing the brain. Mix the solution around the brain gently by pipetting with a 20 µL pipette tip.
      NOTE: Using a pipette rather than forceps in this step avoids stressing the brain.
   4. Take 2 µL of the fibrinogen working solution from the drop and apply it on the coverslip of the glass bottom dish as a small circle. Add 0.8 µL of thrombin and mix very quickly with a pipette tip. The fibrinogen is converted to fibrin and starts to polymerize.
   5. Take the brain that sits in the fibrinogen working solution (step 3.3.1) between a pair of forceps and position it on the matrix once a white matrix of fibrin is visible. Orient the brain anterior side down (for imaging clock neurons). Push it down as close as possible to the cover glass. Fibrin clot is composed of layers of fibrin. Pick one layer and fold it on top of the brain with small and gentle movements without detaching the matrix.
      1. Repeat 2 to 3 times from different parts of the clot to stabilize the brain.
         NOTE: When handling the brain with forceps, be careful not to dry it or impose it a physical stress.
   6. Add 20 µL of SAM/antibiotics on top of the embedded brain to stop the action of thrombin.
   7. Repeat the procedure to mount the remaining brains. It is possible to embed up to 5 to 6 brains on a glass bottom dish.
   8. For long-term live imaging, add 600 µL of SAM/antibiotics in the inner well (the glass part). Position a pre-cut PTFE membrane on top of the medium and stick it to the vacuum grease applied on the plastic part of the bottom (3.1.5) (Figure 1A).
   9. For pharmacological assays, fill the dish with 2 mL of SAM/antibiotics (Figure 1B).
      NOTE: It is very important to avoid evaporation of the medium, as the osmolality of the medium is critical to the viability of neurons. Therefore, for long-term imaging experiments, it is necessary to seal the culture dish with PTFE membrane. Whereas for short-term experiments, sealing membrane is not required as long as the dish is filled with 2 mL of medium and monitored in a humidified chamber.

4. Time-lapse Image Acquisition

1. Place the glass bottom dish on the Petri dish holder inside the stage-top humidity chamber.
2. Go to z-stack panel in the Acquire tab and set the z-section steps from the microscope software (2 µm × ~40 in the experiments shown in Figure 2 and Figure 3, and Movie 1). Set the beginning of a z-stack at the plane farthest from the coverslip and the end at the surface of the brain explant, close to the coverslip. Though brain movements are negligible, add extra z-sections at the beginning and end of the z-stack.
3. Go to Mark & Find panel and set-up multi-position image acquisition. With a 40X objective, 1 brain hemisphere can be captured within a field of view
4. Go to Acquisition mode panel and select xyzt (z-stack time-lapse). Go to Time Acquisition panel and set the preferred time interval and frequency parameters.
   NOTE: Acquire time-lapse images with desired frequency. Note that long-term live imaging (24-72 h) with time lapse interval shorter than 2 h tends to results in fewer cycling neurons, probably because it reduces the viability of neurons. The data volume expands as the rate of image acquisition increases, which makes data analysis and storage more challenging.

5. Pharmacological Treatment of brain Explants with Short-term Live Imaging

NOTE: The following procedure is essentially the same as that for long-term imaging but does not require a PTFE membrane. To avoid exposing the brains to blue light when the chemical is added to the culture, the microscope should be placed in the dark room with red light.

1. Go to Time Acquisition panel and set the desired time interval and number of scan to obtain the baseline data before drug application. Start Scan.
2. After the baseline scan is completed, lift the scan head of the microscope system. Remove the lid of the stage-top humidity controller and very carefully remove the lid of the glass bottom dish without moving it.
3. Remove the appropriate amount of culture medium if necessary. Add the experimental solution in the medium and, carefully and very gently, mix with a sterile Pasteur pipette without touching the brain explants.
   NOTE: For bath application of PDF, use 2 µM PDF stock solution (in 10% DMSO).
4. Replace the lids of the glass bottom dish and the humid chamber, and the scan head. If using, reposition the black opaque cloth around the microscope chamber.
5. Check if the brains are at their original positions in live scan mode. Adjust if necessary.
6. Go to Time Acquisition panel and set the desired time interval and number of scan. Start Scan.
   NOTE: Here we tested time-lapse images acquired every hour for up to 10 h.

Wyniki

Here, we show the representative results of the long-term recording of a circadian fluorescent reporter in ex vivo larval brain culture, and the live imaging results of PDF bath application on reporter expression.

Non-wandering L3 larvae expressing the molecular clock reporter PER-TDT and UAS-mCD8::YFP driven by a clock neuron driver Clk(856)-gal4 (Figure 1C) were entrained in LD. ...

Dyskusje

Here we described the method for long-term fluorescence time-lapse microscopy of cultured larval brains. The success of this type of experiments depends on multiple factors, such as the health of the culture, method for immobilization of the brain explant, fluorescence intensity and signal-to-noise ratio of the reporter, temporal and spatial resolution, and accessibility to the explant. These factors can be mutually exclusive. For example, increasing time-lapse frequency affects the health of the culture, and the immobil...

Materiały

Name	Company	Catalog Number	Comments
KH2PO4	Sigma-Aldrich	P5655	I am not sure they are exactly the same ones we have in the lab. I chose "suitable for insect cell culture" whenever available
CaCl2	Sigma-Aldrich	C7902
MgSO4.7H2O	Sigma-Aldrich	230391
NaCl	Sigma-Aldrich	S5886
NaHCO3	Sigma-Aldrich	S5761
D-(+) Glucose	Sigma-Aldrich	G7021
Yeast extract	Sigma-Aldrich	Y1000
Insulin	Sigma-Aldrich	I0516-5ML
Penicillin-Streptomycin	Sigma-Aldrich	P4333
BIS-TRIS	Sigma-Aldrich	B4429
L-(−)-Malic acid	Sigma-Aldrich	M7397
D-(+)-Trehalose dihydrate	Sigma-Aldrich	T0167
Succinic acid	Sigma-Aldrich	S9512
Fumaric acid	Sigma-Aldrich	F8509
α-Ketoglutaric acid	Sigma-Aldrich	K1128
Non-heat-inactivated, Foetal Calf Serum (FCS) Mycoplasma and Virus screened	BioConcept Ltd. Amimed	2-01F30-I
HEPES-KOH, pH 7.4	E&K Scientific Products	EK-654011
KCl	Sigma-Aldrich	P5405
NaH2PO4	Sigma-Aldrich	S5011
Sucrose	Sigma-Aldrich	S7903
Fibrinogen from bovine plasma	Calbiochem (Merck)	341573-1GM	CAUTION: Harmful by inhalation, in contact with skin and if swallowed. Manipulate under laminar flow
Thrombin from bovine plasma	Sigma-Aldrich	T9549	CAUTION: Health Hazard, use gloves
PDF, NH2-NSELINSLLSLPKNMNDA-OH	Chi Scientific	custom made
Vaccum grease	Sigma-Aldrich	18405
35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated	MatTek	P35G-1.5-20-C
Corning Falcon Easy-Grip Tissue Culture Dishes	fisherscientific	08-772A
Sterile 500 mL Steritop-GP 33 mm threaded bottle top filter, 0.22 μm	Millipore	SCGPS05RE
Polytetrafluoroethylene (PTFE) film	Dupont	200A Teflon FEP Film
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 33 mm, gamma sterilized	Millipore	SLHV033RS
Millex-GV Syringe Filter Unit, 0.22 µm, PVDF, 33 mm, gamma sterilized	Millipore	SLGV033RS
Three-well glass dissection dish	Any company
Fine forceps, size 5, Dumont	Fine Science Tools	11254-20
Tandem scanner inverted TCS SP5 confocal microscope, with resonant scanner and HyD photo-multiplier detectors	Leica microsystem CMS GmbH
Temperature control chamber	Life Imaging Services	The CUBE & BOX temperature control system, custom designed
Stage-top humidity controller	Life Imaging Services	custom made
Water Immersion Micro Dispenser: dispenser, extended micro-pump MP6 series and Autoimmersion Objective Controller software	Leica microsystem CMS GmbH
SUM-stack creation and 3D correction drift plugin	ImageJ software
10x iterative deconvolution	AutoQuant and Imaris software