culturing drosophila third instar larvae for brain metabolite studies

مراقبة نقل مستقلب الدماغ في ذبابة الفاكهة باستخدام مستشعرات الحنق

The brain has high energy requirements due to the high cost of restoring ion gradients in neurons caused by neuronal electric signal generation and transmission, as well as synaptic transmission1,2. This high energy demand has long been thought to be met by the continuous oxidation of glucose to produce ATP3. Specific transporters at the blood-brain barrier transfer the glucose in the blood to the brain. Constant glycemic levels ensure that the brain receives a steady supply of glucose4. Interestingly, growing experimental evidence suggests that molecules derived from glucose metabolism, such as lactate and pyruvate, play an important role in the brain cells&#39; energy production5,6. However, there is still some debate about how important these molecules are for energy production and which cells in the brain produce or use them7,8. The lack of appropriate molecular tools with the high temporal and spatial resolution required for this task is a significant issue that has prevented this controversy from being completely resolved.
The development and application of several engineered fluorescent metabolic sensors have resulted in a remarkable increase in our understanding of where and how metabolites are produced and used, as well as how the metabolic fluxes occur during basal and high neuronal activity9. Genetically encoded metabolic sensors based on F&#246;rster resonance energy transfer (FRET) microscopy, such as ATeam (ATP), FLII12Pglu700&#181;&#948;6 (glucose), Laconic (lactate), and Pyronic (pyruvate), have contributed to our understanding of brain energy metabolism10,11,12,13. However, due to the high costs and sophisticated equipment required to conduct experiments on live animals or tissues, results in vertebrate models are still primarily limited to cell cultures (glial cells and neurons).
The emerging use of the Drosophila model to express these sensors has revealed that key metabolic features are conserved across species and their function can be easily addressed with this tool. More importantly, the Drosophila model has shed light on how glucose and lactate/pyruvate are transported and metabolized in the fly brain, the link between monocarboxylate consumption and memory formation, and the remarkable demonstration of how increases in neural activity and metabolic flux overlap14,15,16,17. The method presented here for measuring monocarboxylate, glucose, and ATP levels using genetically encoded FRET sensors expressed in the larval brain allows researchers to learn more about how the brain of Drosophila uses energy, which can be applied to the brains of other animals.
We show that this method is effective for detecting lactate and glucose in glial cells and neurons, and that a monocarboxylate transporter (Chaski) is involved in lactate import into glial cells. We also demonstrate a simple method for studying metabolite changes during increased neuronal activity, which can be easily induced by bath application of a GABAA receptor antagonist. Finally, we show that this methodology can be used to measure monocarboxylate and glucose transport in other metabolically significant tissues, such as fat bodies.

تسليط الضوء على المؤلف: التقدم في أبحاث استقلاب طاقة الدماغ باستخدام نموذج ذبابة الفاكهة

تصور أحادي الكربوكسيل والمستقلبات الأخرى ذات الصلة في دماغ يرقات ذبابة الفاكهة خارج الجسم الحي باستخدام أجهزة استشعار مشفرة وراثيا

The brain is a tissue with a high energy consumption that mostly uses glucose as fuel. On the other hand, experimental data suggests that other metabolites like ketone bodies and monocarboxylates might be useful as energy sources. However, there is a still debate on which brain cells are the producers and primary consumers.Lactate has been demonstrated to be a relevant metabolite in vertebrates to drive brain activity in vivo. Our group and others have shown in Drosophila the importance of monocarboxylate molecules to fuel the high energy requirements in neurons, as well as the need of glial-derived lactate and ketone bodies in memory formation. We have described monocarboxylate transporters in the Drosophila brain and determined that the transfer of lactate from glial cells to neurons is necessary to maintain the neural activity during high-demand periods.The main advantage is to have the possibility to determine the intracellular dynamic of glucose and its metabolites, such as lactate, pyruvate, and ATB in glial cells and neurons during basal and high neural activity. Additionally, to measure the transfer of these molecules in other living tissues, such as the fat bodies. The use of the Drosophila as in vivo model for the study of brain energy metabolism using a simple setup.This will allow the modeling of metabolic diseases or to better understand the energy management in the normal brain or during the development of neurodegenerative diseases.

Watch this Scientific Journal Video about Culturing Drosophila Third Instar Larvae for Brain Metabolite Studies at JoVE.com

استزراع يرقات ذبابة الفاكهة الثالثة لدراسات مستقلب الدماغ

للبدء ، ضع طبق بتري 60 ملم مغطى ب 1٪ أغاروز و PBS في غرفة وضع البيض. ضع قطرة من الخميرة السائلة في وسط الطبق لتشجيع وضع البيض. بعد ذلك ، أدخل 300 ذباب جيبسوفيلا في الغرفة.حافظ على الذباب عند 25 درجة مئوية لمدة ثلاثة أيام. استبدل طبق بتري في الخميرة المذابة الطازجة مرتين يوميا. في اليوم الرابع ، دع الذباب يضع البيض لمدة أربع ساعات.بعد استبدال الطبق ، اسمح للذباب بوضع البيض لمدة ثلاث ساعات أخرى على الطبق الطازج. بعد ثلاث ساعات ، ضع البلاك مع البيض على حرارة 25 درجة مئوية لمدة 24 ساعة. من هذه اللوحة ، اجمع 50 إلى 100 يرقة حديثة الفقس وضعها في قنينة بلاستيكية تحتوي على علف قياسي.

culturing-drosophila-third-instar-larvae-for-brain-metabolite-studies

Research

JoVE Journal

Neuroscience

Culturing Drosophila Third Instar Larvae for Brain Metabolite Studies

78 Views.  Universidad de Chile. للبدء ، ضع طبق بتري 60 ملم مغطى ب 1٪ أغاروز و PBS في غرفة وضع البيض. ضع قطرة من الخميرة السائلة في وسط الطبق لتشجيع وضع البيض. بعد ذلك ، أدخل 300 ذباب جيبسوفيلا في الغرفة.حافظ على الذباب عند 25 درجة مئوية لمدة ثلاثة أيام. استبدل طبق بتري في الخميرة المذابة الطازجة مرتين يوميا. في ...

Video: استزراع يرقات ذبابة الفاكهة الثالثة لدراسات مستقلب الدماغ

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the production of ATP from glucose and its metabolites, such as the monocarboxylates lactate and pyruvate. It is still unclear how this process is regulated or who the key players are, particularly in Drosophila.
Using genetically encoded F&#246;rster resonance energy transfer-based sensors, we present a simple method for measuring the transport of monocarboxylates and glucose in glial cells and neurons in an ex-vivo Drosophila larval brain preparation. The protocol describes how to dissect and adhere a larval brain expressing one of the sensors to a glass coverslip. 
We present the results of an entire experiment in which lactate transport was measured in larval brains by knocking down previously identified monocarboxylate transporters in glial cells. Furthermore, we demonstrate how to rapidly increase neuronal activity and track metabolite changes in the active brain. The described method, which provides all necessary information, can be used to analyze other Drosophila living tissues.

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain preparation.

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the ...

To begin, place a 60-millimeter Petri dish covered with 1%agarose and PBS in an egg-laying chamber. Put a drop of liquid yeast in the center of the plate to encourage egg-laying. Next, introduce 300 gypsophila flies into the chamber.Keep the flies at 25 degrees Celsius for three days. Replace the Petri dish in freshly dissolved yeast twice daily. On the fourth day, let the flies lay eggs for four hours.After replacing the dish, allow the flies to lay eggs for three more hours on the fresh plate. After three hours, place the plaque with the eggs at 25 degrees Celsius for 24 hours. From this plaque, collect 50 to 100 newly-hatched larvae and place them in a plastic vial containing standard feed.

Dissection of Third Instar Drosophila Larva for Brain Metabolite Imaging

To begin, place 25 millimeter glass cover slips into the wells of a six-well cell culture plate. Place a drop of polylysine in the center of each cover slip. After incubation is complete, wash each cover slip three times with distilled water.Then wash the cover slips two times with a calcium-free saline solution. Next, install the covers in a recording chamber and fill it with calcium-free saline. Collect the wandering third instar larvae from the desired genetic cross of drosophila.Transfer the larvae to a dish well, then wash the larvae three times with distilled water. Place the larvae in another glass dissection dish well containing 750 microliters of ice-cold, calcium-free saline solution. Now place a larvae under a stereo microscope.With a pair of forceps, make a transverse cut across the back of the abdomen. Push the jaw with the forceps to turn the larvae inside out. Observe the ventral nerve cord next to the jaw.Carefully remove the imaginal discs and remaining brain coddle tissues. Next, separate the ventral nerve cord with the central brain and optic lobes from the rest of the tissue. Transfer the ventral nerve cord to the recording chamber containing calcium-free saline solution.To avoid interference, attach the remaining nerves to the bottom of the cover slip with forceps. To perform experiments on fat bodies, proceed to isolate the fat tissues from the turned-out larva. Place the isolated fat bodies expressing the fret sensors in the recording chamber.To acquire images of tissues expressing sensors, turn on the illumination system of the microscope. Place the recording chamber containing ventral nerve cords or fat bodies on the stage of a fluorescent microscope. To view the GCaMP6f fluorescence, set the excitation wavelength to 488 nanometers.For metabolites such as laconic, pyronic, ATP, or glucose sensors, set the excitation to 440 nanometers. Now set the image acquisition at regular intervals for GCaMP6f and metabolite sensor. Place the water immersion objective, ensuring it remains submerged.Next, connect the recording chamber to a perfusion system. Use a low-flux peristaltic pump to extract the liquid from the chamber and maintain a constant flow of three milliliters per minute. Position the solution containing tubes 25 centimeters above the microscope stage.Before any stimulation, obtain a stable fluorescence baseline by allowing the recording solution to flow for five to 10 minutes. Now, replace the flow solution with the stimulation solution containing glucose, pyruvate, or lactate for five minutes. Capture the images of the stimulated ventral nerve cord that was exposed to 80 micromolar picrotoxin to assess neuronal activity.Laconic sensors in both glial cells and motor neurons respond to one millimolar lactate at a similar rate at the start of the pulse. However, the motor neurons reach a higher increase over the baseline during the five minute pulse. The glucose sensor's signal increased at a similar rate in the glial cells and neurons when exposed to five millimolar glucose.During the glucose pulse, however, the signal in neurons increases more than in glial cells. Knocking out Chaski transporter reduced lactate transport in glial cells. Picrotoxin-induced increases in neuronal activity resulted in a transitory drop in ATP levels in the soma of motor neurons.The laconic sensor was observed to be well-expressed in fat bodies. Increased fluorescence of the glucose sensors was seen when the fat bodies were exposed to increasing glucose concentration. Increased lactate and pyruvate concentrations resulted in a proportional increase in laconic and pyronic fluorescence.