Jonah Patel
Drosophila, or fruit flies, are commonly used in laboratories to study neurodegenerative diseases due to their short lifespans and simple husbandry. The climbing assay, or negative geotaxis assay, is a popular method for studying the effects of genetic mutations and environmental conditions on Drosophila climbing behaviour. This assay involves placing a known number of flies in a vial and tapping the vial against a hard surface to cause the flies to fall to the bottom. The flies' natural tendency is to then climb to the top of the vial, and the number of flies that climb past a marker in a set time is recorded. The climbing assay is a cost-effective method that does not require extensive training to obtain reproducible results. However, the traditional climbing assay has been criticised for its inability to detect mild locomotive defects.
| Characteristics | Values |
|---|---|
| Climbing assay popularity | Accessibility, ease of experimental setup, inexpensive, simple data collection |
| Climbing assay disadvantages | Tedious, time-consuming, requires large numbers of flies, performance variability |
| Climbing assay modifications | Increased threshold distance to 17.5 cm, increased experiment duration to 2 min |
| Drosophila climbing behaviour | Reduced following carbon dioxide exposure, impaired by genetic deficiencies |
| Drosophila climbing assays | Negative geotaxis assay, Rapid Iterative Negative Geotaxis (RING) assay, DART setup |
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What You'll Learn
Climbing assay sensitivity
The climbing assay is a popular method for studying neurodegenerative disorders, such as Parkinson's disease, due to the fruit fly's powerful genetic tools, inexpensive husbandry, and short lifespan. However, the traditional climbing assay has limitations in terms of sensitivity and variability. The standard assay may not detect mild locomotive defects, especially during the early stages of disease progression, as it typically measures the ability of flies to climb a height of 2 to 5 cm in 10 to 20 seconds. This has led to the development of more sensitive alternatives, such as the DART system, which can detect more subtle differences in phenotype and motor performance decline.
The climbing assay's popularity stems from its accessibility and ease of implementation. Experimental setups are simple and inexpensive, and data collection only requires basic image capture tools and software. However, the traditional manual methods are often tedious and time-consuming, leading to the development of automated quantification systems like FreeClimber. These automated systems eliminate systematic biases and provide high-throughput analysis.
The climbing assay procedure involves placing a known number of flies in a vial and tapping the vial against a hard surface to make the flies fall to the bottom. The flies' natural tendency to climb, known as negative geotaxis, is then observed as they attempt to climb up the sides of the vial. The number of flies that reach a set distance or height within a given time limit is recorded, either manually or through video recording.
To address the limitations of the traditional climbing assay, researchers have made modifications to increase sensitivity and reduce the number of flies needed. One such modification is to increase the threshold distance to be climbed to 17.5 cm and extend the experiment duration to 2 minutes, allowing for the detection of milder defects. Additionally, the DART system, a MATLAB-based software, has been shown to be more sensitive than the climbing assay in detecting motor performance decline induced by adult-onset neuronal expression of amyloid beta (Aβ) peptides.
The sensitivity of climbing assays can also be affected by external factors such as carbon dioxide (CO2) anaesthesia, which has been found to impair the climbing abilities of Drosophila melanogaster. Exposure to CO2, even for as little as five minutes, can result in climbing deficits that last up to 24 hours or longer, depending on the duration of exposure. Therefore, it is important to consider the potential impact of CO2 anaesthesia on the climbing performance of the flies when designing and interpreting climbing assay experiments.
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CNS and neurodegenerative diseases
The fruit fly, Drosophila, is a commonly used model organism for studying neurodegenerative diseases due to its short lifespan, simple husbandry, and powerful genetic tools. Drosophila exhibits a natural tendency to climb, which is known as negative geotaxis, and this behaviour can be quantified using a climbing assay. This assay can be used to study the effects of genetic mutations and environmental conditions on climbing behaviour, as well as to detect mild locomotor dysfunctions that may be indicative of neurodegenerative disorders.
The climbing assay typically involves placing a known number of flies in a vial and tapping the vial against a hard surface to cause the flies to fall to the bottom. The flies will then attempt to climb up the sides of the vial against gravity, and the number of flies that climb past a marker within a certain time period is recorded. This assay has been modified to increase sensitivity and reduce the number of flies needed, such as by increasing the threshold distance and duration of the experiment.
The expression of aggregative proteins associated with human neurodegenerative diseases in the Drosophila CNS, such as amyloid beta (Aβ), Tau, TDP-43, or expanded Htt, results in an accelerated decline in motor function with age. This decline in motor function is commonly used as a measure of neural output and disease progression in Drosophila models of neurodegeneration.
Neurodegenerative diseases of the CNS, including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis, are characterised by neuronal damage and inflammation. Inflammatory cells and their mediators can trigger basic mechanisms that drive neurodegeneration, such as apoptosis, and the immune system can be harnessed to clear deleterious products and cells. Regulatory T cells, for example, may play a role in controlling inflammatory processes within the CNS, and immunotherapeutic strategies are being explored as potential treatments for neurodegenerative diseases.
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Drosophila climbing behaviour
Drosophila, or fruit flies, are commonly used to study neurodegenerative diseases due to their short lifespans, simple husbandry, and powerful genetic tools. One way to measure disease progression is through the decline in motor performance, which is often quantified using a climbing assay.
The climbing assay is based on the negative geotaxis response, where flies have an innate tendency to climb up and against gravity. The traditional climbing assay involves placing a known number of flies in a vial and tapping the vial against a hard surface to cause the flies to fall to the bottom. The flies will then attempt to climb up, and the number of flies that pass a marker on the vial during a set time is recorded. This can also be recorded using a camera, and the data is used to calculate a performance index.
However, the traditional climbing assay has limitations, including the inability to detect mild locomotive defects due to the height to which the flies are required to climb. To address this, a modified version of the assay has been designed, increasing the threshold distance and duration. This modified assay is more sensitive and can detect milder defects, making it useful for screening candidate drugs for Drosophila mutants with locomotion defects.
Additionally, the standard climbing assay has been found to lack sensitivity and suffer from high variability, requiring large numbers of flies. As an alternative, a MATLAB-based software called DART has been proposed, which is more sensitive to motor performance decline and can be used with smaller cohorts of flies.
Another factor that can impact Drosophila climbing behaviour is carbon dioxide (CO2) anaesthesia, which is commonly used in laboratories for sorting or other work. CO2 exposure has been found to have unwanted physiological effects, including altered respiratory and muscle physiology, which can impact motor function behaviours such as climbing ability. Even a short exposure to 100% CO2 for 5 minutes can cause climbing deficits that last up to 24 hours, with longer exposures resulting in deficits that last for days.
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Climbing assay setup
Drosophila is commonly used to study neurodegenerative diseases due to its powerful genetic tools, cheap and simple husbandry, and short lifespan. The climbing assay is a widely-used measure of disease progression, which is usually quantified using a simple climbing assay.
The climbing assay setup typically involves the following steps:
Drosophila collection and preparation:
Firstly, collect 20 Drosophila flies using CO2 anaesthetization. Place the flies in a collection vial containing food and store the vials horizontally to prevent the flies from getting trapped in any liquids at the bottom. Incubate the flies for at least 21 hours at 22°C and 45% humidity, with a 12-hour light/dark cycle.
Assay setup:
The assay can be performed in a glass graduated cylinder sealed with a wax barrier film. Groups of flies are then transferred into the cylinder or empty vials, which are tapped to make the flies fall to the bottom. The natural tendency of the flies is to climb upwards, against gravity.
Data collection:
The number of flies that climb past a marker on the vial or cylinder during an allotted time is recorded. This can be done using a camera to record the flies' climbing behaviour, and the data is used to generate a simple percentage or performance index.
Assay variations:
To increase sensitivity and overcome the limitations of traditional climbing assays, some variations have been proposed. One method involves increasing the threshold distance to 17.5 cm and the experiment duration to 2 minutes, allowing for the detection of milder defects. Another variation is the DART setup, which is more sensitive to motor performance decline and can be used with smaller cohorts of flies.
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Assay results and analysis
The climbing assay is a widely used method for studying the effects of genetic mutations and environmental conditions on Drosophila climbing behaviour. The assay takes advantage of the natural tendency of flies to climb, known as negative geotaxis. The traditional climbing assay measures the number of flies that successfully climb over a height of 2 to 5 cm in 10 to 20 seconds. However, this assay has been found to lack sensitivity and suffer from high variability, requiring large numbers of flies. To address this issue, a modified version of the assay has been developed, which increases the threshold distance to 17.5 cm and the experiment duration to 2 minutes, allowing for the detection of milder defects and evaluation over time.
The results of the climbing assay are typically analysed by calculating the percentage of flies that reach a set distance above the base within a given time limit. Alternatively, a performance index or mean position at a time cut-off can be calculated. The data collection process is relatively straightforward, requiring only simple image capture tools and basic software. However, the analysis can be tedious and time-consuming, especially when dealing with large numbers of flies.
Several automated platforms have been developed to quantify climbing performance in Drosophila, such as FreeClimber, which uses detectors and trackers to identify the x,y-coordinates of flies across frames and evaluate their position versus time. These automated platforms offer the advantage of being able to identify subtle differences between genotypes and work with longitudinal datasets.
The climbing assay has been used to study the effects of genetic mutations associated with neurodegenerative diseases, such as amyloid beta (Aβ), Tau, TDP-43, and expanded Htt, on motor function decline. It has also been used to assess the effects of environmental factors, such as carbon dioxide (CO2) anaesthesia, on climbing ability. For example, studies have shown that exposure to CO2 can inhibit climbing ability for up to 24 hours, with longer exposure times resulting in deficits that last for days.
Overall, the climbing assay is a valuable tool for studying the effects of genetic and environmental factors on Drosophila climbing behaviour and motor function. The assay is accessible, cost-effective, and can provide insights into the progression of neurodegenerative diseases.
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Frequently asked questions
A climbing assay is a test that measures the climbing ability of Drosophila melanogaster, or fruit flies.
Drosophila is commonly used to study neurodegenerative diseases due to its short lifespan, powerful genetic tools, and simple husbandry.
The climbing assay is performed by placing a known number of flies in a vial and tapping the vial against a hard surface to knock the flies to the bottom. The flies will then attempt to climb up the sides of the vial, and the number of flies that reach a set distance within a certain time limit is recorded.
The climbing assay has been criticised for its lack of sensitivity and high variability, which requires a large number of flies to be tested. The assay is also time-consuming and tedious.
