Research Proposal: Cold Nociception in Drosopila

Over the fall 2022 semester, I had the wonderful opportunity to participate in a Course-based Undergraduate Research Experience (CURE) at Georgia State University. I was able to conduct original research on cold nociception in Drosophila melanogaster under the supervision of Dr. Jamin Letcher. 

Georgia State University is home to some of the most knowledgeable experts in this field of research, making it a true honor to dive further into this topic under their guidance.

I would have not been able to complete my research without the collaboration of my incredible lab partner Anukruthi Venukadasula. Together we designed the following research proposal, executed various experiments, and performed data analysis. 

Nociception is the physiological response to a noxious, potentially damaging, stimulus. This behavior can be observed in Drosophila melanogaster, a commonly studied animal model, due to its simple nervous system. Drosophila larvae respond to extreme cold temperatures by presenting full-body contraction (CT). Prior research presents evidence of the involvement of Class III (CIII) neurons, known as nociceptors, which play an essential role in cold nociception. However, the specific interneurons downstream from the CIII neurons that participate in the cold nociception circuitry are still yet to be discovered. This study aims to understand the involvement of two neuronal populations, Ecdysone-induced protein 93F (EIP93F) and CG4168, in the cold nociception circuit. These neuronal populations are of interest due to their overlap with CIII md neurons in the ventral nerve cord. In this study, we hypothesize that EIP93F and CG4168 neurons are necessary for contraction behavior in Drosophila larvae in response to cold noxious stimuli. We also hypothesize that EIP93F and CG4168 neurons are sufficient for contraction behavior in Drosophila larvae in the absence of cold noxious stimuli. To investigate the necessity of our neuronal populations in cold nociception, we will silence the neurons using tetanus toxin (TNT) and then examine their response to noxious cold using a cold plate assay. We will investigate the sufficiency of our neuronal populations to evoke contraction without direct cold stimulus. To do so, we will cross the GAL4-neurons genotypes with the UAS-ChETA genotypes and activate them using optogenetics.

Aim 1: In our first experiment, we intend to test the necessity of the neuronal populations expressing EIP93F and CG4168 in cold nociception of Drosophila melanogaster. In order to assess the necessity of these neurons, we will cross our lines GAL4Eip93F and GAL4CG4168 with a UAS-TNT-E2 line. This process silences the GAL4 neurons and allows us to test the behavioral response without the activity of these neurons. We can observe the effects of silencing these neuronal populations by analyzing the contraction behaviors of GAL4-UAS-TNT larvae in response to cold plate assays.

Aim 2: In our second experiment, we aim to provide evidence that the neuronal populations expressing EIP93F and CG4168 are sufficient for contraction behavior in Drosophila melanogaster. We will use optogenetics to assess if our neuronal populations can produce contraction behaviors when activated by blue light. We will cross our GAL4Eip93F and GAL4CG4168 flies a UAS-ChETA-YFP line, a modified channelrhodospin-2 that responds to blue light, using the GAL4/UAS system. If the larvae contract when the neurons are activated via optogenetics, it will demonstrate sufficiency.

Nociception is the innate response to a potentially dangerous, noxious stimulus (1). Across species, extremely cold temperatures result in specified behaviors due to the damage to tissue at the injury site (2). The specified behavioral responses result in hypersensitized sensory neurons at the injury site for as long as the noxious stimulus lasts (1). For example, in Drosophila melanogaster, extremely cold temperatures may cause detrimental effects on fertility and probable death at early life stages (3). Commonly, as the animal senses a noxious stimulus, it produces a fight-or-flight response through physiological behaviors that allow it to mitigate the trigger (4). In Drosophila larvae, the predominant behavioral response to a noxious stimulus is a segmental CT, namely, forward movement (anterior to posterior segments) and backward movement (posterior to anterior segments) (5). The drawing back of the anterior segment is also known as head withdrawal (HW) (2). It is essential to understand the behaviors of animals to noxious triggers to avoid injury or death to the organism (4).

The two neuronal populations of interest in this study are those expressing EIP93F and CG4168. According to FlyBase.org, EIP93F is a protein-coding gene in Drosophila, and the homologous human gene is LCOR. LCOR controls maternal hormones like estrogen that signal fetal development and metabolism (6). CG4168 is another protein-coding gene in Drosophila and is homologous to various human genes. Although the protein encoded by CG4168 is poorly understood, one of the human orthologs is IGFALS. IGFALS encodes a protein that binds to insulin-like growth factor (IGF) (7). It is essential to understand that increased estrogen and insulin levels may contribute to breast cancer (8). Understanding the neural circuitry of EIP93F neurons and CG4168 neurons in Drosophila may lead to future experiments to understand the involvement of LCOR and IGFALS.

This study utilizes the Gal4/UAS system, a sufficient genetic tool to induce controlled gene expression in Drosophila larvae (9). The promoter region on Gal4 determines the spatial and temporal expression of Gal4 (9). Drosophila flies involve the Gal4-promoter driver line and the UAS-transgene line. The UAS-transgene line is crossed with the Gal4-promoter driver line, where the GAL4- protein sequence recognizes the UAS sequence and turns on the gene downstream of UAS (9).

This study utilizes optogenetics and the GAL4/UAS binary system to observe the neural circuitry of the transgenic Drosophila. Channelrhodopsin2 (ChR2) is a light-activated cation channel that provides the means of noninvasive activation of specified neurons to observe the neural circuitry (10). The modified ChR2 in this experiment offers a highly rapid, reliable, and precise photoactivation in the Drosophila larvae (10). In this experiment, we combine the Gal4/UAS system with optogenetics to activate modulatory neurons to observe the nociceptive responses in Drosophila larvae (10).

Drosophila melanogaster responds to noxious stimuli through peripheral sensory neurons in the epidermis layer of the larva (4). It is known that the peripheral nervous system of larvae includes four classes of type II multi-dendritic (md) neurons, and the CIII and CIV neurons are called nociceptors (2). Optogenetic experimental methods show that Class III nociceptors are crucial for sensing cold noxious stimuli (3). However, whether cold nociception involves specific interneurons is unknown, and to what extent they are involved (1). This study aims to understand the involvement of the neuronal populations EIP93F and CG4168 and whether they are activated by Class III neurons, in cold nociception. The study also attempts to provide evidence on the necessity and sufficiency of the gene lines EIP93F and CG4168. To investigate the necessity of our neuronal populations in cold nociception, we will silence the neurons using tetanus toxin (TNT) and then examine their response to noxious cold using a cold plate assay. We will investigate the sufficiency of our neuronal populations to evoke contraction without direct cold stimulus. To do so, we will cross the GAL4-neurons genotypes with the UAS-ChETA genotypes and activate them using optogenetics.

In order to complete our experiment, we used stocks containing flies expressing CG4168 and Ecdysone-induced protein 93F (Eip93F). These neuronal populations are of interest because they overlap the same region of the ventral nerve cord as the CIII axon terminals. This suggests they may be a part of the neuronal circuit downstream of the CIII md neurons. We also utilized a stock of Oregon-R flies, which is a wild type that can be used as a control for observing normal CT behavior.

To address our first hypothesis mentioned in our specific aims, we will begin by collecting female virgin flies from a UAS-TNT-E2 line. After collecting approximately 30 virgins, we will cross 15 female UAS-TNT-E2 flies each with 7 males from our GAL4Eip93F line and 7 males from our GAL4CG4168 line which produces +;UAS-TNT-E2/GAL4Eip93F;+ and +;UAS-TNT-E2/GAL4CG4168;+ flies to silence the neuronal populations. This process allows us to see if these neuronal populations are necessary for contraction behavior induced by noxious cold exposure.

Once the larvae mature to the third instar, we will complete cold plate assays in order to assess their contraction behavior. The cold plate assays will be performed on Oregon-R flies which serve as a control, +;UAS-TNT-E2/GAL4Eip93F;+ flies, and +;UAS-TNT-E2/GAL4CG4168;+ flies. To complete the cold plate assay, we will begin by setting the temperature of the cold plate to 6 degrees ºC (2). Then, we will prep our black aluminum sheets by misting them with distilled water. We will move some of our larvae from their tubes to a petri dish with a wet Kimwipe where we will remove any food particles and locate the third instars. Next, 3-4 larvae will be gently moved onto the metal arena at 25ºC (2). After the larvae begin to locomote, the black aluminum sheet will be transferred to the cold plate arena set to 6 ºC. Behavior in response to the cold plate exposure will be recorded for 45 seconds using a Nikon camera placed above the cold plate (2). Once the recording period is complete, the larvae will be disposed of and the experimental instruments will be cleaned.

After completing the data collection, the videos will be transferred to a computer where we will convert our videos into uncompressed raw files using Video to Video Converter and then use FIJI to measure the area of the larvae within the videos. We have set a threshold of a 10% loss in area to determine if a contraction occurred during the time period. We will then generate a table in Excel containing all of our samples to compare contraction behavior across our different control and experimental groups.

To address our second hypothesis mentioned in the specific aims, we will complete another set of crosses to produce flies that can be manipulated utilizing optogenetics. Optogenetics can allow for the activation of specific neuronal populations using light of a specific wavelength and can initiate CT behavior in larvae (2). We will use flies from a UAS-ChETA-YFP line, which allows us to activate our neuronal populations using blue light. Utilizing the GAL4-UAS system, we will cross GAL4Eip93F flies and GAL4CG4168 flies with UAS-ChETA-YFP flies. After completing the crosses, the experimental flies will be supplied with food containing all-trans-retinal (ATR), which is a required cofactor for the activation and function of ChETA(2).

Once we have obtained third instar larvae, we will be able to observe the effects of optogenetic activation for our specific neuronal populations. To do so, we will clean food particles off a single larva, move it onto a misted aluminum plate, and then wait for locomotion. We will then expose the larvae to light with a wavelength of 480 nm to activate the neuronal populations(2). Light exposure will include 3 consecutive 5-second pulses of blue light activation followed by a 10-second period of no light(2). Data will be captured on a Nikon camera, and then analyzed for contraction behavior using the same steps as described for the cold plate assays.

Some potential limitations of this study include human error involved in the handling of the larvae and the analysis of data. If the larvae selected are too young (second instar) or too old (pupae), we may not observe the expected contraction behavior. When the larvae are moved onto the metal arena, they may still have food particles attached if not properly cleaned, which could lead to an inaccurate area when completing data analysis. If larvae are not fully locomoting prior to cold plate/light exposure, the results may be inaccurate as a lack of contraction would not be in response to noxious cold or light stimulation. In addition, if the metal arena is not properly misted or the larvae leave water trails during locomotion, this extra water could potentially show up as additional area in the video recordings.

An alternative way we could investigate the necessity of our neuronal populations is by using a cold probe assay, which provides a noxious cold stimulus that is local (2). This provides a more precise stimulation than the global cold plate assay and can display more unique behavioral responses, which could provide deeper insight into how we can manipulate our neuronal populations (2).

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