Identification method and application of Drosophila larval neurons
By constructing and hybridizing fruit flies with specific genes and using the neuronal library of the CATMAID platform, the problems of inefficiency and cumbersomeness of traditional thermal stimulation markers are solved, and the rapid and efficient identification of neuronal identity of fruit flies larval is achieved.
Patent Information
- Application Number
- CN202211674429.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-26
- Publication Date
- 2025-06-17
- Estimated Expiration
- 2042-12-26
AI Technical Summary
When traditional thermal stimulation marking method is used to obtain the three-dimensional morphological structure of fruit fly larval neurons, there are problems such as high mortality rate, cumbersome experimental process and low labeling efficiency.
By constructing a homozygous tool for co-expressing the phiC31 gene and SPARC-jGCaMP7 gene, and hybridizing with the paternal fruit fly strain, the first generation of fruit fly larvae that randomly recombinantly expresses the jGCaMP7 protein was obtained, and the neuron library was obtained in combination with the CATMAID platform, and neurons that coincided with the first generation of fruit fly larvae neurons were screened to determine their identity.
It has achieved efficient identification of the identity of fruit fly larval neurons in a short period of time, which is simple and quick to operate and improves experimental efficiency.
Smart Images
Figure CN116034953B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of neurobiology, and particularly to a method for identifying Drosophila larval neurons and its application. Background Art
[0002] Drosophila is a classical model organism in the field of neurobiology research. In the study of the neural circuit of Drosophila, the generation of a behavior often depends on the interaction of multiple neurons. Therefore, it is necessary to study the functions of each neuron on the neural circuit to determine the neural mechanism of behavior generation.
[0003] In order to study the functions of each neuron, it is first necessary to obtain the morphology of Drosophila larval neurons and identify the identities of neurons. The traditional method for obtaining the three-dimensional morphological structure of Drosophila larval neurons is the heat stimulation labeling method. This method requires putting Drosophila embryos or first-instar larvae into a water bath for heat shock, and obtaining the best labeling efficiency by continuously adjusting and optimizing the heat stimulation time to label an appropriate number of target neurons. However, heat stimulation will increase the mortality rate of Drosophila larvae, and the experimental process is cumbersome with low labeling efficiency. Summary of the Invention
[0004] Based on this, it is necessary to provide a method for identifying Drosophila larval neurons and its application to solve the above problems. This identification method can identify the identities of Drosophila larval neurons in a short time, with simple and fast operation and high experimental efficiency.
[0005] The present invention provides a method for identifying Drosophila larval neurons, comprising the following steps:
[0006] Construct a homozygous transgenic Drosophila co-expressing phiC31 gene and SPARC-jGCaMP7 gene, and provide a paternal Drosophila strain;
[0007] Determine the whole-neuron expression map of the paternal Drosophila strain, and select the homozygous transgenic Drosophila with different SPARC-jGCaMP7 genes according to the whole-neuron expression map of the paternal Drosophila strain;
[0008] Hybridize the homozygous transgenic Drosophila with the paternal Drosophila strain to obtain the first-generation Drosophila larvae randomly recombinantly expressing jGCaMP7 protein, and obtain the 3D morphology of the neurons of the first-generation Drosophila larvae in the whole brain of the first-generation Drosophila larvae;
[0009] Use the CATMAID platform to obtain all the neurons of the body segments of the Drosophila larval model in the whole-brain connectome atlas database of the Drosophila larval model as a neuron library;
[0010] Screen the neurons in the neuron library that match the spatial positions of the cell bodies, axons, and dendrites of the neurons of the first-generation offspring of Drosophila larvae to determine the neuronal identities of the first-generation offspring of Drosophila larvae.
[0011] In one embodiment, in the step of determining the whole-neuron expression map of the paternal Drosophila strain, all neurons of the paternal Drosophila strain are specifically expressed with green fluorescent protein GFP to obtain the whole-neuron expression map.
[0012] In one embodiment, the homozygous tool Drosophila with different SPARC-jGCaMP7 genes includes phiC31 with moderately randomly labeled neurons; SPARC-I-jGCaMP7 homozygous tool Drosophila or phiC31 with sparsely randomly labeled neurons; SPARC-S-jGCaMP7 homozygous tool Drosophila.
[0013] In one embodiment, when the number of whole neurons of the paternal Drosophila strain is less than or equal to 20, the phiC31; SPARC-I-jGCaMP7 homozygous tool Drosophila is selected, and when the number of whole neurons is greater than 20, the phiC31; SPARC-S-jGCaMP7 homozygous tool Drosophila is selected.
[0014] In one embodiment, in the step of obtaining the 3D morphology of the whole brain of the first-generation offspring of Drosophila larvae, the 3D morphology of the whole brain of the first-generation offspring of Drosophila larvae includes the spatial position distribution of the cell bodies, axons, and dendrites of the neurons of the first-generation offspring of Drosophila larvae in the whole brain of the first-generation offspring of Drosophila larvae.
[0015] In one embodiment, the steps of obtaining the 3D morphology of the whole brain of the first-generation offspring of Drosophila larvae include: obtaining the brain of the first-generation offspring of Drosophila larvae, labeling the neurons expressing the jGCaMP7 protein with green fluorescent protein, labeling the body segments of the first-generation offspring of Drosophila larvae with Fas2 antibody, and scanning the brain of the first-generation offspring of Drosophila larvae to obtain the 3D morphology of the neurons.
[0016] In one embodiment, in the step of obtaining the 3D morphology of the whole brain of the first-generation offspring of Drosophila larvae, the relative positions of the cell bodies, axons, and dendrites of the neurons of the first-generation offspring of Drosophila larvae with respect to the dorsal and ventral sides are determined according to the Fas2 antibody labeling results and the GFP labeling results.
[0017] In one embodiment, the body segment of the Drosophila larva model is selected from the first abdominal segment.
[0018] In one embodiment, in the step of obtaining all the neurons of the somites of the Drosophila larva model by using the CATMAID platform as a neuron library, all the neurons with a somite depth range of 2100 - 2700 of the Drosophila larva model are obtained as the neuron library in the direction from the head to the tail of the Drosophila larva model.
[0019] An application of the above identification method in the identification of Drosophila larva neurons.
[0020] The method for identifying Drosophila larva neurons provided by the present invention involves crossing a homozygous tool Drosophila with a paternal Drosophila strain to obtain the first-generation Drosophila larva with randomly labeled neurons. Then, the three-dimensional morphology of the neurons in the brain of the first-generation Drosophila larva is obtained through immunofluorescence and laser confocal methods. Combining with the CATMAID (The Collaborative Annotation Toolkit for Massive Amounts of Image Data) platform, a neuron library including all the neurons of the somites of the Drosophila larva model is obtained. By comparing with the neurons of the first-generation Drosophila larva, the identity of the neurons of the first-generation Drosophila larva is finally determined in the neural network connectome. This identification method has a simple process, convenient operation, and high identification efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 is a flowchart of the method for identifying Drosophila larva neurons provided by the present invention;
[0022] Figure 2 is a projection morphology diagram of one neuron of the first-generation Drosophila larva in the XY plane in Example 1;
[0023] Figure 3 is a projection morphology diagram of one neuron of the first-generation Drosophila larva in the XZ plane in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0024] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to relevant embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.
[0026] As Figure 1 shown, the method for identifying Drosophila larval neurons provided by the present invention includes the following steps:
[0027] S10, constructing a homozygous tool Drosophila co-expressing phiC31 gene and SPARC-jGCaMP7 gene as the maternal Drosophila strain, and providing a paternal Drosophila strain;
[0028] S20, determining the whole-neuron expression map of the paternal Drosophila strain, and selecting homozygous tool Drosophila with different SPARC-jGCaMP7 genes according to the whole-neuron expression map of the paternal Drosophila strain;
[0029] S30, crossing the homozygous tool Drosophila with the paternal Drosophila strain to obtain the first-generation Drosophila larvae randomly recombinantly expressing jGCaMP7 protein, and obtaining the 3D morphology of the neurons of the first-generation Drosophila larvae in the whole brain of the first-generation Drosophila larvae;
[0030] S40, using the CATMAID platform to obtain all the neurons of the Drosophila larval segments in the Drosophila larval model whole-brain connectome atlas database as the neuron library;
[0031] S50, screening the neurons in the neuron library that match the spatial positions of the cell bodies, axons, and dendrites of the neurons of the first-generation Drosophila larvae, and determining the neuron identities of the first-generation Drosophila larvae.
[0032] In step S10, it can be understood that since the phiC31 gene is located on the X chromosome, in order to randomly label the Drosophila larvae obtained by hybridization with the calcium indicator, phiC31; SPARC-jGCaMP7 double homozygous tool Drosophila is selected as the maternal Drosophila strain to cross with the paternal Drosophila strain.
[0033] PhiC31 is a recombinase that can recognize the phage attP site and the bacterial attB site and mediate irreversible and efficient integration between the sites. SPARC is a plasmid vector of UAS-attP-attB-effector. After the two genes are hybridized, PhiC31 can recombine the attP and attB sites in the plasmid vector.
[0034] According to the number of base pairs of attP, the SPARC-jGCaMP7 gene is divided into the SPARC-I-jGCaMP7 gene that moderately labels neurons and the SPARC-S-jGCaMP7 gene that sparsely labels neurons, and thus the obtained homozygous tool Drosophila is divided into two types, phiC31; SPARC-I-jGCaMP7 Drosophila and phiC31; SPARC-S-jGCaMP7 Drosophila.
[0035] In one embodiment, constructing a homozygous tool Drosophila expressing both the phiC31 gene and the SPARC-jGCaMP7 gene as the maternal Drosophila strain specifically includes the following steps: integrating the phiC31 gene expressing the phiC31 recombinase and the SPARC-jGCaMP7 gene expressing SPARC-jGCaMP7 driven by UAS into the same Drosophila strain through hybridization to construct the phiC31;SPARC-jGCaMP7 double homozygous tool Drosophila expressing both genes as the maternal Drosophila strain.
[0036] In step S20, to determine the whole-neuron expression map of the paternal Drosophila strain, the whole-neuron expression pattern of the paternal Drosophila strain can be searched from the Bloomington Drosophila Stock Center, or the GAL4 / UAS transgenic system can be used to specifically express green fluorescent protein (GFP) in all neurons of the paternal Drosophila strain to obtain the whole-neuron expression map of the paternal Drosophila strain; preferably, green fluorescent protein is specifically expressed in all neurons of the paternal Drosophila strain to obtain the whole-neuron expression map.
[0037] In the process of determining the type of the homozygous tool Drosophila according to the number of neurons labeled in the paternal Drosophila strain, when the number of neurons is less than or equal to 20, select the phiC31;SPARC-I-jGCaMP7 homozygous tool Drosophila as the mother; when the number of neurons is greater than 20, select the phiC31;UAS-SPARC-S-jGCaMP7 homozygous tool Drosophila as the mother.
[0038] In step S30, due to the random recombination of SPARC and the recombinase phiC31, the obtained first-generation hybrid Drosophila larvae will express the calcium indicator jGCaMP7 in some random neurons as the first-generation hybrid Drosophila larvae. When the number of neurons in the paternal Drosophila strain is relatively large, such as greater than or equal to 20, the SPARC-S-jGCaMP7 gene of the selected maternal Drosophila strain will sparsely label the neurons randomly marked in the first-generation hybrid Drosophila larvae, reducing the situation where the morphology of a single neuron cannot be recognized due to too many labeled neurons. When the number of neurons in the paternal Drosophila strain is relatively small, such as less than 20, the SPARC-I-jGCaMP7 gene of the selected maternal Drosophila strain will label more neurons randomly marked in the first-generation hybrid Drosophila larvae, but the morphology of a single neuron can still be recognized.
[0039] In one embodiment, the steps of obtaining the 3D morphology of the whole brain of the first-generation hybrid Drosophila larvae include:
[0040] Obtaining the brain of the first-generation hybrid Drosophila larvae, specifically, the isolated brain of the Drosophila larvae can be obtained by dissection. Preferably, the first-generation hybrid Drosophila larvae are preferably selected from the third-instar first-generation hybrid Drosophila larvae;
[0041] Label the neurons of the first-generation Drosophila larvae expressing the calcium indicator jGCaMP7 with green fluorescent protein, and label the body segments of the first-generation Drosophila larvae with Fas2 (Fasciclin 2) antibody. Among them, the Fas2 antibody is an affinity interaction protein that can be highly expressed in proliferative epithelial cells and differentiated non-proliferative cells. Therefore, the Fas2 antibody can display each body segment of the first-generation Drosophila larvae;
[0042] Scan the brain of the first-generation Drosophila larvae. Specifically, the laser confocal imaging technology can be used to scan the brain of the first-generation Drosophila larvae layer by layer to obtain the 3D morphology of the neurons, including the position of the cell body, the projection and spatial distribution of axons and dendrites.
[0043] The specific steps to obtain the 3D morphology of the neurons include: determining the dorsal and ventral sides in the 3D morphology of the neurons according to the Fas2 antibody staining imaging results. Among them, the side close to the back of the first-generation Drosophila larvae is the dorsal side, and the side close to the abdomen of the first-generation Drosophila larvae is the ventral side. It is stipulated that the direction from the head to the tail of the first-generation Drosophila larvae is the positive direction of the Z axis, the direction from the abdomen to the back of the first-generation Drosophila larvae is the positive direction of the Y axis, and the direction from the left brain to the right brain of the first-generation Drosophila larvae is the positive direction of the X axis. Establish a spatial rectangular coordinate system. Obtain the projection morphology of the neurons in the XZ plane according to the green fluorescent protein staining imaging results, extract the projection morphology of the neurons of the first-generation Drosophila larvae in the XY plane through Image J software, determine the dorsal and ventral sides of the brain tissue of the first-generation Drosophila larvae according to the Fas2 antibody staining imaging, and determine whether the cell body is close to the dorsal or ventral side of the brain tissue of the first-generation Drosophila larvae, whether the axon is close to the dorsal or ventral side of the brain tissue of the first-generation Drosophila larvae, and the relative positions of the axon and dendrites to the dorsal and ventral sides according to the projection morphology of the neurons in the XY plane and combined with the Fas2 antibody staining imaging. Then, determine morphological characteristics such as whether the axon projects to the ipsilateral or contralateral side of the position it belongs to, whether the axon projects to the head segment or the tail segment, and the number and spatial distribution of dendrites according to the projection state in the XZ plane.
[0044] In step S40, the CATMAID platform specifically refers to the electron microscopy brain atlas dataset of the Drosophila larva model.
[0045] Step S40 specifically includes: using the CATMAID platform, searching for neurons in the depth range of 2100 - 2700 of the body segments of the Drosophila larva model in the direction from the head to the tail of the Drosophila larva model. Since the neurons are symmetrically distributed on the left and right, it is only necessary to detect the central nervous system on the left or right half side. Starting from 2100 images, circle all the neurons that appear in the electron microscopy connectome images, and repeat the circle selection every 20 images until the 2700th image to end the circle selection. The repeated neurons will not be displayed repeatedly, so as to obtain all the neurons in the first abdominal segment of the Drosophila larva model as the neuron library, and each neuron in it corresponds to an identity of the neuron.
[0046] Step S50 specifically includes: comparing the 3D morphology of the neurons to be identified obtained from the first-generation Drosophila larvae with the neuron library. First, find a group of neurons with roughly similar morphologies in the neuron library according to the projection morphology of the neurons to be identified in the XZ plane to obtain a batch of candidate neurons. Then, determine the relative positions of the cell body, axon, dendrites with the dorsal or ventral side according to the projection morphology of the neurons to be identified in the XY plane, and eliminate a batch of candidate neurons. Finally, determine the identity of the neurons to be identified in the first-generation Drosophila larvae according to the number of dendritic projections and distribution details. If no similar neurons to be identified in the first-generation Drosophila larvae can be found in this neuron library, search for neurons in other body segments of the Drosophila larva model as a new neuron library until similar neurons to the neurons to be identified are found in the new neuron library.
[0047] The present invention also provides an application of the above identification method in the identification of Drosophila larval neurons. This identification method has a simple process, convenient operation, and high identification efficiency.
[0048] Hereinafter, the identification method and application of Drosophila larval neurons will be further described through the following specific examples.
[0049] Example 1
[0050] Provide a paternal Drosophila strain with 18 neurons labeled with green fluorescent protein. Use the homozygous tool Drosophila phiC31; SPARC-I-jGCaMP7 as the maternal parent and cross it with the paternal Drosophila strain to obtain the first-generation third-instar Drosophila larvae that randomly recombinantly express the jGCaMP7 protein.
[0051] Dissect the first-generation third-instar Drosophila larvae to obtain the isolated brains of the first-generation third-instar Drosophila larvae. Label the neurons expressing the jGCaMP7 protein in the first-generation third-instar Drosophila larvae with green fluorescent protein, label the body segments of the first-generation third-instar Drosophila larvae with Fas2 antibody, and use the laser confocal imaging technology to scan the brains of the first-generation third-instar Drosophila larvae layer by layer to obtain the 3D morphology of the neurons of the first-generation third-instar Drosophila larvae.
[0052] Taking the back of the first-generation third-instar Drosophila larvae as the dorsal side and the abdomen of the first-generation third-instar Drosophila larvae as the ventral side, it is stipulated that the direction from the head to the tail of the first-generation third-instar Drosophila larvae is the positive direction of the Z-axis, the direction from the abdomen to the back of the first-generation third-instar Drosophila larvae is the positive direction of the Y-axis, and the direction from the left brain to the right brain of the first-generation third-instar Drosophila larvae is the positive direction of the X-axis. A spatial rectangular coordinate system is established. The dorsal-ventral side in the 3D morphology of the neurons of the first-generation third-instar Drosophila larvae is determined according to the Fas2 antibody staining imaging. The projection morphology of the neurons of the first-generation third-instar Drosophila larvae in the XZ plane is obtained according to the green fluorescent protein staining imaging. The projection morphology of the neurons of the first-generation third-instar Drosophila larvae in the XY plane is extracted by ImageJ software, and the morphological characteristics such as the relative positions and spatial distributions of the neuron cell bodies, axons, and dendrites are determined.
[0053] Using the CATMAID platform, neurons in the left half of the central nervous system of the Drosophila larva model are searched within the Z-axis depth range of 2100 - 2700. Starting from 2100 images, all the neurons that appear in the electron microscopy connectome are circled, and the circling is repeated every 20 images until the circling ends at 2700 images. All the neurons in the first abdominal segment of the Drosophila larva model are obtained as the neuron library.
[0054] According to the projection states of the neuron to be identified in the XZ and XY planes, neurons in the neuron library that match the spatial positions of the cell body, axon, and dendrite of the neuron to be identified in the first-generation third-instar Drosophila larvae are screened to determine the identity of the neuron to be identified in the first-generation third-instar Drosophila larvae.
[0055] Figure 2 and Figure 3 are the projection morphologies of one of the neurons in the first-generation Drosophila larvae in the XY and XZ planes. It can be seen from Figure 2 and Figure 3 the morphological characteristics such as the relative positions and spatial distributions of the neuron cell bodies, axons, and dendrites.
[0056] Example 2
[0057] A paternal Drosophila strain with 25 neurons labeled with green fluorescent protein is provided. The phiC31; SPARC-S-jGCaMP7 homozygous tool Drosophila is used as the maternal parent and crossed with the paternal Drosophila strain to obtain the first-generation third-instar Drosophila larvae that randomly recombinantly express the jGCaMP7 protein.
[0058] The first-generation third-instar Drosophila larvae are dissected to obtain the isolated brain of the first-generation third-instar Drosophila larvae. The neurons expressing the jGCaMP7 protein in the first-generation third-instar Drosophila larvae are labeled with green fluorescent protein, the body segments of the first-generation third-instar Drosophila larvae are labeled with Fas2 antibody, and the brain of the first-generation third-instar Drosophila larvae is scanned layer by layer using laser confocal imaging technology to obtain the 3D morphology of the neurons of the first-generation third-instar Drosophila larvae.
[0059] Taking the back of the first-generation third-instar Drosophila larvae as the dorsal side and the abdomen of the first-generation third-instar Drosophila larvae as the ventral side, it is stipulated that the direction from the head to the tail of the first-generation third-instar Drosophila larvae is the positive direction of the Z-axis, the direction from the abdomen to the back of the first-generation third-instar Drosophila larvae is the positive direction of the Y-axis, and the direction from the left brain to the right brain of the first-generation third-instar Drosophila larvae is the positive direction of the X-axis. A spatial rectangular coordinate system is established. According to the Fas2 antibody staining imaging, the dorsal-ventral side in the 3D morphology of the neurons of the first-generation third-instar Drosophila larvae is determined. According to the green fluorescent protein staining imaging, the projection morphology of the neurons of the first-generation third-instar Drosophila larvae in the XZ plane is obtained. The projection morphology of the neurons of the first-generation third-instar Drosophila larvae in the XY plane is extracted by ImageJ software, and the morphological characteristics such as the relative positions and spatial distributions of the neuron cell bodies, axons, and dendrites are determined.
[0060] Using the CATMAID platform, neurons in the left half of the central nervous system of the Drosophila larval model are searched within the Z-axis depth range of 2100 - 2700. Starting from 2100 images, all the neurons that appear in the electron microscopy connectome are circled, and the circling is repeated every 20 images until 2700 images are completed. All the neurons in the first abdominal segment of the Drosophila larval model are obtained as the neuron library.
[0061] According to the projection states of the neurons to be identified in the XZ and XY planes, neurons in the neuron library that match the spatial positions of the cell bodies, axons, and dendrites of the neurons to be identified in the first-generation third-instar Drosophila larvae are screened to determine the identities of the neurons to be identified in the first-generation third-instar Drosophila larvae.
[0062] Example 3
[0063] A paternal Drosophila strain with 18 neurons labeled with green fluorescent protein is provided. The homozygous tool Drosophila phiC31;SPARC-I-jGCaMP7 is used as the female parent and crossed with the paternal Drosophila strain to obtain the first-generation third-instar Drosophila larvae that randomly recombinantly express the jGCaMP7 protein.
[0064] The first-generation third-instar Drosophila larvae are dissected to obtain the isolated brains of the first-generation third-instar Drosophila larvae. The neurons expressing the jGCaMP7 protein in the first-generation third-instar Drosophila larvae are labeled with green fluorescent protein, the body segments of the first-generation third-instar Drosophila larvae are labeled with Fas2 antibody, and the brains of the first-generation third-instar Drosophila larvae are scanned layer by layer using laser confocal imaging technology to obtain the 3D morphology of the neurons of the first-generation third-instar Drosophila larvae.
[0065] Taking the back of the first-generation third-instar Drosophila larvae as the dorsal side and the abdomen of the first-generation third-instar Drosophila larvae as the ventral side, it is stipulated that the direction from the head to the tail of the first-generation third-instar Drosophila larvae is the positive direction of the Z-axis, the direction from the abdomen to the back of the first-generation third-instar Drosophila larvae is the positive direction of the Y-axis, and the direction from the left brain to the right brain of the first-generation third-instar Drosophila larvae is the positive direction of the X-axis. A spatial rectangular coordinate system is established. The dorsal-ventral side in the 3D morphology of the neurons of the first-generation third-instar Drosophila larvae is determined according to the Fas2 antibody staining imaging. The projection morphology of the neurons of the first-generation third-instar Drosophila larvae in the XZ plane is obtained according to the green fluorescent protein staining imaging. The projection morphology of the neurons of the first-generation third-instar Drosophila larvae in the XY plane is extracted by ImageJ software to determine the morphological characteristics such as the relative positions and spatial distributions of the neuron cell bodies, axons, and dendrites.
[0066] Use the CATMAID platform to search for neurons in the right half of the central nervous system of the Drosophila larva model within the range of Z-axis depth from 2700 to 2900. Starting from 2700 images, all the neurons that appear in the electron microscopy connectome are circled, and the circling is repeated every 20 images until 2900 images to obtain all the neurons in the second abdominal segment of the Drosophila larva model as the neuron library.
[0067] According to the projection states of the neurons to be identified in the XZ and XY planes, the neurons in the neuron library that match the spatial positions of the cell bodies, axons, and dendrites of the neurons to be identified in the first-generation third-instar Drosophila larvae are screened to determine the identities of the neurons to be identified in the first-generation third-instar Drosophila larvae.
[0068] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.
[0069] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.
Claims
1. A method for identifying Drosophila larval neurons, characterized in that, Comprising the following steps: Construct a homozygous tool Drosophila co-expressing phiC31 gene and SPARC-jGCaMP7 gene as the maternal Drosophila strain, and provide a paternal Drosophila strain; Determine the whole-neuron expression map of the paternal Drosophila strain, and select the homozygous tool Drosophila with different SPARC-jGCaMP7 genes according to the whole-neuron expression map of the paternal Drosophila strain; Hybridize the homozygous tool Drosophila and the paternal Drosophila strain to obtain the first-generation Drosophila larvae randomly recombinantly expressing jGCaMP7 protein, and obtain the 3D morphology of the neurons of the Drosophila larvae in the whole brain of the Drosophila larvae; Use the CATMAID platform to obtain all neurons of the body segments of the Drosophila larval model in the whole-brain connectome atlas database of the Drosophila larval model, and the body segments of the Drosophila larval model are selected from the first abdominal segment; Screen the neurons in the neuron library that match the spatial positions of the cell bodies, axons and dendrites of the neurons of the Drosophila larvae of the first generation, and determine the neuron identities of the Drosophila larvae of the first generation.
2. The method for identifying Drosophila larval neurons according to claim 1, characterized in that, In the step of determining the whole-neuron expression map of the paternal Drosophila strain, all neurons of the paternal Drosophila strain are specifically expressed with green fluorescent protein to obtain the whole-neuron expression map.
3. The method for identifying Drosophila larval neurons according to claim 1, characterized in that, The homozygous tool Drosophila with different SPARC-jGCaMP7 genes includes phiC31; SPARC-I-jGCaMP7 homozygous tool Drosophila with moderately randomly labeled neurons or phiC31; SPARC-S-jGCaMP7 homozygous tool Drosophila with sparsely randomly labeled neurons.
4. The method for identifying Drosophila larval neurons according to claim 3, characterized in that, When the number of whole neurons of the paternal Drosophila strain is less than or equal to 20, select the phiC31; SPARC-I-jGCaMP7 homozygous tool Drosophila, and when the number of whole neurons is greater than 20, select phiC31; SPARC-S-jGCaMP7 homozygous tool Drosophila.
5. The method for identifying Drosophila larval neurons according to any one of claims 1-4, characterized in that, In the step of obtaining the 3D morphology of the whole brain of the Drosophila larvae of the first generation, the 3D morphology of the whole brain of the Drosophila larvae of the first generation includes the spatial position distribution of the cell bodies, axons and dendrites of the neurons of the Drosophila larvae of the first generation in the whole brain of the Drosophila larvae of the first generation.
6. The method for identifying Drosophila larval neurons according to claim 5, characterized in that, The step of obtaining the 3D morphology of the whole brain of the Drosophila larvae of the first generation includes: obtaining the brain of the Drosophila larvae of the first generation, labeling the neurons of the Drosophila larvae of the first generation expressing jGCaMP7 protein with green fluorescent protein GFP, labeling the body segments of the Drosophila larvae of the first generation with Fas2 antibody, and scanning the brain of the Drosophila larvae of the first generation to obtain the 3D morphology of the neurons.
7. The method for identifying Drosophila larval neurons according to claim 6, characterized in that, In the step of obtaining the 3D morphology of the whole brain of the Drosophila larvae of the first generation, determine the relative positions of the cell bodies, axons, dendrites of the neurons of the Drosophila larvae of the first generation with the dorsal and ventral sides according to the labeling results of Fas2 antibody and GFP labeling.
8. The method for identifying Drosophila larval neurons according to claim 1, characterized in that, In the step of obtaining all neurons of the somites of the Drosophila larva model as a neuron library by using the CATMAID platform, all neurons with a somite depth range of 2100-2700 of the Drosophila larva model are obtained as the neuron library in the direction from the head to the tail of the Drosophila larva model.
9. Application of the identification method according to any one of claims 1-8 in the identification of Drosophila larval neurons.
Citation Information
Patent Citations
Method for genome editing
CN102858985A
Building method of Drosophila melanogaster model for screening and researching mitochondria disease virulence gene, and application of same
CN106376527A