Method for observing shape and number of synapses in dentate gyrus of sea horse based on image analysis

Abstract

The invention discloses a method for observing the shape and number of synapses in a dentate gyrus region based on image analysis, and relates to the technical field of neurobiology. The method comprises the following steps: S1, tissue treatment: after an experimental animal is anesthetized, pouring a buffer solution and a stationary solution into the heart in stages, separating hippocampus tissue, trimming and fixing, pre-cooling, slicing and preserving at low temperature; s2, dyeing and marking: performing targeted marking on the dentate gyrus area of the sea horse by adopting DiI particles through a fine tungsten steel through needle, and preserving at low temperature after incubation; s3, fixing and mounting: fixing for the second time, and mounting with an anti-fluorescence quenching agent after DAPI nuclear dyeing; s4, confocal imaging is carried out, exclusive parameter layer scanning is set, and qualified dendritic spine images are screened; and image quantitative analysis can be further carried out, and accurate quantification is realized according to classification standards. The method is standard in operation, greatly improves sample quality and experimental result credibility, optimizes experimental efficiency and controllability and reduces technical threshold by means of targeted marking and standardized processes, and is suitable for relevant basis and application research of the hippocampal synapses.

Description

Technical Field

[0001] This invention relates to the field of neurobiology, and in particular to a method for observing the morphology and number of synapses in the dentate gyrus of the hippocampus based on image analysis. Background Technology

[0002] The hippocampus, as a core brain region regulating learning and memory, contains dendritic spines, key structures of excitatory synapses. Changes in the morphology, density, and plasticity of these spines are core biological indicators reflecting synaptic functional status, possessing irreplaceable research value in areas such as the pathogenesis of neurodegenerative diseases and the development of targeted drugs. Accurate high-fidelity labeling, clear imaging, and quantitative analysis of dendritic spines are crucial technological requirements for advancing research in these fields.

[0003] Existing dendritic spine observation techniques have formed various technical paths around the core process of labeling, tissue processing, imaging, and analysis. However, they generally suffer from technical bottlenecks: the labeling stage lacks precise targeting and operational controllability; the standardization of fixation and slicing in tissue processing is insufficient; imaging parameter settings rely on experience-based judgment, leading to poor result stability; and the analysis process lacks unified quantitative standards and systematic specifications. These factors affect the reliability, repeatability, and cross-laboratory comparability of experimental results, making it difficult to meet the practical needs of high-precision research. Therefore, developing a dendritic spine observation technique that combines stability, standardization, and normativity has become an urgent technical challenge in this field. Summary of the Invention

[0004] The purpose of this invention is to overcome the technical defects in the prior art where the setting of imaging parameters for observing dendritic spines of hippocampal neurons relies on empirical judgment, resulting in poor stability of the observation results. This invention provides a method for observing the morphology and number of synapses in the dentate gyrus of the hippocampus based on image analysis.

[0005] This invention provides a method for observing the morphology and number of synapses in the dentate gyrus of the hippocampus based on image analysis, comprising the following steps:

[0006] S1. Tissue processing: After anesthetizing the experimental animals, the heart was perfused with buffer and fixative in stages. The hippocampal tissue was separated, trimmed and fixed, and then sectioned and stored at low temperature under pre-cooling conditions.

[0007] S2. Staining and labeling: The dentate gyrus region of hippocampal tissue sections was targeted and labeled with specific labeling materials, and then incubated and stored at low temperature.

[0008] S3. Fixation and mounting: After secondary fixation and nuclear staining of the stained sections, mount them with an anti-fluorescence quencher.

[0009] S4. Confocal Imaging: Set imaging parameters, locate the target area, perform layer scanning, and select to obtain clear images of dendritic spines.

[0010] Furthermore, the specific labeling material mentioned in step S2 is DiI particles, which are targeted and placed in the dentate gyrus region of the hippocampus using a fine tungsten carbide needle.

[0011] Furthermore, in step S3, the nuclear staining is performed using DAPI, and the secondary fixation is performed using 3-5% paraformaldehyde at room temperature for 20-40 minutes.

[0012] Furthermore, the imaging parameters in step S4 are: 60x or 100x oil immersion lens, pinhole 1-1.2 Airy Unit, Z-axis layer scanning distance 0.3-0.5μm, and layer scanning with magnification of 2-3x after locking onto the target.

[0013] Furthermore, the criteria for screening the dendritic spines in step S4 are: length > 20 μm, no crossing, and exclusion of degenerative and enlarged segments.

[0014] Furthermore, it also includes image quantitative analysis, in which the clear dendritic spine image obtained in step S4 is imported into professional software for preprocessing, and based on the dendritic spine classification standard, the counting software is used to achieve accurate quantitative analysis of different types of dendritic spines.

[0015] Furthermore, the classification criteria for dendritic spines are as follows: ① Mushroom type: head diameter > 0.5 μm or head-to-neck diameter ratio > 1.5; ② Short and thick type: length < 0.5 μm, head-to-neck diameter ratio ≤ 1 or no obvious head; ③ Slender type: length 0.5-1 μm, head diameter < 0.5 μm or no obvious head or head-to-neck diameter ratio < 1.5; Fibropodia type: head and neck diameters are equal, length > 3 μm, neck diameter 0.1-0.4 μm or head-to-neck diameter ratio < 2.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. The operation specifications of this invention, through targeted labeling of the dentate gyrus and standardized tissue processing procedures, effectively improve sample quality and fidelity, preserve the original morphology of dendritic spines to the greatest extent, and avoid problems such as label diffusion and tissue deformation in traditional techniques, thus laying a high-quality sample foundation for subsequent accurate analysis.

[0018] 2. This invention, by utilizing dedicated confocal imaging parameters and dendritic spine screening, classification and quantification standards, significantly improves the reliability and repeatability of experimental results, reduces subjective operational errors, ensures the comparability of data between different operators and different laboratories, and makes experimental conclusions more convincing.

[0019] 3. This invention relies on standardized operation throughout the entire process to optimize experimental efficiency and controllability, reduce the risk of experimental failure, shorten the experimental cycle, and lower the technical implementation threshold. It can stably carry out experiments without relying on the experience of operators, and is suitable for various basic and applied research related to hippocampal synapses. Attached Figure Description

[0020] Figure 1 Brain slices (150 μm) containing the hippocampus after being sliced ​​using a vibratory microtome;

[0021] Figure 2 Brain slices under an upright microscope;

[0022] Figure 3 It is a tungsten carbide needle, and the tip is used to pick up dye particles;

[0023] Figure 4 This is an overall fluorescence imaging image of the hippocampal dentate gyrus, where the blue fluorescent areas are marked by DAPI staining to correspond to the hippocampal dentate gyrus, and the red fluorescent parts are displayed by 561-channel imaging to show neurons in this region.

[0024] Figure 5 The brain slices were stained under an upright microscope. The granules in the dentate gyrus of the hippocampus were dyed with DIL.

[0025] Figure 6 A diagram of the overall neurons in the dentate gyrus of the hippocampus;

[0026] Figure 7 The neuronal segments under the maximum projection of Image J are (M mushroom-shaped spine, T filamentous spine, S thick and short spine). Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: A Method for Observing Synaptic Morphology and Number in the Dentate Gyrus of the Hippocampus Based on Image Analysis

[0029] This embodiment aims to elaborate on a method for observing the morphology and number of synapses in the dentate gyrus of the hippocampus based on image analysis. It employs a standardized experimental procedure, clearly defined operating parameters, and dedicated image analysis tools to observe the morphology and number of synapses in the dentate gyrus of the hippocampus, providing experimental evidence for neurobiological research. The following detailed description of each step, in conjunction with the accompanying drawings, is provided:

[0030] I. Experimental Materials

[0031] 1. Experimental reagents

[0032] Isoflurane, 0.1M PBS powder, Dil staining agent, paraformaldehyde, anti-fluorescence quenching mounting medium, DAPI, 0.5% heparin sodium solution, sodium chloride, agar powder.

[0033] 2. Preparation methods of main reagents

[0034] 0.1M PBS: Take 2L of 0.1M PBS powder, add 2L of ultrapure water, stir thoroughly to dissolve, and set aside.

[0035] 1.5% paraformaldehyde solution: Weigh 7.5g of paraformaldehyde, add 500ml of 0.1M PBS solution, stir until completely dissolved and set aside.

[0036] 4% paraformaldehyde solution: Weigh 20g of paraformaldehyde, add 500ml of 0.1M PBS solution, mix thoroughly to dissolve, and set aside.

[0037] Agar block: Weigh 8g of agar powder and 1.8g of sodium chloride, add 200ml of ultrapure water (dd water), heat in a microwave oven until boiling, and stir continuously until the solute is completely dissolved. Pour into a glass petri dish and let cool naturally. Store at 4℃ for later use.

[0038] 3. Experimental instruments

[0039] Scalp vein needles, 50ml centrifuge tubes, 12-well plates, inverted glass culture dishes, filter paper, paintbrushes, upright microscopes, fine tungsten carbide needles and handles, vibratory microtome, laser confocal microscopes, shakers, Parbuch pipettes, adhesive slides, coverslips, scalpels, ultrapure water systems, surgical instruments, light-proof aluminum foil, culture dishes, and small animal respiratory anesthesia machines.

[0040] II. Specific Steps

[0041] S1: Material Selection and Fixing

[0042] 1. Anesthesia: Anesthesia was performed using a small animal respiratory anesthesia machine. The specific steps are as follows: First, induction anesthesia was performed by placing the mice in an isoflurane anesthesia chamber and adjusting the isoflurane concentration to 3%-5% using the small animal respiratory anesthesia machine until the mice lost consciousness and their breathing stabilized. Then, maintenance anesthesia was initiated, with the isoflurane concentration maintained at 1.0%-1.5% for mice and 2%-3% for rats. During maintenance anesthesia, an anesthesia mask was placed over the mouse's head, and anesthetic gas was continuously supplied. Simultaneously, the mice were placed supine on a foam board, and their limbs were fixed with needles. Subsequent procedures were performed under maintenance anesthesia.

[0043] 2. Staged cardiac perfusion:

[0044] ① Use surgical scissors to cut the skin and abdominal muscles along the midline of the mouse's abdomen. Use hemostats to clamp the xiphoid process and lift it upwards. Cut the abdomen open to the left and right along the incision to expose the diaphragm. Carefully cut the diaphragm with surgical scissors to expose the thoracic cavity. Use hemostats to lift the tissue and place it in the direction of the head. Use forceps in the left hand to clamp the atrium. Use surgical scissors in the right hand to gently separate the thymus tissue and clearly expose the aorta.

[0045] ② Insert a scalp needle slowly into the aorta along the apex of the heart, clamp the needle with forceps to fix it, and at the same time use surgical scissors to cut open the right atrial appendage as a channel for the outflow of perfusion fluid to ensure a smooth perfusion process;

[0046] ③ Adjust the perfusion volume according to the age of the mice: for young mice, slowly perfuse about 10 ml of 0.1 M PBS anticoagulation buffer containing heparin sodium, and for adult mice, perfuse about 20 ml. When the liver is observed to change from red to pale white, switch to 1.5% paraformaldehyde fixative. Continue perfusing about 15 ml for young mice and about 30 ml for adult mice. Keep the perfusion rate constant during the process to avoid tissue damage.

[0047] 3. Tissue Isolation and Preservation: After perfusion, the head was severed and the skull was cut open. The mouse brain tissue was quickly removed, and the hippocampus tissue was carefully isolated in 0.1M PBS buffer, removing excess connective tissue. The isolated hippocampal tissue was placed in a 50ml centrifuge tube containing 1.5% paraformaldehyde and stored overnight at 4°C to ensure adequate tissue fixation.

[0048] S2: Slice preparation

[0049] 1. Tissue cleaning and trimming: Remove the hippocampal tissue stored at 4°C and wash it once with 0.1M PBS buffer to remove residual fixative. Line an inverted glass culture dish with filter paper, add a small amount of pre-cooled 0.1M PBS, place the hippocampal tissue on the filter paper, remove the cerebellar tissue with ophthalmic scissors, and trim the left and right ends of the brain to preserve the complete hippocampal structure.

[0050] 2. Tissue Fixation and Sectioning: Take one side of the trimmed hippocampus tissue and fix it on the sample stage of the vibratory microtome using agar blocks and glue, ensuring the tissue is stable and does not move. Add pre-cooled 0.1M PBS buffer to the sample chamber of the microtome to completely saturate the tissue. Set the section thickness to 150μm and start the instrument for sectioning.

[0051] 3. Sectioning and Preservation: The hippocampal tissue sections are shown in the attached figure. Figure 1 As shown, a 150μm brain slice containing the hippocampus was prepared by a vibratory microtome and gently transferred to a 12-well plate with a brush. An appropriate amount of 0.1M PBS buffer was added to each well to prevent the slice from drying out. The 12-well plate was then stored at 4°C until the subsequent staining step.

[0052] S3: Staining mark

[0053] 1. Section transfer: Carefully transfer the hippocampal tissue section from the 12-well plate to a clean adhesive slide using a brush. Use absorbent paper to remove any excess 0.1M PBS buffer around the section, being careful not to dry it out completely (too much moisture will make it difficult for the dye to bind to the tissue, while too little moisture will cause the tissue section to dehydrate).

[0054] 2. Targeted staining: Place the slide under an upright microscope; the brain slices observed at this time are shown in the attached image. Figure 2 As shown, a handheld fine tungsten carbide needle (with) Figure 3 Take an appropriate amount of Dil dye particles (the particles should not be too large) and precisely target them in the dentate gyrus (DG area) of the hippocampal tissue section under a microscope to ensure that the dye particles are in full contact with the DG area tissue.

[0055] 3. Incubation and preservation: After placing the dye, incubate the slides at room temperature for 5-10 minutes to allow the dye to initially penetrate. Then, use a brush to transfer the slides back into the 12-well plate, add an appropriate amount of 0.1M PBS buffer to each well, and incubate the 12-well plate at 4°C. The slides must be mounted and photographed within seven days.

[0056] S4: Fixing and Sealing

[0057] 1. Secondary fixation: Take out the stained sections after incubation at 4℃ and fix them with 4% paraformaldehyde fixative for 30 minutes at room temperature to further stabilize the staining effect and prevent the dye from falling off in subsequent operations.

[0058] 2. Washing and Nuclear Staining: After fixation, place the slides on a shaker and wash twice with 0.1M PBS buffer, 5 minutes each time, to remove residual fixative. After washing, add an appropriate amount of DAPI staining solution to the slides and stain at room temperature for 5 minutes to stain the cell nuclei. Figure 4 The blue area is the hippocampal DG region marked by DAPI.

[0059] 3. Re-washing and mounting: After DAPI staining, wash the sections twice with 0.1M PBS buffer, 5 minutes each time, to remove unbound staining solution. Finally, add an appropriate amount of anti-fluorescence quenching mounting medium to the sections, covering the entire hippocampal tissue area, and slowly cover with a coverslip, avoiding air bubbles, to complete the mounting process. In the mounted sections, the particles in the DG region exhibit specific fluorescence due to Dil dye labeling, as shown in the attached image. Figure 5 The image shows a stained brain slice under an upright microscope.

[0060] S5: Confocal Imaging

[0061] 1. Imaging parameter settings: Place the mounted slide on the stage of the laser confocal microscopy system, select a 60x or 100x oil immersion lens, and adjust the pinhole size to 1-1.2 Airy Units to ensure imaging resolution.

[0062] 2. Target Area Localization: Activate the microscope's multi-channel switching function, first switching to the DAPI channel (blue fluorescence). Use this channel to quickly locate the overall position of the hippocampal DG region (as shown in the attached image). Figure 4 As shown, the blue area represents the hippocampal DG region; then switch to channel 561 (red fluorescence) to locate and lock onto the target neuron in the DG region. Figure 4 The red portion represents neurons in the DG region. Figure 6 This refers to the entirety of neurons in the DG region.

[0063] 3. Image Screening and Tomographic Scanning: When screening dendritic spine segments of target neurons, the following conditions must be met: ① Segment length > 20 μm; ② No intersection with other dendritic spine segments; ③ Exclude degenerated and enlarged dendritic spine segments. After successful screening, magnify the target segment 2-3 times, set the Z-axis tomographic scan distance to 0.3-0.5 μm, and activate the Z-axis tomographic scan function to obtain clear three-dimensional image data of the dendritic spines.

[0064] S6: Image Quantization Analysis

[0065] 1. Image Preprocessing: Import the Z-stack image acquired by confocal imaging into ImageJ image analysis software. Use the software's "Maximum Projection" function to create a two-dimensional maximum projection image, such as... Figure 7 As shown, the neuronal segments under the maximum projection of Image J include M mushroom-shaped spines, T filamentous spines, and S thick short spines. Image preprocessing was performed to highlight the morphological features of the dendritic spines.

[0066] 2. Classification and Quantitative Analysis: Open the Cell Counter counting software, import the preprocessed image, and accurately count and measure the morphological parameters of different types of dendritic spines according to the classification criteria shown in Table 1.

[0067] Table 1 Classification criteria for different types of dendritic thorns

[0068]

[0069] 3. Data statistics: Record parameters such as the number, length, and diameter of different types of dendritic spines, and use statistical software for data processing and analysis.

[0070] III. Experimental Results

[0071] The number of dendritic spines of different types recorded in the image quantitative analysis was statistically analyzed. The number of dendritic spines of each type was divided by the length of the corresponding dendritic segment to obtain the number of dendritic spines of that type per μm length. Multiplying this value by 10 yields the number of different types of dendritic spines per 10 μm length, i.e., the dendritic spine density. Simultaneously, the total number of dendritic spines of all types was counted and divided by the length of the corresponding dendritic segment to obtain the total dendritic spine density. This ultimately achieves a quantitative representation of the synaptic morphology and number characteristics of the hippocampal dentate gyrus.

[0072] Through the above specific implementation methods, the morphology and number of synapses in the dentate gyrus of the hippocampus can be systematically observed and analyzed. This method is standardized in operation and has clear parameters, making it suitable for basic and applied research related to hippocampal synapses in the field of neurobiology.

[0073] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for observing the morphology and number of synapses in the dentate gyrus of the hippocampus based on image analysis, characterized in that, Includes the following steps: S1. Tissue processing: After anesthetizing the experimental animals, the heart was perfused with buffer and fixative in stages. The hippocampal tissue was separated, trimmed and fixed, and then sectioned and stored at low temperature under pre-cooling conditions. S2. Staining and labeling: The dentate gyrus region of hippocampal tissue sections was targeted and labeled with specific labeling materials, and then incubated and stored at low temperature. S3. Fixation and mounting: After secondary fixation and nuclear staining of the stained sections, mount them with an anti-fluorescence quencher. S4. Confocal Imaging: Set imaging parameters, locate the target area, perform layer scanning, and select to obtain clear images of dendritic spines.

2. The method according to claim 1, characterized in that, The specific labeling material mentioned in step S2 is DiI particles, which are targeted and placed in the dentate gyrus region of the hippocampus using a fine tungsten carbide needle.

3. The method according to claim 1, characterized in that, The nuclear staining in step S3 uses DAPI, and the secondary fixation uses 3-5% paraformaldehyde, fixed at room temperature for 20-40 minutes.

4. The method according to claim 1, characterized in that, The imaging parameters in step S4 are: 60x or 100x oil immersion lens, pinhole 1-1.2 Airy Unit, Z-axis layer scanning distance 0.3-0.5μm, and layer scanning with magnification of 2-3x after locking onto the target.

5. The method according to claim 1, characterized in that, The criteria for screening the dendritic spines in step S4 are: length > 20 μm, no crossing, and exclusion of degenerative and enlarged segments.

6. The method according to claim 1, characterized in that, It also includes image quantification analysis, in which the clear dendritic spine image obtained in step S4 is imported into professional software for preprocessing, and based on the dendritic spine classification standard, the counting software is used to achieve accurate quantitative analysis of different types of dendritic spines.

7. The method according to claim 6, characterized in that, The classification criteria for dendritic spines are as follows: ① Mushroom type: head diameter > 0.5 μm or head-to-neck diameter ratio > 1.5; ② Short and thick type: length < 0.5 μm, head-to-neck diameter ratio ≤ 1 or no obvious head; ③ Slender type: length 0.5-1 μm, head diameter < 0.5 μm or no obvious head or head-to-neck diameter ratio < 1.5; Filamentous pseudopodia type: head and neck diameters are equal, length > 3 μm, neck diameter 0.1-0.4 μm or head-to-neck diameter ratio < 2.

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