Use of rabies virus mutants in traditional two-photon deep brain cortex neurons

Abstract

The application discloses application of a rabies virus mutant in traditional two-photon microscope deep brain cortex neuron imaging, and the rabies virus mutant is CVS-N2c-ΔG-GCaM6s. The application also discloses a calcium imaging virus containing the rabies virus mutant. The rabies virus mutant in the application is used as a retrograde labeling virus, has the advantages of high brightness of fluorescent expression of the labeled neuron, and has fast expression speed, thereby greatly saving time cost. In addition, the rabies virus mutant has the ability of labeling the complete structure of a cell, can completely record the functional signal from a dendritic spine to a cell body, provides a suitable method for studying input-output integration of single neuron information, especially deep neuron. The rabies virus mutant can realize high-quality functional imaging of deep neuron under the conventional two-photon microscope, and has the technical characteristics of high safety and low use cost.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a rabies virus mutant in traditional two-photon imaging of deep neurons in the cerebral cortex; particularly high-quality imaging of the cell bodies and subcellular structures of deep neurons in the cerebral cortex. Background Technology

[0002] The brain is the most important organ in the human body, and neuroscientists have long been dedicated to its study. The mammalian cortex has six layers and forms the crucial structural basis for the brain's processing of sensory information. To explore the mechanisms by which the brain processes information, we must understand both how groups of neurons respond to external stimuli and how individual neurons integrate inputs from different times and spaces along their dendrites to produce outputs.

[0003] With the development of two-photon technology, in vivo studies of synaptic integration have determined the synaptic activity patterns of dendrites in layers 2 / 3 and 4 of the visual and auditory cortex during information reception. Studies have also investigated dendritic integration patterns in layer 5 of the somatosensory cortex during behavioral patterns. Because layer 6 is located at the deepest part of the cortex, current imaging methods have limitations in achieving high-resolution imaging of neurons in layer 6. However, layer 6 is the first layer to develop in the neocortical structure, playing a crucial role not only in the formation of the cerebral cortical neural network but also in irreplaceable functions such as gain regulation and active perception. Therefore, studying the information processing mechanisms of neurons in layer 6 is vital for understanding the information processing mechanisms of the cerebral cortex. Achieving high-quality functional imaging of neurons in layer 6 is a critical technical challenge that urgently needs to be overcome.

[0004] Currently, there are three main methods for visualizing neurons in deep brain tissue:

[0005] 1) Replace two-photon excitation with three-photon excitation. For the same biological fluorophore, longer wavelength excitation light can reduce scattering through the tissue; the same principle applies to using redshifted fluorophores (Cal 590 dye) to improve imaging depth.

[0006] 2) Correcting the wavefront of the excitation light, i.e., using adaptive optics technology to correct optical wavefront distortions caused by brain tissue, thereby achieving deeper imaging.

[0007] 3) Deep tissue imaging is achieved by surgically removing superficial brain tissue.

[0008] Current imaging methods for studying deep cortical tissues all have certain limitations. Blind insertion methods based on in vivo electrophysiological data struggle to determine the type of cells recorded. Two-photon imaging allows direct observation of neurons, but its nonlinear physics and the inhomogeneity of scattering in brain tissue limit imaging depth (primarily within 500 μm). Theoretically, adaptive optics and three-photon imaging systems allow for greater imaging depth. However, firstly, these methods require high laser power or complex additional instruments; secondly, they are optimized from the probe end, and for the biological brain tissue being examined, excessively high laser power can damage neurons. Currently, the primary method for labeling neurons to record functional activity uses recombinant adeno-associated viruses, which exhibit weak fluorescence expression. Therefore, high-quality functional imaging of deep tissues remains limited by the labeling methods for neurons. Summary of the Invention

[0009] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide a rabies virus mutant that is low-cost, highly safe, and capable of imaging deep cortical neurons under conventional two-photon microscopy, and its applications.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] The application of rabies virus mutants in imaging deep neurons of the cerebral cortex under conventional two-photon microscopy.

[0012] In some preferred embodiments, the rabies virus mutant is CVS-N2c-ΔG-GCaM6s.

[0013] In some preferred embodiments, the deep cortical neurons are CT neurons in L6 of the cerebral cortex.

[0014] In some preferred embodiments, the application targets are wild-type transgenic animal strains of rodents, mammals, etc., that express recombinases or tetracycline transcription activators, and / or non-human primates.

[0015] A calcium imaging virus for CT neurons in the L6 cerebral cortex, the calcium imaging virus comprising an effective amount of the aforementioned rabies virus mutant.

[0016] In some preferred embodiments, the calcium imaging virus also includes a pharmaceutically acceptable vector.

[0017] Compared with the prior art, the present invention has at least the following advantages:

[0018] 1) The rabies virus mutant in this invention, as a retrograde labeling virus, avoids potential damage to the imaging area caused by dye loading / virus injection into the target area, which is also a great benefit to the improvement of imaging quality. The labeled neurons have the advantage of high fluorescence intensity. Compared with neurons labeled with recombinant adeno-associated virus (AAV), neurons labeled with CVS virus have stronger fluorescence intensity and significantly improved imaging signal-to-noise ratio. Moreover, its expression speed is fast. Compared with retrograde dye (CTB) labeling, which requires 7 days of expression, and retrograde AAV virus labeling, which requires 21 days of expression, the number of cells and cell density of CVS virus-labeled cells reach the level of CTB expression for 7 days or AAV expression for 21 days in 3 days, which greatly saves time. Furthermore, it has the ability to label the complete structure of cells and can completely record functional signals from dendritic spines to cell bodies. This provides a suitable method for studying the input-output integration of information of a single neuron, especially for deep neurons.

[0019] 2) The rabies virus mutant of this invention can be used for imaging deep neurons in the cerebral cortex without requiring the construction of complex instruments as in the case of three-photon excitation or adaptive optics systems. High-quality functional imaging of CT neurons in the L6 layer of the cerebral cortex can be achieved under conventional two-photon microscopy, offering advantages such as high safety and low cost. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0021] Figure 1 This is a comparison diagram of the labeling effect of the rabies virus mutant (CVS) AAV virus in this invention;

[0022] Figure 2 This is a comparison diagram showing the labeling speed of the rabies virus mutant (CVS) with CTB dye and AAV virus in this invention;

[0023] Figure 3 This invention provides a complete functional signaling diagram of the rabies virus mutant (CVS) from dendritic spines to cell body.

[0024] Figure 4 This is a high-quality functional imaging image of deep neurons in the cerebral cortex achieved by the rabies virus mutant (CVS) under a conventional two-photon microscope in this invention;

[0025] Figure 5 The rabies virus mutant (CVS) used in this invention is used to specifically label CT neurons in layer 6 of the mouse auditory cortex. Detailed Implementation

[0026] To make the technical means, creative features, objectives and effects of the present invention easier to understand, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the following embodiments are merely preferred embodiments of the present invention and not all of them.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] In the following implementations, the rabies virus mutant used was CVS-N2c-ΔG-GCaM6s (BC-RV-CVS715), which was purchased from Brinkes Company in Shenzhen, China.

[0029] Example 1

[0030] Expression rate of rabies virus mutants:

[0031] In this embodiment, the rabies virus mutant (CVS), CTB dye, and retrograde AAV are labeled, specifically as follows:

[0032] In vivo injection: CVS-N2c-ΔG-GCaM6s (BC-RV-CVS715) was injected into wild-type mice MGB at a volume of 100 nL, and perfusion sections were performed at 1, 2, 3, 7 and 21 days of virus expression.

[0033] CTB (purchased from Invitrogen, USA, C34775) was injected into wild-type mouse MGB at a volume of 100 nL, and perfusion sections were performed 7 days after dye expression.

[0034] Retrograde AAV virus (purchased from Wuhan Shumi Company, China, PT-0100) was injected into wild-type mice MGB at a volume of 100 nL, and perfusion sections were performed 21 days after virus expression.

[0035] The results are as follows Figure 2 As shown, a. the effects of CVS expression for 1, 2, 3, 7, and 21 days; b. the effect of AAV expression for 21 days; c. the effect of CTB dye expression for 7 days; d. cell count results show that CVS expression for 3 days achieves the same cell count as CTB expression for 7 days or AAV expression for 21 days; e. cell density results show that CVS expression for 3 days achieves the same cell density as CTB expression for 7 days or AAV expression for 21 days. Figure 2It can be seen that the rabies virus mutant (CVS) is expressed quickly. In contrast, retrograde dye (CTB) labeling requires 7 days of expression and retrograde AAV virus labeling requires 21 days of expression. The number of cells and cell density of CVS virus labeling reached the level of CTB expression for 7 days or AAV expression for 21 days in just 3 days, which greatly saves time and costs.

[0036] Example 2

[0037] A rabies virus mutant (BC-RV-CVS715) was used to specifically label corticothalamic (CT) neurons in layer 6 of the mouse auditory cortex, specifically as follows: Figure 5 As shown, a. Schematic diagram of in vivo injection; b. Cell body distribution map 3 days after CVS injection, CTB-555 is used for injection site localization; c. Enlarged view of cell body distribution; d. Foxp2 staining map, Foxp2 is a marker specifically expressed by neurons in layer 6.

[0038] In vivo injection: The high-quality imaging CVS-N2c-ΔG-GCaM6s (BC-RV-CVS715) that achieves deep neuronal cell body and subcellular structure was injected at a volume of 100 nL into the MedialGeniculate Body (MGB) brain region (AP-3.1 mm, ML 2.0 mm, DV-2.8 mm) of wild-type mice. After 3 days of viral expression, the mice were morphologically sectioned to determine the specificity of the virus.

[0039] Morphological sections: First, the blood was replaced with physiological saline, and then fixed with 4% paraformaldehyde (PFA) solution. After dehydration in 15% sucrose PBS and fixation for 24 hours, mouse brains were sliced ​​in the sagittal plane using a cryostat to obtain 50 μm thick sections. Slices with fluorescent markers and fiber optic implantation traces were selected using a fluorescence microscope, and cell nuclei were stained with DAPI (1:10000, D9564, Sigma-Aldrich) solution. Images were obtained using a confocal microscope (Zeiss, LSM700) at 20× and 40× magnification, with numerical apertures of 0.7 and 1.25, respectively. To determine the viral injection site, the virus was contaminated with CTB-555. To determine that the labeled neurons were located in layer 6 of the auditory cortex, more precise localization was required using immunohistochemical staining. Here, FOXP2 antibody was used to label and stain pyramidal neurons in layer 6 of the auditory cortex. The primary antibody used was: goat-derived anti-FOXP2 (1:500, Abcam, ab1307). The secondary antibody used was: Alexa Fluor 594 donkey anti-goat (1:1000, Invitrogen, A-11058).

[0040] Data Analysis: For grayscale analysis, the distance from the dura mater to the sixth layer / white matter boundary of labeled cells within the Region of Interest (ROI) was measured. The proportional distance was then obtained by normalizing the distance to cortical thickness with a step size of 1%, with the dura mater and the sixth layer / white matter boundary representing 0% and 100%, respectively. Grayscale values ​​were measured using the PlotProfile (pixel intensity count) function of Image J. The number of cells per step was averaged across different animals. Cells located near the edge of the analysis region were excluded from the analysis.

[0041] Test results are as follows Figure 1 As shown, a. 3D reconstruction of CVS-labeled neurons using two-photon imaging; b. Imaging effect of CVS-labeled neurons in each layer of the cortex; c. Imaging effect of AAV-labeled neurons in each layer of the cortex; d. The green curve represents the relative gray value (fluorescence intensity - background intensity) of the fluorescence intensity of the CVS-labeled structure, and the blue curve represents the relative gray value of the fluorescence intensity of the AAV-labeled structure; e. Statistical values ​​show that from the 2nd / 3rd layer onwards, the fluorescence intensity of the CVS-labeled structure is significantly stronger than that of the AAV-labeled structure. As can be seen from the figures, the rabies virus mutant-labeled neurons in this application have the advantage of high fluorescence expression brightness. Compared with neurons labeled with recombinant adeno-associated (AAV) virus, the CVS-labeled neurons have stronger fluorescence intensity and a significantly improved imaging signal-to-noise ratio.

[0042] Example 3

[0043] Rabies virus mutants are used for two-photon imaging;

[0044] In vivo injection: The packaged high-quality imaging CVS-N2c-ΔG-GCaM6s (BC-RV-CVS715) for deep neuronal cell bodies and subcellular structures was injected at a volume of 100 nL into the MedialGeniculate Body (MGB) brain region (AP-3.1 mm, ML 2.0 mm, DV-2.8 mm) of wild-type mice. Two-photon imaging was performed 3 days after viral expression.

[0045] Two-photon imaging: Mice were first anesthetized with isoflurane diluted in pure oxygen (1-2%) for surgery and placed on a temperature-controlled plate (37.5-38°C). After injection of the local anesthetic lidocaine, the skin and muscle covering the primary auditory cortex were removed. Next, a homemade plastic skull fixation device was attached to the skull containing the primary auditory cortex using cyanoacrylate adhesive. The skull was then removed using a hand drill to expose the surface of the primary auditory cortex (center point: 2.5 mm posterior to the anterior fontanelle, 4.5 mm lateral to the midline). The mice were then transferred to a two-photon recording table, and the anesthesia system was removed to allow the mice to awaken. They were allowed to fully awaken for half an hour. The exposed mouse primary auditory cortex was covered with a continuously circulating artificial cerebrospinal fluid (CSF) at pH 7.4 (composition: 125 mM NaCl, 4.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, and 20 mM glucose; circulated with 95% pure oxygen and 5% carbon dioxide). The two-photon imaging experiments in this study used a self-made two-photon microscopy system (Lotos 1.0, Suzhou Institute of Biomedical Engineering and Technology) based on a 12 kHz resonant scanner, similar to some previously reported experimental systems. The excitation light used in the two-photon imaging was generated via a mode-locked Ti:sapphire laser (Mai-Tai DeepSee, Spectrophysic), and transmitted to the sample through a 40x immersion objective lens with a numerical aperture of 0.8. During the two-photon calcium imaging experiment, the excitation wavelength was modulated to 920 nm. The average laser energy delivered to the brain ranges from 30 to 160 mW, depending on the depth of the imaging plane. In all calcium imaging experiments, image data were collected at a resolution of 600 × 600 pixels per frame and a sampling rate of 40 Hz. In most cases, at least two or more imaging planes were collected for each experimental animal.

[0046] Data Analysis: All calcium imaging and electrophysiological data analysis was performed offline using self-developed software (LabVIEW 2012), Igor Pro 5.0 (Experimental Data Graphics Company), and Matlab 2014a (Mexico). To extract cellular fluorescence signals, the region of interest (ROI), i.e., the space occupied by the cell, was manually selected using the software. For each selected cellular region, the fluorescence values ​​of all pixels within it were averaged to obtain the cell's fluorescence value (f) at that time point. The relative fluorescence change Δf / f = (f - f0) / f0 represents the cell's calcium signal. The baseline fluorescence value (f0) refers to the fluorescence value at the 25th percentile within a sliding time window. For all two-photon calcium imaging data, neuronal calcium signals were automatically detected by the program. The baseline detection sliding time window was 1 second, followed by a 500-millisecond calcium signal detection time window. In short, a linear fitting model was first used to detrend the baseline calcium signal; three times the baseline standard deviation was defined as the noise level. The amplitude of the calcium signal is defined as the average fluorescence value within a 200ms time window centered on the highest value. Furthermore, the derivative of the calcium signal within the detection time window is used to measure the rate of rise of the calcium signal. A signal is considered valid only if both its amplitude and rise rate are above the corresponding noise level. The detected calcium signal curve is then processed by exponential IIR filtering (time window: 200ms) and subtracted from the original curve; the resulting curve serves as the baseline for the next calcium signal detection.

[0047] Test results are as follows Figure 3 and Figure 4 As shown, where Figure 3 In this context, a. complete structural reconstruction of a single cell; b. signal recording of dendritic spines, trunk, and cell body in a single cell. From... Figure 3 It is known that the rabies virus mutant (CVS) has the ability to label intact cellular structures and can completely record functional signals from dendritic spines to the cell body. This provides a suitable method for studying the input-output integration of information in individual neurons, especially for deep neurons. Figure 4 In the diagram, a. Population cell imaging; ba diagram shows the spontaneous signals of neurons in anesthesia and wakefulness. ba diagram shows the signals of neurons in response to white noise in anesthesia and wakefulness; from... Figure 4 As can be seen, by applying the rabies virus mutant (CVS) labeling method in this application, we do not need to construct additional complex instruments to achieve imaging of deep neurons, as is required with three-photon excitation or adaptive optics systems. High-quality functional imaging of deep neurons can be achieved using a conventional two-photon microscope.

[0048] In summary, the rabies virus mutant (CVS) of this invention can label the entire cellular structure, enabling high signal-to-noise ratio CT neuronal functional imaging in the L6 cerebral cortex under conventional two-photon microscopy. CVS virus is a retrograde virus that can specifically label circuits. This labeling method shows great promise in assisting two-photon observation of neurons, and can significantly improve the imaging depth and resolution of deep neurons.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Use of a rabies virus mutant for imaging deep layer neurons in the cerebral cortex under a conventional two-photon microscope, characterized in that, The rabies virus mutant specific labeling deep layer neurons in the cerebral cortex, wherein the rabies virus mutant is CVS-N2c-ΔG-GCaM6s, the deep layer neurons in the cerebral cortex are CT neurons in the cerebral cortex L6, wherein the rabies virus mutant is CVS-N2c-ΔG-GCaM6s labeled neurons fluorescence intensity becomes stronger, and the imaging is using a 12KHz resonance scanner based two-photon microscopy imaging system.

2. Use according to claim 1, characterized in that, The subject of the application is a rodent, a transgenic animal line expressing a recombinase or a tetracycline transcriptional activator and / or a non-human primate.

3. A calcium imaging virus of CT neurons in cerebral cortex L6, characterized in that, The calcium imaging virus comprises an effective amount of the rabies virus mutant of claim 1 or 2.

4. The calcium imaging virus of claim 3, wherein, The calcium imaging virus further comprises a pharmaceutically acceptable carrier.

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