Identification and electrophysiology recording method and system for rapidly issuing interneurons and application of identification and electrophysiology recording method and system

Abstract

The invention discloses an identification and electrophysiology recording method and system for rapidly issuing interneurons and application of the identification and electrophysiology recording method and system. The recognition method comprises the following steps: trimming brain tissues to obtain a brain block containing a target area, and then preparing an in-vitro brain slice; based on the preset morphological information and the preset position information of the target neurons, searching from the in-vitro brain slice by using a patch clamp electrophysiology system and identifying suspected target neurons through comparison; and performing electrophysiological recording on the suspected target neurons and other neurons, and comparing to determine whether the suspected target neurons are target neurons or not. The electrophysiological recording method comprises the following steps: finding the quickly issued interneurons by adopting the identification method, and then carrying out electrophysiological recording analysis. According to the method, under the condition of not depending on a fluorescence microscope and a transgenic animal with a special marker, the interneurons rapidly distributed in the hippocampal CA1 region can be accurately recognized, recorded and analyzed, the experimental process can be simplified, the experimental cost can be reduced, and the experimental efficiency and flexibility can be improved.

Description

Technical Field

[0001] The present invention relates to the field of neuroscience technology, and in particular to a method and system for identifying and electrophysiologically recording fast-spiking interneurons and applications thereof. Background Art

[0002] Fast-spiking parvalbumin-positive interneurons (FS PV) + INs are an important type of GABAergic neurons that participate in regulatory mechanisms in multiple brain regions and play a key role in the nervous system. In current nervous system research, the common method to study these neurons is to use patch clamp electrophysiological techniques on isolated brain slices to record their electrophysiological signals. This method usually requires the use of transgenic animal models. + INs are specially marked to facilitate identification and recording under a fluorescence microscope. For example, CN110082516A discloses a multi-dimensional method for acquiring neuronal information, which is a method for combining patch clamp technology with gene chip technology to acquire multi-dimensional information of a single neuron. Specifically, the electrophysiological characteristics of the target neuron are recorded using patch clamp technology, and the target single cell sample is obtained through the glass electrode in the patch clamp system, and then the molecular marker information is acquired using gene chip technology, thereby organically combining patch clamp technology and gene chip technology. This method can simultaneously acquire the morphological characteristics, distribution characteristics, electrophysiological characteristics, and molecular markers of the same target neuron, that is, multi-dimensional cell information, which can be applied to neuron typing, can classify neurons in more dimensions, and can also reveal the functions of neurons in more detail.

[0003] Although this transgenic animal model is relatively mature in neuroscience research, it also has some problems. First, the breeding process of transgenic animals is complicated and costly; second, the patch clamp experiment must rely on a fluorescence microscope, which not only limits the scope of application of the experiment, but also reduces the flexibility and efficiency of the operation. The whole process is quite labor-intensive and costly. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a method, system and application of identifying and electrophysiologically recording fast-spiking interneurons, which can enable researchers to study the FS PV in the CA1 region of the hippocampus without relying on expensive fluorescence microscopes and specially labeled transgenic animals. + The precise identification, recording and analysis of interneurons also simplifies the experimental process, significantly reduces experimental costs, and improves experimental efficiency and flexibility.

[0005] To achieve the above objectives and other related objectives, the present invention provides a method for identifying fast-firing interneurons, comprising:

[0006] Taking brain tissue, trimming it to obtain a brain block containing the target area, and preparing the brain block containing the target area into an in vitro brain slice;

[0007] Based on the preset morphological information and preset position information of the target neuron, using a patch clamp electrophysiological system to observe and search for the target neuron in the isolated brain slice, and obtaining the measured morphological information and measured position information of the neuron;

[0008] Analyzing and comparing the preset morphological information and preset position information with the measured morphological information and measured position information, and identifying suspected target neurons from the isolated brain slice according to the comparison result;

[0009] Use the patch clamp electrophysiological system to perform electrophysiological recordings on suspected target neurons and other neurons to obtain measured electrophysiological information;

[0010] Analyzing and comparing the preset electrophysiological information of the target neuron with the measured electrophysiological information, determining whether the suspected target neuron is the target neuron according to the comparison result, and identifying the target neuron from other neurons;

[0011] The target area is the hippocampus, and the target neurons are fast-spiking interneurons.

[0012] Further, based on the preset morphological information and preset position information of the target neuron, a patch clamp electrophysiological system is used to observe and search for the target neuron from the isolated brain slice, and the measured morphological information and measured position information of the neuron are obtained; the preset morphological information and preset position information are analyzed and compared with the measured morphological information and measured position information, and the suspected target neuron is identified from the isolated brain slice according to the comparison result, including:

[0013] After finding the cell band in the CA1 region of the hippocampus, searching for the target neurons and pyramidal neurons along the cell band, distinguishing and determining suspected target neurons according to preset morphological information and preset position information of the target neurons and pyramidal neurons;

[0014] The preset morphological information of the target neuron and the pyramidal neuron includes the following preset morphological features: the cell body of the fast-spiking interneuron is larger than that of the pyramidal neuron and has multipolar dendrites; the cell body of the pyramidal neuron is mostly cone-shaped, and a main dendrite extends from the tip of the cone;

[0015] The preset position information of the target neuron and the pyramidal neuron includes the following preset position features: the fast-firing interneurons are independently distributed on both side edges of the pyramidal neuron strip, and are in contrast to the large number of regularly arranged pyramidal neurons.

[0016] Furthermore, the electrophysiological information includes electrophysiological characteristics of neuronal cell membranes and / or neuronal action potential characteristics.

[0017] Furthermore, the method of using a patch clamp electrophysiological system to perform electrophysiological recordings on suspected target neurons and other neurons to obtain the measured electrophysiological properties of neuronal cell membranes includes: sealing and breaking the membranes of suspected target neurons and other neurons, and using a whole-cell recording mode to observe and record to obtain the measured electrophysiological properties of neuronal cell membranes.

[0018] Furthermore, the electrophysiological characteristics of the neuron cell membrane include at least one of a resting membrane potential, a capacitance value (Cm) and a membrane resistance (Rm).

[0019] Furthermore, the preset neuron cell membrane electrophysiological characteristics of the fast-spiking interneuron include at least one of the following A1 to A3:

[0020] A1, resting membrane potential is -71 to -72 mV;

[0021] A2, capacitance value is 120~180pF;

[0022] A3. Membrane resistance is 60~90MΩ.

[0023] Furthermore, the method of using a patch clamp electrophysiological system to perform electrophysiological recordings on suspected target neurons and other neurons to obtain the measured neuronal action potential characteristics includes: recording in current clamp mode, injecting current into suspected target neurons and other pyramidal neurons to induce them to generate action potentials, and observing and recording to obtain the measured neuronal action potential characteristics.

[0024] Furthermore, the neuron action potential characteristics include at least one of a discharge pattern, a discharge frequency, a half-peak width, and a hyperpolarization afterpotential.

[0025] Furthermore, the preset neuron action potential characteristics of the fast-spiking interneuron include at least one of the following items B1 to B5:

[0026] B1. Under an injection current of 100-400 pA, the fast-firing interneurons exhibit clustered discharges (i.e., clustered discharge mode);

[0027] B2. At an injected current of >1000 pA, the action potential (AP) of the fast-firing interneuron continues to discharge, and the discharge frequency is >100 Hz;

[0028] B3. Under the same injection current, the firing frequency of the action potential of the fast-firing interneuron is higher than the firing frequency of the action potential of the pyramidal neuron;

[0029] B4. Under the same injection current, the half-width of a single action potential of the fast-spiking interneuron is smaller than the half-width of a single action potential of the pyramidal neuron;

[0030] B5. Under the same injection current, the after-hyperpolarization potential (AHP) of a single action potential of the fast-firing interneuron is greater than the after-hyperpolarization potential of a single action potential of the pyramidal neuron.

[0031] Further, analyzing and comparing the preset electrophysiological information of the target neuron with the measured electrophysiological information, determining whether the suspected target neuron is the target neuron according to the comparison result, and identifying the target neuron from other neurons, including:

[0032] The preset neuron cell membrane electrophysiological characteristics are analyzed and compared with the measured neuron cell membrane electrophysiological characteristics, and / or the preset neuron action potential characteristics are analyzed and compared with the measured neuron action potential characteristics, and whether the suspected target neuron is the target neuron is determined based on the comparison results, and the target neuron is identified from other neurons.

[0033] Furthermore, the method also includes: taking all or part of the fresh brain and immersing it in oxygen-saturated and frozen sectioning solution to separate and obtain brain tissue.

[0034] Furthermore, the method further comprises: preparing the brain block containing the target area into an in vitro brain slice and then transferring it into artificial cerebrospinal fluid with continuous oxygenation for incubation. The purpose of this operation is to wait for the neurons on the brain slice to recover their state function so as to facilitate the patch clamp experiment.

[0035] Furthermore, the brain tissue is derived from rodents, including but not limited to mice, rats, etc.

[0036] The present invention also provides an electrophysiological recording method of fast-firing interneurons, comprising: using the above-mentioned identification method to find the fast-firing interneurons, and then performing electrophysiological recording and analysis on the fast-firing interneurons.

[0037] The present invention also provides a fast-firing interneuron identification system, including an isolated brain slice, a patch clamp electrophysiological system, and a neuron analysis module;

[0038] The in vitro brain slice is prepared from a brain block containing the target area, and the brain block containing the target area is obtained by trimming brain tissue;

[0039] The patch clamp electrophysiological system is used to observe and find target neurons from the isolated brain slice, and obtain measured morphological information and measured position information of the neurons, and to perform electrophysiological recording on suspected target neurons and other neurons to obtain measured electrophysiological information;

[0040] The neuron analysis module is used to analyze and compare the preset morphological information and preset position information with the measured morphological information and measured position information, and identify suspected target neurons from the isolated brain slice according to the comparison results; and to analyze and compare the preset electrophysiological information of the target neuron with the measured electrophysiological information, determine whether the suspected target neuron is the target neuron according to the comparison results, and identify the target neuron from other neurons;

[0041] The target area is the hippocampus, and the target neurons are fast-spiking interneurons.

[0042] The present invention also provides the application of the above-mentioned method for identifying fast-spiking interneurons and / or the above-mentioned method for electrophysiological recording of fast-spiking interneurons and / or the above-mentioned system for identifying fast-spiking interneurons in neuroscience research.

[0043] As described above, the method, system and application of the present invention for identifying and electrophysiologically recording fast-spiking interneurons have the following beneficial effects:

[0044] The present invention adopts a specific sampling and trimming method to obtain a brain block containing the hippocampus and make it into an in vitro brain slice; then, by analyzing and comparing the cell morphology and position characteristics of FS interneurons and pyramidal neurons, a part of the suspected target neurons, namely FS interneurons, are identified, but some cannot be determined to be FS interneurons; further electrophysiological recording is performed, and then by analyzing and comparing the cell membrane characteristics and electrophysiological characteristics of the two types of neurons, the suspected target neurons identified are further verified and determined to be FS interneurons, and more FS interneurons can also be found. Based on this, researchers can study FS PV in the CA1 region of the hippocampus without relying on expensive fluorescence microscopes and specially labeled transgenic animals. +The method and system of the present invention can significantly reduce the experimental cost and improve the efficiency and flexibility of the experiment. At the same time, the present invention also provides a cost-effective and easy-to-operate solution for research teams of different sizes by simplifying the experimental process. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Shown is a schematic diagram of the preparation of rat hippocampal CA1 brain tissue in Example 1 of the present invention.

[0046] Figure 2 Shown is the neuronal band in the hippocampal CA1 region observed in Example 1 of the present invention.

[0047] Figure 3 The figure shows the FS interneurons labeled with biocytin in Example 1 of the present invention.

[0048] Figure 4 Shown are the action potentials induced under different injection currents in Example 1 of the present invention.

[0049] Figure 5 It shows a comparison diagram of the AP of FS interneurons (red curve) and the AP of pyramidal neurons (black curve) in Example 1 of the present invention. DETAILED DESCRIPTION

[0050] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0051] In the present invention, unless otherwise specified, the term "plurality" means two or more.

[0052] The character " / " indicates that the objects before and after are in an "or" relationship. For example, A / B means: A or B.

[0053] The term "and / or" is a description of the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or, A and B.

[0054] An embodiment of the present invention provides a method for identifying fast-spiking interneurons, comprising:

[0055] Based on the preset morphological information and preset position information of the target neuron, using a patch clamp electrophysiological system to observe and search for the target neuron in the isolated brain slice, and obtaining the measured morphological information and measured position information of the neuron;

[0056] Analyzing and comparing the preset morphological information and preset position information with the measured morphological information and measured position information, and identifying suspected target neurons from the isolated brain slice according to the comparison result;

[0057] Use the patch clamp electrophysiological system to perform electrophysiological recordings on suspected target neurons and other neurons to obtain measured electrophysiological information;

[0058] Analyzing and comparing the preset electrophysiological information of the target neuron with the measured electrophysiological information, determining whether the suspected target neuron is the target neuron according to the comparison result, and identifying the target neuron from other neurons;

[0059] The target area is the hippocampus, and the target neurons are fast-spiking interneurons.

[0060] The above implementation method obtains a brain block containing the hippocampus by trimming brain tissue and makes it into an in vitro brain slice; then, by analyzing and comparing the cell morphology and position characteristics of FS interneurons and pyramidal neurons, a part of the suspected target neurons, namely FS interneurons, are identified, but some cannot be determined to be FS interneurons; further electrophysiological recording is performed, and then by analyzing and comparing the cell membrane characteristics and electrophysiological characteristics of the two types of neurons, the suspected target neurons identified are further verified and determined to be FS interneurons, and more FS interneurons can also be found. Based on this, researchers can study FS PV in the CA1 region of the hippocampus without relying on expensive fluorescence microscopes and specially labeled transgenic animals. + On this basis, researchers can efficiently conduct in-depth electrophysiological recording analysis of this specific neuron, and then study the neural system circuits, ion channel regulation mechanisms, etc., and significantly reduce experimental costs and improve experimental efficiency and flexibility by simplifying the experimental process.

[0061] In some embodiments, brain tissue is taken and trimmed to obtain a brain block containing the target area, and the brain block containing the target area is prepared into an in vitro brain slice, comprising the following steps:

[0062] S1. Lay the removed brain tissue ventral side down.

[0063] S2, separate the cerebrum and cerebellum, retain the brain tissue;

[0064] S3, remove the olfactory bulbs and prefrontal lobe areas of the left and right brain;

[0065] S4, separate the left and right brains and place them separately;

[0066] S5, cut off the caudal sides of the left and right brain;

[0067] S6, cutting off the raphe part and the lateral cerebral cortex part perpendicular to the incisions in steps S3 and S5 to obtain a brain block containing the target area;

[0068] S7. Turn the brain block containing the target area toward the midline and fix it for slicing to prepare in vitro brain slices.

[0069] In a specific embodiment, in step S2, a vertical cut is made to separate the cerebrum and cerebellum, and the cerebral tissue is retained.

[0070] In a specific embodiment, in step S3, the olfactory bulbs and prefrontal lobe regions of the left and right brains are cut vertically and tilted at a preset angle with the incision direction of step S2 as the center line. Exemplarily, the preset angle may be 30°, for example.

[0071] In a specific embodiment, in step S4, a vertical cut is made along the midline to separate the left and right brains.

[0072] In a specific embodiment, in step S5, the incision direction in step S2 is used as the center line, and the left and right caudal sides of the brain are cut vertically at a preset angle. Exemplarily, the preset angle may be 30°, for example.

[0073] The inventors of the present application have found through many experiments that in the research related to the identification and electrophysiological recording of FS interneurons, the brain blocks obtained by trimming the brain tissue of mice and other rodents according to the sampling and trimming methods recorded in the prior art, making in vitro brain slices and using them as research samples, cannot find FS interneurons well and perform electrophysiological recordings. Therefore, the inventors of the present application conducted in-depth research on the sampling and trimming methods, and finally clarified the appropriate sampling and trimming methods, and gave the best implementation method, namely the sampling and trimming methods provided in the above embodiments. The brain blocks containing the hippocampus obtained based on the sampling and trimming methods provided in the above embodiments are made into in vitro brain slices and used as research samples, which helps to quickly find FS interneurons through the patch clamp electrophysiological system.

[0074] In some embodiments, based on preset morphological information and preset position information of the target neuron, a patch clamp electrophysiological system is used to observe and search for the target neuron from the isolated brain slice, and the measured morphological information and measured position information of the neuron are obtained; the preset morphological information and preset position information are analyzed and compared with the measured morphological information and measured position information, and the suspected target neuron is identified from the isolated brain slice according to the comparison result, including:

[0075] After finding the cell band in the CA1 region of the hippocampus, searching for the target neurons and pyramidal neurons along the cell band, distinguishing and determining suspected target neurons according to preset morphological information and preset position information of the target neurons and pyramidal neurons;

[0076] The preset morphological information of the target neuron and the pyramidal neuron includes the following preset morphological features: the cell body of the fast-spiking interneuron is larger than that of the pyramidal neuron and has multipolar dendrites; the cell body of the pyramidal neuron is mostly cone-shaped, and a main dendrite extends from the tip of the cone;

[0077] The preset position information of the target neurons and pyramidal neurons includes the following preset position features: the fast-sending interneurons are independently distributed on the two side edges of the pyramidal neuron strips, and are in contrast to the large number of regularly arranged pyramidal neurons. The morphological characteristics and positional characteristics of the target neurons and pyramidal neurons mentioned in the above embodiments, especially the morphological characteristics and positional characteristics of the FS interneurons, are discovered and summarized by the inventors of the present application after many actual experiments and long-term research. Although previous studies have also analyzed and explained the morphological characteristics and positional characteristics of FS interneurons and / or pyramidal neurons, in actual experiments, the inventors of the present application found that according to the contents recorded in the prior art, without relying on fluorescent microscopes and specially labeled transgenic animals, it is impossible to quickly and accurately identify FS PV from the CA1 region of the hippocampus. + Interneurons, in-depth analysis of the reasons may also be: FS PV in the hippocampal CA1 area + Intermediate neurons are relatively few in number, accounting for far less than 10% of the total number of cells in the hippocampus. They are difficult to find without fluorescent labeling to stain the target neurons and then observing them using a fluorescent microscope.

[0078] In some embodiments, the electrophysiological information includes electrophysiological properties of neuronal cell membranes and / or neuronal action potential properties.

[0079] In some embodiments, the method of using a patch clamp electrophysiological system to perform electrophysiological recordings on suspected target neurons and other neurons to obtain measured neuronal cell membrane electrophysiological properties includes: sealing and breaking the membranes of suspected target neurons and other neurons, and using whole-cell recording mode to observe and record to obtain measured neuronal cell membrane electrophysiological properties.

[0080] In some embodiments, the electrophysiological properties of the neuronal cell membrane include at least one of resting membrane potential, capacitance value, and membrane resistance.

[0081] In some embodiments, the preset neuron cell membrane electrophysiological properties of the fast-spiking interneuron include at least one of the following A1 to A3:

[0082] A1, resting membrane potential is -71 to -72 mV;

[0083] A2, capacitance value is 120~180pF;

[0084] A3. Membrane resistance is 60~90MΩ.

[0085] The membrane characteristics of the FS interneurons mentioned in the above embodiments are discovered and summarized by the inventors of the present application after multiple actual experiments and long-term research. Although previous studies have also analyzed and explained the membrane characteristics of FS interneurons, in actual experiments, the inventors of the present application found that according to the content recorded in the prior art, it is impossible to accurately judge whether the identified target neuron is a FS interneuron, or the membrane characteristics of some FS interneurons recorded in the prior art are slightly different from or significantly different from those obtained by the inventors of the present application through actual experimental records and analysis. Moreover, the human brain or primates and rodents have obvious differences in brain tissue size and total number of neurons, and the proportion of FS interneurons in different brain regions is also different, so the regulatory ability is also different, so it is normal to have certain differences. Based on the complexity of nervous system research, the same template cannot be simply applied to study all brain regions, and it is necessary to make adjustments in actual experiments.

[0086] In some embodiments, a patch clamp electrophysiological system is used to perform electrophysiological recordings on suspected target neurons and other neurons to obtain measured neuronal action potential characteristics, including: recording in current-clamp mode, injecting current into suspected target neurons and other pyramidal neurons to induce them to generate action potentials, and observing and recording to obtain measured neuronal action potential characteristics.

[0087] In some embodiments, the neuronal action potential characteristics include at least one of a discharge pattern, a discharge frequency, a half-peak width, and a hyperpolarization afterpotential.

[0088] The embodiment of the present invention further verifies and determines that the identified target neuron is a FS interneuron by recording, analyzing and comparing the electrophysiological characteristics of the two neurons, thereby enabling researchers to study the FS PV in the CA1 region of the hippocampus without relying on expensive fluorescence microscopes and specially labeled transgenic animals. + Interneurons are precisely identified.

[0089] In some embodiments, the preset neuron action potential characteristics of the fast-spiking interneuron include at least one of the following B1 to B5:

[0090] B1. Under an injection current of 100-400 pA, the fast-firing interneurons exhibit clustered discharges (i.e., clustered discharge mode);

[0091] B2. At an injected current of >1000 pA, the action potential of the fast-firing interneuron continues to discharge, and the discharge frequency is >100 Hz;

[0092] B3. Under the same injection current, the firing frequency of the action potential of the fast-firing interneuron is higher than the firing frequency of the action potential of the pyramidal neuron;

[0093] B4. Under the same injection current, the half-peak width of a single action potential of the fast-spiking interneuron is smaller than the half-peak width of a single action potential of the pyramidal neuron;

[0094] B5. Under the same injection current, the after-hyperpolarization potential of a single action potential of the fast-spiking interneuron is greater than the after-hyperpolarization potential of a single action potential of the pyramidal neuron.

[0095] The electrophysiological characteristics of the FS interneurons and pyramidal neurons mentioned in the above embodiments are found and summarized by the inventors of the present application after multiple actual experiments and long-term research. Although previous studies have also analyzed and explained the electrophysiological characteristics of FS interneurons and pyramidal neurons, in actual experiments, the inventors of the present application found that according to the content recorded in the prior art, it is impossible to accurately judge whether the identified target neuron is a FS interneuron, or the electrophysiological characteristics of some FS interneurons and pyramidal neurons recorded in the prior art are slightly different from or significantly different from those obtained by the inventors of the present application through actual experimental records and analysis. Moreover, the human brain or primates and rodents have obvious differences in brain tissue size and total number of neurons, and the proportion of FS interneurons in different brain regions is also different, so the regulatory ability is also different, so it is normal to have certain differences. Based on the complexity of nervous system research, the same template cannot be simply applied to study all brain regions, and it is necessary to make adjustments in actual experiments.

[0096] In some embodiments, analyzing and comparing the preset electrophysiological information of the target neuron with the measured electrophysiological information, determining whether the suspected target neuron is the target neuron according to the comparison result, and identifying the target neuron from other neurons includes:

[0097] The preset neuron cell membrane electrophysiological characteristics are analyzed and compared with the measured neuron cell membrane electrophysiological characteristics, and / or the preset neuron action potential characteristics are analyzed and compared with the measured neuron action potential characteristics, and whether the suspected target neuron is the target neuron is determined based on the comparison results, and the target neuron is identified from other neurons.

[0098] In some embodiments, the method further comprises: taking all or part of the fresh brain and immersing it in oxygen-saturated and frozen slicing liquid to separate and obtain brain tissue. Wherein, illustratively, the slicing liquid can be selected from the following formula: 110mM sucrose, 60mM NaCl, 3mM KCl, 5mM glucose, 28mM NaHCO3, 1.25mM NaH2PO4, 0.5mM CaCl2, 7mM MgCl2; the osmotic pressure of the slicing liquid can be 310-320, but is not limited thereto.

[0099] In some embodiments, the method further comprises: preparing the brain block containing the hippocampus into an in vitro brain slice and then transferring it into an artificial cerebrospinal fluid with continuous oxygenation for incubation. The purpose of this operation is to wait for the neurons on the brain slice to recover their state function so as to facilitate the patch clamp experiment. Among them, exemplarily, the artificial cerebrospinal fluid can be selected from the following formula: 119mM NaCl, 2.5mM KCl, 1mM NaH2PO4, 26mM NaHCO3, 2.5mM CaCl2, 1.3mM MgCl2, 11mM Glucose; the osmotic pressure of the artificial cerebrospinal fluid can be 290-300, but is not limited thereto.

[0100] In some embodiments, the incubation time is 0.5 to 1.5 hours.

[0101] In some embodiments, the incubation temperature is 34-37°C.

[0102] In some embodiments, the brain tissue is derived from rodents, including but not limited to mice, rats, etc. The method provided by the present invention is currently only applicable to rodents because it has not been verified on primates or humans, so this judgment cannot be made, but it does not mean that it is not applicable to rodents. Further research based on the present invention may develop a method for identifying and electrophysiologically recording FS interneurons suitable for primates or humans, providing new technical means for related research.

[0103] Another embodiment of the present invention also provides an electrophysiological recording method of fast-spiking interneurons, comprising: using the identification method described in the above-mentioned embodiment and / or example to find the fast-spiking interneurons, and then performing electrophysiological recording and analysis on the fast-spiking interneurons.

[0104] Another embodiment of the present invention also provides a system for identifying fast-firing interneurons, including an isolated brain slice, a patch clamp electrophysiological system, and a neuron analysis module;

[0105] The in vitro brain slice is prepared from a brain block containing the target area, and the brain block containing the target area is obtained by trimming brain tissue;

[0106] The patch clamp electrophysiological system is used to observe and find target neurons from the isolated brain slice, and obtain cell morphological characteristics and position information;

[0107] The neuron information analysis and comparison module is used to observe and search for target neurons from the isolated brain slice, and obtain measured morphological information and measured position information of the neurons, and to perform electrophysiological recording on suspected target neurons and other neurons to obtain measured electrophysiological information;

[0108] The neuron analysis module is used to analyze and compare the preset morphological information and preset position information with the measured morphological information and measured position information, and identify suspected target neurons from the isolated brain slice according to the comparison results; and to analyze and compare the preset electrophysiological information of the target neuron with the measured electrophysiological information, determine whether the suspected target neuron is the target neuron according to the comparison results, and identify the target neuron from other neurons;

[0109] The target area is the hippocampus, and the target neurons are fast-spiking interneurons.

[0110] It should be noted that the identification system of the fast-spiking interneurons provided in the above embodiment and the identification method of the fast-spiking interneurons provided in the above embodiment belong to the same concept, wherein the specific manner in which each module and system performs operations has been described in detail in the method embodiment and will not be repeated here. In practical applications, the identification system of the fast-spiking interneurons provided in the above embodiment can distribute the above functions to different functional modules as needed, that is, divide the internal structure of the system into different functional modules to complete all or part of the functions described above, and this is not limited here.

[0111] Another embodiment of the present invention also provides a method for identifying fast-spiking interneurons as described in the above embodiments and / or examples and / or an electrophysiological recording method for fast-spiking interneurons as described in the above embodiments and / or examples and / or an application of a system for identifying fast-spiking interneurons as described in the above embodiments and / or examples in neuroscience research.

[0112] The following specific examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range according to the description herein, and are not limited to the specific values ​​​​exemplified below.

[0113] Example 1

[0114] In this example, rats were used as an example to prepare in vitro brain slices, observe and record using a patch clamp electrophysiological system, and analyze and compare the cell morphological characteristics, cell membrane properties, and electrophysiological properties to accurately identify FS PV in the hippocampal CA1 region. + Intermediate neuron. The specific implementation process is as follows:

[0115] 1. Preparation of ex vivo brain slices: Use isoflurane to deeply anesthetize the rat, then quickly decapitate and transfer the brain into oxygen-saturated frozen slice solution (the slice solution formula is: 110mM sucrose, 60mM NaCl, 3mM KCl, 5mM Glucose, 28mM NaHCO3, 1.25mM NaH2PO4, 0.5mM CaCl2, 7mM MgCl2, osmotic pressure is 310-320). Figure 1 As shown, the brain was gently removed from the slicing solution and placed on a cold platform. The brain tissue was carefully trimmed with a blade to ensure that the brain block contained the hippocampus ( Figure 1 The middle-colored rectangular block area is the brain block containing the hippocampus) is complete, and the specific steps are as follows:

[0116] (1) Place the removed brain tissue with the ventral side facing down on a low-temperature platform;

[0117] (2) Cut vertically to separate the cerebrum and cerebellum, retaining the brain tissue;

[0118] (3) Incline the knife vertically at a 30° angle to remove the olfactory bulbs and prefrontal lobe areas of the left and right brain;

[0119] (4) Cut vertically along the midline to separate the left and right brains and gently place them apart;

[0120] (5) Incline the left and right sides of the brain vertically at a 30° angle;

[0121] (6) The raphe part and the lateral cerebral cortex part are cut perpendicularly to the incisions in steps S3 and S5 to obtain a brain block containing the hippocampus, i.e. Figure 1 The part shown by the red rectangle.

[0122] Gently turn the brain block containing the hippocampus toward the midline and fix it on the sample tray of the vibration slicer. Use the vibration slicer to slice the trimmed brain block to prepare 300μM brain slices, and move into continuously oxygenated artificial cerebrospinal fluid (artificial cerebrospinal fluid formula: 119mM NaCl, 2.5mM KCl, 1mM NaH2PO4, 26mM NaHCO3, 2.5mM CaCl2, 1.3mM MgCl2, 11mM Glucose, osmotic pressure of 290-300), incubate at 35℃ for 30 minutes, and then transfer to room temperature for use.

[0123] 2. Operation of the patch clamp electrophysiology system: Turn on the patch clamp system equipment and use artificial cerebrospinal fluid with continuous oxygenation to circulate the recording tank to ensure that the environment of the brain slice is stable. Use a pipette to carefully draw the brain slice and lay it flat in the recording tank. Use a metal pressure net to gently press the brain slice to ensure that it is fixed in place. Adjust the microscope to ensure that the hippocampal CA1 area appears clearly in the field of view. Use infrared differential interference contrast (DIC-IR) to assist the microscope to start looking for cells to prepare for electrophysiological recording ( Figure 2 ).

[0124] 3. Cell morphology: After clearly observing the cell band in the CA1 region of the hippocampus, we searched along the cell band. FS interneurons are usually independently distributed on both sides of the pyramidal neuron band, which is in contrast to the large number of pyramidal neurons that are regularly arranged. FS interneurons have larger cell bodies and multipolar dendrites ( Figure 3 ), which is a prominent feature of FS interneurons, while pyramidal neurons have a cone-shaped cell body and a main dendrite emanating from the tip of the cone.

[0125] 4. Cell membrane properties: FS interneurons were recorded using an Axon Multiclamp 700B amplifier (Molecular Devices, Union City, CA, USA). After forming a high-resistance seal (resistance > 1 GΩ), the membrane was broken and the whole-cell recording mode was entered to observe the membrane properties of the recorded FS interneurons. The resting membrane potential was approximately -72 mV, the capacitance was approximately 120 to 180 pF, and the membrane resistance was approximately 60 to 90 MΩ.

[0126] 5. Neuronal action potential characteristics: After forming a whole-cell recording, select the current clamp mode for recording. Continuously inject current into the neuron to induce it to produce action potentials. Under low injection current (100-400pA), FS interneurons can have cluster discharges. Under high injection current (>1000pA), it can have continuous high-frequency (>100Hz) action potential discharges ( Figure 4). At the same injection current, the action potentials of FS interneurons have a higher firing frequency than those of pyramidal neurons. The single action potentials of FS interneurons have a smaller half-peak width and a larger after-hyperpolarization potential ( Figure 5 ).

[0127] In addition, it should be noted that the present invention found that pyramidal neurons did not have cluster discharges through in vitro electrophysiological experiments, but this does not mean that the pyramidal neuron electrophysiological signals recorded in in vivo electrophysiological recording experiments, or according to other recording methods, or using other recording systems, also do not have cluster discharges. In other words, in in vivo electrophysiological recording experiments, or using a different recording method from the present invention, or using a different recording system from the present invention, the recorded neuronal electrophysiological signals are likely to be different from those of the present invention, and there may even be significant substantial differences.

[0128] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for identifying fast-firing interneurons, characterized in that: include: Taking brain tissue, trimming it to obtain a brain block containing the target area, and preparing the brain block containing the target area into an in vitro brain slice; Based on the preset morphological information and preset position information of the target neuron, using a patch clamp electrophysiological system to observe and search for the target neuron in the isolated brain slice, and obtaining the measured morphological information and measured position information of the neuron; Analyzing and comparing the preset morphological information and the preset position information with the measured morphological information and the measured position information, and identifying suspected target neurons from the isolated brain slice according to the comparison result; Use the patch clamp electrophysiological system to perform electrophysiological recordings on suspected target neurons and other neurons to obtain measured electrophysiological information; Analyzing and comparing the preset electrophysiological information of the target neuron with the measured electrophysiological information, determining whether the suspected target neuron is the target neuron according to the comparison result, and identifying the target neuron from other neurons; The target area is the hippocampus, and the target neurons are fast-spiking interneurons.

2. The identification method according to claim 1, characterized in that: Based on the preset morphological information and preset position information of the target neuron, a patch clamp electrophysiological system is used to observe and search for the target neuron from the isolated brain slice, and the measured morphological information and measured position information of the neuron are obtained; the preset morphological information and preset position information are analyzed and compared with the measured morphological information and measured position information, and the suspected target neuron is identified from the isolated brain slice according to the comparison result, including: After finding the cell band in the hippocampal CA1 region, searching for the target neurons and pyramidal neurons along the cell band, and distinguishing and determining the suspected target neurons according to the preset morphological information and preset position information of the target neurons and pyramidal neurons; The preset morphological information of the target neuron and the pyramidal neuron includes the following preset morphological features: the cell body of the fast-spiking interneuron is larger than that of the pyramidal neuron and has multipolar dendrites; the cell body of the pyramidal neuron is mostly cone-shaped, and a main dendrite extends from the tip of the cone; The preset position information of the target neuron and the pyramidal neuron includes the following preset position features: the fast-firing interneurons are independently distributed on both side edges of the pyramidal neuron strip, and are in contrast to the large number of regularly arranged pyramidal neurons.

3. The identification method according to claim 1, characterized in that: The electrophysiological information includes electrophysiological characteristics of neuronal cell membranes and / or neuronal action potential characteristics.

4. The identification method according to claim 3, characterized in that: The method of using a patch clamp electrophysiological system to perform electrophysiological recording on suspected target neurons and other neurons to obtain the measured electrophysiological characteristics of neuronal cell membranes includes: sealing and breaking the membranes of suspected target neurons and other neurons, and observing and recording in a whole-cell recording mode to obtain the measured electrophysiological characteristics of neuronal cell membranes; And / or, the method of using a patch clamp electrophysiological system to perform electrophysiological recording on suspected target neurons and other neurons to obtain the measured neuronal action potential characteristics includes: recording in current clamp mode, injecting current into suspected target neurons and other pyramidal neurons to induce them to generate action potentials, and observing and recording to obtain the measured neuronal action potential characteristics.

5. The identification method according to claim 3 or 4, characterized in that: The electrophysiological properties of the neuron cell membrane include at least one of resting membrane potential, capacitance value and membrane resistance; And / or, the neuron action potential characteristics include at least one of a discharge pattern, a discharge frequency, a half-peak width, and a hyperpolarization afterpotential.

6. The identification method according to claim 5, characterized in that: The preset neuron cell membrane electrophysiological characteristics of the fast-spiking interneuron include at least one of the following A1 to A3: A1, resting membrane potential is -71 to -72 mV; A2, capacitance value is 120~180pF; A3, membrane resistance is 60~90MΩ; And / or, the preset neuron action potential characteristic of the fast-spiking interneuron includes at least one of the following items B1 to B5: B1. Under an injection current of 100-400 pA, the fast-firing interneurons exhibit clustered discharges; B2. At an injected current of >1000 pA, the action potential of the fast-firing interneuron continues to discharge, and the discharge frequency is >100 Hz; B3. Under the same injection current, the firing frequency of the action potential of the fast-firing interneuron is higher than the firing frequency of the action potential of the pyramidal neuron; B4. Under the same injection current, the half-peak width of a single action potential of the fast-spiking interneuron is smaller than the half-peak width of a single action potential of the pyramidal neuron; B5. Under the same injection current, the after-hyperpolarization potential of a single action potential of the fast-spiking interneuron is greater than the after-hyperpolarization potential of a single action potential of the pyramidal neuron.

7. The identification method according to claim 1, characterized in that: Analyzing and comparing the preset electrophysiological information of the target neuron with the measured electrophysiological information, determining whether the suspected target neuron is the target neuron according to the comparison result, and identifying the target neuron from other neurons, including: The preset neuron cell membrane electrophysiological characteristics are analyzed and compared with the measured neuron cell membrane electrophysiological characteristics, and / or the preset neuron action potential characteristics are analyzed and compared with the measured neuron action potential characteristics, and whether the suspected target neuron is the target neuron is determined based on the comparison results, and the target neuron is identified from other neurons.

8. A method for electrophysiological recording of fast-firing interneurons, characterized in that: include: The fast-spiking interneuron is found by using the identification method according to any one of claims 1 to 7, and then electrophysiological recording and analysis are performed on the fast-spiking interneuron.

9. A fast-firing interneuron recognition system, characterized in that: Includes isolated brain slices, patch clamp electrophysiology system and neuron analysis module; The in vitro brain slice is prepared from a brain block containing the target area, and the brain block containing the target area is obtained by trimming brain tissue; The patch clamp electrophysiological system is used to observe and find target neurons from the isolated brain slice, and obtain measured morphological information and measured position information of the neurons, and to perform electrophysiological recording on suspected target neurons and other neurons to obtain measured electrophysiological information; The neuron analysis module is used to analyze and compare the preset morphological information and preset position information with the measured morphological information and measured position information, and identify suspected target neurons from the isolated brain slice according to the comparison result; and, for analyzing and comparing the preset electrophysiological information of the target neuron with the measured electrophysiological information, determining whether the suspected target neuron is the target neuron according to the comparison result, and identifying the target neuron from other neurons; The target area is the hippocampus, and the target neurons are fast-spiking interneurons.

10. Application of the method for identifying fast-spiking interneurons as described in any one of claims 1 to 7 and / or the method for electrophysiological recording of fast-spiking interneurons as described in claim 8 and / or the system for identifying fast-spiking interneurons as described in claim 9 in neuroscience research, wherein the application is not for disease diagnosis or treatment purposes.

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