An experimental method for synchronously detecting animal pain behavior and neuronal activity

Abstract

The present invention relates to the fields of biology and medicine, and in particular to an experimental method for synchronously detecting the correlation between animal pain behavior and neuronal activity, characterized in that it comprises at least a stimulation unit (1), a processing unit (2) and an imaging unit (3), wherein the stimulation unit (1) and the imaging unit (3) are electrically connected to the processing unit (2) respectively, the stimulation unit (1) is used to perform stimulation intensity or depth operation on an experimental subject (4), and is used to provide a stimulation intensity or depth signal to a processor (2); the imaging unit (3) is used to obtain and record the impact information when the stimulation intensity or depth is performed on the experimental subject (4); the processor (2) is used to synchronously record the imaging unit (3) and the stimulation unit (1), and is used to locally amplify and extract the synchronous record so that the details of the changes in the stimulation of the experimental subject (4) correspond to the stimulation intensity or depth signal; and an experimental method for quantitatively detecting the correlation between mechanical pain response and neuronal activity in a specific brain region is provided, which is simple to operate, low in cost and has good repeatability.

Description

Technical Field

[0001] The invention relates to the fields of biology and medicine, and in particular to an experimental method for synchronously detecting the correlation between animal pain behavior and neuron activity. Background Art

[0002] Experiments to detect the correlation between animal pain behavior and neuronal activity are very important in the field of neuroscience and medicine. Existing methods use calcium imaging technology to combine animal in vitro and in vivo experiments to study whether the specific behavior of animals is related to the activity of neurons in a specific brain area.

[0003] By using in vivo calcium imaging technology to observe the relationship between the withdrawal reflex, licking and shaking of the feet caused by Von Frey filament stimulation and the functional activities of neurons in specific brain regions, the recording system was used to synchronously record the calcium signal activity of neurons in a specific brain region when the mice showed pain behaviors. However, how to establish the relationship between pain response and neuronal functional activities, synchronously record the calcium ion signals of group neurons / specific neurons and animal pain behavior, and form a quantitative relationship of pain, is of great significance for the exploration of the functional activities of specific neurons / group neurons in specific brain regions involved in mechanical pain response. Summary of the invention

[0004] The purpose of the present invention is to provide an experimental method for quantitatively correlating mechanical pain response with neuronal activity in specific brain regions, which is simple to operate, low in cost and has good repeatability.

[0005] The object of the present invention is achieved by providing an experimental method for synchronously detecting animal pain behavior and neuronal activity, characterized in that it at least comprises: a stimulation unit (1), a processing unit (2) and an imaging unit (3), wherein the stimulation unit (1) and the imaging unit (3) are electrically connected to the processing unit (2) respectively, the stimulation unit (1) is used to perform stimulation intensity or depth operation on an experimental subject (4); and is used to provide a stimulation intensity or depth signal to the processor (2); the imaging unit (3) is used to obtain and record the impact information when the stimulation intensity or depth is performed on the experimental subject (4); the processor (2) is used to synchronously record the imaging unit (3) and the stimulation unit (1), and is used to locally amplify and extract the synchronous record so that the details of the changes in the stimulation of the experimental subject (4) are consistent with the stimulation. The stimulation unit (1) at least comprises a stimulation needle (101), a conductor filament (102) wound on the stimulation needle rod, a clamping sleeve (103) for clamping the stimulation needle rod, and a handle (104) connected to the clamping sleeve for clamping the stimulation needle rod; the conductor filament (102) is wound along the length of the stimulation needle rod, and both ends of the conductor filament (102) are electrically connected to the bridge interface of the processing unit (2); the conductor filament (102) wound on the stimulation needle rod will deform when the stimulation needle (101) performs a stimulation intensity or depth operation on the experimental subject (4); the processing unit (2) detects the output of the bridge circuit (105) through the bridge interface, and converts the deformation of the stimulation needle (101) into a signal that changes with time and is provided to the processing unit (2).

[0006] The imaging unit (3) uses calcium imaging to observe the corresponding relationship between the stimulation unit (1) and the different depths and intensities of the stimulation of the experimental subject (4), thereby combining the stimulation electrical information with the neuronal activity.

[0007] The imaging unit (3) adopts a combination of calcium imaging and optical imaging. The corresponding relationship between the stimulation unit (1) and the different depths and intensities of the stimulation of the experimental subject (4) is observed through calcium imaging, and the stimulation electrical information is combined with the neuronal activity; the facial expressions of the stimulation unit (1) and the different depths and intensities of the stimulation of the experimental subject (4) are obtained through optical imaging, and the stimulation electrical information is combined with the neuronal activity and the facial expressions.

[0008] The processing unit (2) or the fixation and imaging unit (3) or the fixation and stimulation unit (1) may exist independently.

[0009] The processing unit (2) outputs the image information that changes with time, formed by the dynamic change details of the experimental subject (4), and the signal that the deformation size of the stimulation needle (101) changes with time to the display unit (6) at the same time.

[0010] The stimulation unit (1) further comprises a button group (109) for sending a command to the processing unit (2), with the time of sending the command being used as a file for recording the above information.

[0011] The processing unit (2) further comprises: outputting the recorded information in fragments.

[0012] A flexible material, such as silicone rubber, is provided between the clamping sleeve (103) and the clamping stimulation needle rod, so that a flexible connection is formed between the clamping sleeve (103) and the clamping stimulation needle rod.

[0013] The stimulation needle (101), the conductor filament (102) wound on the stimulation needle rod, and the clamping sleeve (103) clamping the stimulation needle rod are an integrated structure, and the conductor filament (102) and the clamping sleeve (103) form two electrical connection output points.

[0014] The diameter of the stimulation needle (101) and the diameter of the wound conductor filament (102) match each other, and the diameter of the stimulation needle (101) and the diameter of the wound conductor filament (102) form different resistance strain range outputs, so as to form experimental effects of different intensities and depths.

[0015] Advantages of the present invention: The present invention combines stimulation of the experimental subject with calcium imaging, and obtains calcium imaging information and stimulation information through the stimulation process of the experimental subject at different intensities and depths. It can deeply understand and study the exploration process of the functional activities of specific neurons / groups of neurons, and provide a scientific basis for understanding and studying the quantitative correlation between mechanical pain response and neuronal activity in specific brain regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below in conjunction with embodiments and accompanying drawings:

[0017] Figure 1 This is a schematic diagram of the principle of Embodiment 1 of the present invention;

[0018] Figure 2 This is a schematic diagram of the principle of Embodiment 2 of the present invention;

[0019] Figure 3 is a schematic diagram of the structure of a stimulation unit according to an embodiment of the present invention;

[0020] Figure 4 It is a schematic diagram of a stimulation unit processing circuit according to an embodiment of the present invention;

[0021] Figure 5 This is a second schematic diagram of the stimulation unit processing circuit according to an embodiment of the present invention.

[0022] In the figure, 1. stimulation unit; 2. processing unit; 3. imaging unit; 4. experimental subject; 5. calcium imaging; 6. display unit; 7. optical imaging; 101. stimulation needle; 102. conductor filament; 103. clamping sleeve; 104. handle; 105. bridge circuit; 106. signal amplifier; 107. signal processing circuit; 108. interface circuit; 109. button group. DETAILED DESCRIPTION

[0023] Example 1

[0024] like Figure 1 , Figure 2 , Figure 4 As shown, an experimental method for synchronously detecting the correlation between pain behavior and neuronal electrical activity is characterized in that it at least includes: a stimulation unit 1, a processing unit 2 and an imaging unit 3, the stimulation unit 1 and the imaging unit 3 are electrically connected to the processing unit 2 respectively, the stimulation unit 1 is used to perform stimulation intensity or depth operation on the experimental subject 4; it is used to provide stimulation intensity or depth signals to the processing unit 2; the imaging unit 3 is used to obtain and record the impact information when the stimulation intensity or depth is performed on the experimental subject 4; the processing unit 2 is used to synchronously record the imaging unit 3 and the stimulation unit 1, and is used to locally amplify and extract the synchronous record, so that the details of the changes in the stimulation of the experimental subject 4 correspond to the stimulation intensity or depth signals. The stimulation unit 1 includes at least one stimulation needle 101, a conductor filament 102 wound on the stimulation needle rod, a clamping sleeve 103 for clamping the stimulation needle rod, a handle 104 connected to the clamping sleeve for clamping the stimulation needle rod, a bridge circuit 105, a signal amplifier 106, a signal processing circuit 107 and an interface circuit 108; the conductor filament 102 is wound along the length of the stimulation needle rod, and both ends of the conductor filament 102 are electrically connected to the bridge circuit 105 interface of the signal amplifier 106. The conductor filament 102 wound on the stimulation needle rod will deform when the stimulation needle 101 performs stimulation intensity or depth operation on the experimental subject 4. The signal processing circuit 107 obtains the output signal of the signal amplifier 106, converts it into a digital signal, and the interface circuit 108 converts the deformation of the stimulation needle 101 into a signal that changes with time and provides it to the processing unit 2.

[0025] The signal amplifier 106 is a measuring amplifier, the signal processing circuit 107 is a processor with an A / D interface, and the interface circuit 108 is a Bluetooth or wireless transmission circuit.

[0026] The imaging unit 3 uses calcium imaging 5 to observe the corresponding relationship between the stimulation unit 1 and the different depths and intensities of the stimulation of the experimental subject 4, and to combine the stimulation electrical information with the neuronal activity.

[0027] The processing unit 2 outputs the image information formed by the dynamic change details of the experimental subject 4 and the signal of the deformation size of the stimulation needle 101 changing with time to the display unit 6 at the same time.

[0028] The diameter of the stimulation needle 101 and the diameter of the wound conductor filament 102 match each other, and the diameter of the stimulation needle 101 and the diameter of the wound conductor filament 102 form different resistance strain range outputs to form experimental effects of different intensities and depths.

[0029] There is a flexible material, such as silicone rubber, between the clamping sleeve 103 and the clamping stimulation needle rod, so that a flexible connection is formed between the clamping sleeve 103 and the clamping stimulation needle rod.

[0030] Example 2

[0031] like Figure 1 , Figure 3 , Figure 4 As shown, an experimental method for synchronously detecting the correlation between pain behavior and neuronal electrical activity is characterized in that it at least includes: a stimulation unit 1, a processing unit 2 and an imaging unit 3, the stimulation unit 1 and the imaging unit 3 are electrically connected to the processing unit 2 respectively, the stimulation unit 1 is used to perform stimulation intensity or depth operation on the experimental subject 4; it is used to provide stimulation intensity or depth signals to the processing unit 2; the imaging unit 3 is used to obtain and record the impact information when the stimulation intensity or depth is performed on the experimental subject 4; the processing unit 2 is used to synchronously record the imaging unit 3 and the stimulation unit 1, and is used to locally amplify and extract the synchronous record, so that the details of the changes in the stimulation of the experimental subject 4 correspond to the stimulation intensity or depth signals. The stimulation unit 1 includes at least one stimulation needle 101, a conductor filament 102 wound on the stimulation needle rod, a clamping sleeve 103 for clamping the stimulation needle rod, a handle 104 connected to the clamping sleeve for clamping the stimulation needle rod, a bridge circuit 105, a signal amplifier 106, a signal processing circuit 107 and an interface circuit 108; the conductor filament 102 is wound along the length of the stimulation needle rod, and both ends of the conductor filament 102 are electrically connected to the bridge circuit 105 interface of the signal amplifier 106. The conductor filament 102 wound on the stimulation needle rod will deform when the stimulation needle 101 performs stimulation intensity or depth operation on the experimental subject 4. The signal processing circuit 107 obtains the output signal of the signal amplifier 106, converts it into a digital signal, and the interface circuit 108 converts the deformation of the stimulation needle 101 into a signal that changes with time and provides it to the processing unit 2.

[0032] The signal amplifier 106 is a measuring amplifier, the signal processing circuit 107 is a processor with an A / D interface, and the interface circuit 108 is a Bluetooth or wireless transmission circuit.

[0033] The imaging unit 3 adopts a combination of calcium imaging 5 and optical imaging 7. The calcium imaging 5 is used to observe the corresponding relationship between the stimulation unit 1 and the different depths and intensities of the stimulation of the experimental subject 4, and the stimulation electrical information is combined with the neuronal activity; the optical imaging 7 is used to obtain the facial expressions of the stimulation unit 1 and the different depths and intensities of the stimulation of the experimental subject 4, and the stimulation electrical information is combined with the neuronal activity and facial expressions.

[0034] The processing unit 2 outputs the image information formed by the dynamic change details of the experimental subject 4 and the signal of the deformation size of the stimulation needle 101 changing with time to the display unit 6 at the same time.

[0035] The diameter of the stimulation needle 101 and the diameter of the wound conductor filament 102 match each other, and the diameter of the stimulation needle 101 and the diameter of the wound conductor filament 102 form different resistance strain range outputs to form experimental effects of different intensities and depths.

[0036] There is a flexible material, such as silicone rubber, between the clamping sleeve 103 and the clamping stimulation needle rod, so that a flexible connection is formed between the clamping sleeve 103 and the clamping stimulation needle rod.

[0037] Example 3

[0038] like Figure 5 As shown, the stimulation unit 1 also includes a button group 109, so that the image information formed by the dynamic change details of the experimental subject 4 and the signal of the deformation size of the stimulation needle 101 changing with time can be combined and output at any time, or an independent time file can be formed and stored at any time.

[0039] The calcium imaging technology in the present invention refers to a method of monitoring calcium ion concentration in tissues using calcium ion indicators. In the study of the nervous system, in vivo or in vitro studies, calcium imaging technology is widely used to simultaneously monitor changes in calcium ions in hundreds or thousands of neurons, thereby detecting the activity of neurons. With calcium imaging technology, the originally silent neural activity has become a colorful and spectacular image, and scientists can finally watch with their own eyes the neural signals shuttling back and forth in the neural network. Therefore, as soon as this technology appeared, it was sought after by neuroscientists all over the world, and it is still the most direct means for people to observe neural activity.

[0040] In biological organisms, calcium ions generate a variety of intracellular signals, which exist in almost every type of cell and play an important role in many functions, such as the control of cardiac muscle cell contraction and the regulation of the entire cell cycle from cell proliferation to cell death. In the mammalian nervous system, calcium ions are an important type of neuronal intracellular signaling molecules. In the resting state, the intracellular calcium ion concentration of most neurons is 50-100nM, while when neurons are active, the intracellular calcium ion concentration can rise 10-100 times. The increased calcium ions are essential for the exocytosis and release of synaptic vesicles containing neurotransmitters. In other words, the activity of neurons is closely related to the calcium ion concentration inside them. When neurons discharge, a short peak of calcium ion concentration will burst out. The principle of neuronal calcium imaging technology is to use the strict correspondence between calcium ion concentration and neuronal activity, and use special fluorescent dyes or protein fluorescent probes (calcium ion indicators, calcium indicator) to express the calcium ion concentration in neurons through fluorescence intensity, so as to achieve the purpose of monitoring neuronal activity.

[0041] There are two types of calcium ion indicators that are widely used now: chemical indicators and genetically-encoded indicators:

[0042] 1. Chemical calcium ion indicators: refers to small molecules that can chelate calcium ions. All of these small molecules are based on BAPTA (aminophenylethane tetraacetic acid), a homologue of EGTA (ethylene glycol ditetraacetic acid). BAPTA can specifically chelate calcium ions, but not magnesium ions, so it is widely used as a calcium ion chelator. The most widely used chemical calcium ion indicators are: Oregon Green-1, Fura-2, Indo-1, Fluo-3, Fluo-4.

[0043] 2. Genetically encoded calcium indicators: These indicators are fluorescent proteins derived from green fluorescent protein (GFP) and its variants (such as circularly permuted GFP, YFP, CFP), fused with calmodulin (CaM) and myosin light chain kinase M13 domain. The most widely used genetically encoded calcium indicators are: GCaMP, Pericams, Cameleons, TN-XXL and Twitch. Among them, GCaMP6 is now widely used in in vivo calcium imaging studies due to its super sensitivity.

Claims

1. An experimental method for synchronously detecting animal pain behavior and neuronal activity, comprising at least: A stimulation unit (1), a processing unit (2) and an imaging unit (3), wherein the stimulation unit (1) and the imaging unit (3) are electrically connected to the processing unit (2) respectively. The stimulation unit (1) is used to perform stimulation intensity or depth operation on an experimental subject (4); and is used to provide a stimulation intensity or depth signal to the processor (2). The imaging unit (3) is used to obtain and record the impact information when the stimulation intensity or depth is performed on the experimental subject (4). The processor (2) is used to synchronously record the imaging unit (3) and the stimulation unit (1), and is used to locally amplify and extract the synchronous record so that the details of the changes in the stimulation of the experimental subject (4) correspond to the stimulation intensity or depth signal. The stimulation unit (1) comprises at least one stimulation needle (101), a conductor filament (102) wound on the stimulation needle rod, a clamping sleeve (103) for clamping the stimulation needle rod, and a handle (104) connected to the clamping sleeve for clamping the stimulation needle rod. The conductor filament (102) is wound along the length of the stimulation needle rod, and both ends of the conductor filament (102) are electrically connected to the bridge interface of the processing unit (2). The conductive filament (102) wound on the stimulating needle rod will deform when the stimulating needle (101) performs a stimulation intensity or depth operation on the experimental subject (4), and the processing unit (2) detects the output of the bridge circuit (105) through the bridge interface, and converts the deformation of the stimulating needle (101) into a signal that changes with time and is provided to the processing unit (2), wherein: a flexible material silicone rubber is provided between the clamping sleeve (103) and the clamping stimulating needle rod, so that a flexible connection is formed between the clamping sleeve (103) and the clamping stimulating needle rod The stimulation needle (101), the conductor filament (102) wound on the stimulation needle rod, and the clamping sleeve (103) for clamping the stimulation needle rod are an integrated structure, and the conductor filament (102) and the clamping sleeve (103) form two electrical connection output points; the diameter of the stimulation needle (101) and the diameter of the wound conductor filament (102) match each other, and the diameter of the stimulation needle (101) and the diameter of the wound conductor filament (102) form different resistance strain range outputs, so as to form experimental effects of different intensities or depths.

2. The experimental method for synchronously detecting animal pain behavior and neuronal activity according to claim 1, characterized in that: The imaging unit (3) uses calcium imaging to observe the corresponding relationship between the stimulation unit (1) and the different depths and intensities of the stimulation of the experimental subject (4), thereby combining the stimulation electrical information with the neuronal activity.

3. The experimental method for synchronously detecting animal pain behavior and neuronal activity according to claim 1, characterized in that: The imaging unit (3) adopts calcium imaging and (3) to observe the corresponding relationship between the stimulation unit (1) and the different depths and intensities of the stimulation of the experimental subject (4) through calcium imaging, and combine the stimulation electrical information with the neuronal activity; and obtains the facial expressions of the stimulation unit (1) and the different depths and intensities of the stimulation of the experimental subject (4) through optical imaging, and combines the stimulation electrical information with the neuronal activity and the facial expressions.

4. The experimental method for synchronously detecting animal pain behavior and neuronal activity according to claim 1, characterized in that: The processing unit (2) or the fixation and imaging unit (3) or the fixation and stimulation unit (1) may exist independently.

5. The experimental method for synchronously detecting animal pain behavior and neuronal activity according to claim 1, characterized in that: The processing unit (2) outputs the image information that changes with time, formed by the dynamic change details of the experimental subject (4), and the signal that the deformation size of the stimulation needle (101) changes with time to the display unit (6) at the same time.

6. The experimental method for synchronously detecting animal pain behavior and neuronal activity according to claim 1, characterized in that: The stimulation unit (1) further comprises a button group (109) for sending a command to the processing unit (2), with the time of sending the command being used as a file for recording the above information.

7. The experimental method for synchronously detecting animal pain behavior and neuronal activity according to claim 1, characterized in that: The processing unit (2) further comprises: outputting the recorded information in fragments.

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