Neural circuit sensitive to object-position correlation recognition memory impairment caused by microwave radiation and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a neural circuit sensitive to object-position correlation recognition memory impairment caused by microwave radiation and application of the neural circuit. The invention provides application of a reagent or a pharmaceutical composition for activating a PRH-mPFC glutamic acid neural circuit in preparation of a medicine for treating or relieving object-position associated recognition memory ability impairment of an animal caused by microwave radiation. The invention finds that specific microwave radiation can cause the object-position associated recognition memory disorder, a PRH-mPFC glutamic acid neural circuit is related to the microwave radiation-caused object-position associated recognition memory ability damage, and the object-position associated recognition memory disorder caused by microwave radiation can be recovered by activating the PRH-mPFC glutamic acid neural circuit. Therefore, the reagent or the pharmaceutical composition for activating the PRH-mPFC glutamic acid neural circuit can be used for preparing the medicine for treating or relieving the object-position associated recognition memory ability damage of the animal caused by microwave radiation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a neural circuit sensitive to object-location association recognition memory impairment caused by microwave radiation and application thereof. BACKGROUND

[0002] Microwave (MW) is an electromagnetic wave with a frequency of 300 MHz-300 GHz and a wavelength range of 1 m-1 mm. Microwave can cause impairment of animal's ability to perform memory-related tasks, and is mainly concentrated in object recognition memory and spatial learning memory. Hippocampus is a key brain area for memory. Current research on the influence of microwave radiation on memory behavior is mainly focused on the hippocampus. In addition to the hippocampus, some studies have found that microwave radiation can cause histopathological damage to the cerebral cortex and cerebellum, and inhibit the transmission of glutamatergic synapses of pyramidal neurons in the fifth layer of the prefrontal cortex.

[0003] Object-location association memory is the ability to judge whether a specific stimulus has ever appeared at a specific location. As a complex memory task, its impairment can serve as a sensitive indicator of early memory deficits. Object-location association recognition memory can be divided into different sub-components, and object recognition memory and spatial location memory are the two main components of object-location association recognition memory, which play an important role in completing goal-oriented behaviors. Currently, there are few studies on the influence of microwave radiation on neural circuits, and the influence of microwave radiation on object-location association recognition memory has not been reported. Therefore, it is necessary to find a neural circuit sensitive to object-location association recognition memory impairment caused by microwave radiation. SUMMARY

[0004] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide the use of PRH-mPFC glutamate neural circuit in screening drugs for treating or alleviating microwave radiation-induced impairment of animal's object-location association recognition memory. The inventors have found a neural circuit sensitive to object-location association recognition memory impairment caused by microwave radiation, which is PRH-mPFC glutamate neural circuit. This circuit is related to microwave radiation-induced impairment of object-location association recognition memory, and activation of the circuit can restore microwave radiation-induced object-location association recognition memory impairment.

[0005] To this end, the first aspect of the present application provides the use of an agent or a pharmaceutical composition for activating PRH-mPFC glutamate neural circuit in the preparation of a drug for treating or alleviating microwave radiation-induced impairment of animal's object-location association recognition memory.

[0006] Both PRH-mPFC circuit and vHPC-mPFC circuit are involved in object-location associative recognition memory. It has been found that glutamatergic neurons in vHPC-mPFC circuit play an important role in object-location associative recognition memory, and injection of glutamate receptor antagonist into vHPC and mPFC can impair the memory encoding process of short-term and long-term object-location associative recognition memory without affecting memory retrieval.

[0007] The object of the present application is to find a neural circuit sensitive to object-location associative recognition memory impairment caused by microwave radiation. The present application finally finds that microwave radiation can cause object-location associative recognition memory impairment, PRH-mPFC glutamate neural circuit is related to object-location associative recognition memory impairment caused by microwave radiation, and activation of the circuit can restore the object-location associative recognition memory impairment caused by microwave radiation. Therefore, the agent or pharmaceutical composition for activating the PRH-mPFC glutamate neural circuit can be used for preparing a drug for treating or alleviating the object-location associative recognition memory impairment of animals caused by microwave radiation.

[0008] According to an embodiment of the present application, the conditions of the microwave radiation are:

[0009] The microwave carrier frequency is 9-10 GHz, the average power density is 20-40 mW / cm2, the exposure time is 15 minutes each time, and the total exposure time is 3 times, with an interval of 5 minutes each time.

[0010] The second aspect of the present application provides the use of PRH-mPFC glutamate neural circuit in screening a drug for treating or alleviating the object-location associative recognition memory impairment of animals caused by microwave radiation.

[0011] According to an embodiment of the present application, the conditions of the microwave radiation are:

[0012] The microwave carrier frequency is 9-10 GHz, the average power density is 20-40 mW / cm2, the exposure time is 15 minutes each time, and the total exposure time is 3 times, with an interval of 5 minutes each time.

[0013] The third aspect of the present application provides a method for screening a drug for treating or alleviating the object-location associative recognition memory impairment of animals caused by microwave radiation. According to an embodiment of the present application, the method comprises:

[0014] administering the drug to be screened to an animal model of object-location associative recognition memory impairment of animals caused by microwave radiation,

[0015] determining the activation degree of PRH-mPFC glutamate neural circuit of the animal model under the action of different drugs to be screened,

[0016] The drug to be screened is capable of activating the PRH-mPFC glutamate neural circuit of the animal model, which is an indication that the drug to be screened is a drug for treating or relieving the object-place association recognition memory impairment of animals caused by microwave radiation.

[0017] According to an embodiment of the present application, the conditions of the microwave radiation are:

[0018] The microwave carrier frequency is 9-10 GHz, the average power density is 20-40 mW / cm2, the exposure time is 15 minutes each time, and the total exposure time is 3 times, with an interval of 5 minutes each time.

[0019] The fourth aspect of the present application provides a method for preparing an animal model of object-place association recognition memory impairment caused by microwave radiation. According to an embodiment of the present application, the method comprises:

[0020] applying microwave radiation to the animal to obtain an animal model of object-place association recognition memory impairment,

[0021] wherein the conditions of the microwave radiation are:

[0022] The microwave carrier frequency is 9-10 GHz, the average power density is 20-40 mW / cm2, the exposure time is 15 minutes each time, and the total exposure time is 3 times, with an interval of 5 minutes each time.

[0023] The present application discovers a neural circuit sensitive to object-place association recognition memory impairment caused by microwave radiation, PRH-mPFC glutamate neural circuit, through object-place association recognition task, induction of neuronal apoptosis, and chemical genetics, etc. The circuit plays a key role in object-place association recognition memory impairment caused by microwave radiation. Studies have found that microwave radiation under certain conditions can cause object-place recognition memory impairment in mice, and this change is closely related to PRH (perirhinal cortex)-mPFC (medial prefrontal cortex (mPFC)). Activation of the PRH-mPFC glutamate neural circuit can alleviate object-place recognition memory impairment caused by microwave radiation. The present application provides a potential target for treatment after object-place association recognition memory impairment caused by microwave radiation.

[0024] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0025] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0026] Figure 1The dose measurement and temperature detection results of 9.375 GHz microwave radiation are shown, wherein A is a schematic diagram of the platform and the radiation source; B is the position of the mouse in the irradiation box; C is a schematic diagram of the mouse model and the microwave radiation dose distribution; D is the change of the anal temperature of the mouse in the R group during microwave exposure;

[0027] Figure 2 The effect of microwave radiation on the object-place association recognition memory of mice is shown, wherein A is a schematic diagram of the object-place association recognition memory task; B is a column chart of the total exploration time of the mice in the test group 2; C is the statistical analysis result of the discrimination index of the object-place association recognition memory task of the mice, and * indicates P<0.05;

[0028] Figure 3 The effect of microwave radiation on the object-place association recognition memory ability of PRH-mPFC apoptotic mice is shown, wherein A is a schematic diagram of apoptotic virus injection; B is the fluorescence protein expression imaging of the control virus and the apoptotic virus in the PRH brain area, and the scale bar is 200 microns; C is the statistical analysis of the discrimination index of the object-place association recognition memory task of the PRH-mPFC apoptotic mice;

[0029] Figure 4 The effect of microwave radiation on the object-place association recognition memory ability of vHPC-mPFC apoptotic mice is shown, wherein A is a schematic diagram of apoptotic virus injection; B is the fluorescence protein expression imaging of the control virus and the apoptotic virus in the PRH brain area, and the scale bar is 200 microns; C is the statistical analysis of the discrimination index of the object-place association recognition memory task of the vHPC-mPFC apoptotic mice, and * indicates P<0.05;

[0030] Figure 5 The effect of activating the PRH-mPFC glutamate loop on the object-place association recognition memory of mice after microwave radiation is shown, wherein A is a schematic diagram of chemical genetics virus injection; B is the statistical analysis of the discrimination index of the object-place association recognition memory task of the vHPC-mPFC chemical genetics activated mice, and * indicates P<0.05. DETAILED DESCRIPTION

[0031] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0032] It should be noted that the terms "first", "second" are only used for description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can be explicitly or implicitly included one or more features. Further, in the description of the present application, unless otherwise specified, the meaning of "multiple" is two or more.

[0033] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as exactly that endpoint. Any of the ranges or values should be interpreted as being approximate, as encompassing fractions thereof, and ranges ending with a letter should be interpreted to also include that last letter. The endpoints of the ranges and any values are provided as a separate endpoint with this understanding.

[0034] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless explicitly stated otherwise, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the present application belongs.

[0035] In the present document, the terms "comprising" or "comprise" are intended to indicate that the compositions or methods "comprised" can include more than the recited elements, but are not limited to the elements as they are recited.

[0036] In the present document, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0037] According to a specific embodiment of the present application, the present application provides use of an agent or a pharmaceutical composition for activating PRH-mPFC glutamate neural circuit in the preparation of a medicament for treating or alleviating impairment of object-location associative recognition memory ability in an animal caused by microwave radiation.

[0038] The inventors have found that the agent or pharmaceutical composition for activating PRH-mPFC glutamate neural circuit (e.g., hM3D(Gq) + CNO) provided by the present application can be used in the preparation of a medicament for treating or alleviating impairment of object-location associative recognition memory ability in an animal caused by microwave radiation.

[0039] According to a specific embodiment of the present application, the condition of microwave radiation is:

[0040] Microwave carrier frequency 9-10 GHz, average power density 20-40 milliwatts per square centimeter, exposure 15 minutes per time, a total of 3 times, each interval 5 minutes.

[0041] According to a preferred embodiment of the present application, the condition of microwave radiation is:

[0042] Microwave carrier frequency 9.375 GHz, average power density 30 milliwatts per square centimeter, exposure 15 minutes per time, a total of 3 times, each interval 5 minutes.

[0043] According to a specific embodiment of the present invention, the PRH-mPFC glutamate neural circuit can be used to screen drugs for treating or alleviating the damage of object-position association recognition memory ability of animals caused by microwave radiation.

[0044] The inventors discovered a neural circuit that is sensitive to damage to object-position association recognition memory caused by microwave radiation, namely the PRH-mPFC glutamate neural circuit. This circuit plays a key role in damage to object-position association memory caused by microwave radiation and can be used to screen drugs to treat or alleviate damage to object-position association recognition memory ability in animals caused by microwave radiation.

[0045] According to a specific embodiment of the present invention, the conditions of microwave radiation are:

[0046] The microwave carrier frequency was 9-10 GHz, the average power density was 20-40 mW / cm2, the exposure time was 15 minutes per time, and there were 3 exposures in total, with an interval of 5 minutes between each exposure.

[0047] According to a preferred embodiment of the present invention, the conditions of the microwave radiation are:

[0048] The microwave carrier frequency was 9.375 GHz, the average power density was 30 mW / cm2, and the exposure time was 15 minutes per exposure, for a total of 3 exposures, with an interval of 5 minutes between each exposure.

[0049] According to a specific embodiment of the present invention, the present invention provides a method for screening a drug for treating or alleviating microwave radiation-induced damage to an animal's object-position association recognition memory ability, comprising:

[0050] administering the drug to be screened to an animal model in which the object-position association recognition memory ability of the animal is impaired by microwave radiation,

[0051] Determine the activation degree of the PRH-mPFC glutamate neural circuit of the animal model under the action of different drugs to be screened,

[0052] The drug to be screened can activate the PRH-mPFC glutamate neural circuit in the animal model, which is an indication that the drug to be screened is a drug for treating or alleviating the damage to the object-position association recognition memory ability of animals caused by microwave radiation.

[0053] The inventors discovered that by administering the drugs to be screened to an animal model in which the animal's object-position association recognition memory ability is impaired due to microwave radiation, the inventors can determine which drugs to be screened can treat or alleviate the object-position association recognition memory ability impairment induced by microwave radiation in the animal model based on the degree of activation of the PRH-mPFC glutamate neural circuit of the animal model under the action of different drugs to be screened. The ability of the drugs to be screened to activate the PRH-mPFC glutamate neural circuit of the animal model is an indication that the drugs to be screened are drugs that can treat or alleviate the object-position association recognition memory ability impairment induced by microwave radiation in animals.

[0054] According to a specific embodiment of the present invention, the conditions of microwave radiation are:

[0055] The microwave carrier frequency was 9-10 GHz, the average power density was 20-40 mW / cm2, the exposure time was 15 minutes per time, and there were 3 exposures in total, with an interval of 5 minutes between each exposure.

[0056] According to a preferred embodiment of the present invention, the conditions of the microwave radiation are:

[0057] The microwave carrier frequency was 9.375 GHz, the average power density was 30 mW / cm2, and the exposure time was 15 minutes per exposure, for a total of 3 exposures, with an interval of 5 minutes between each exposure.

[0058] According to a specific embodiment of the present invention, the present invention provides a method for preparing an animal model of microwave radiation-induced object-position association recognition memory impairment in animals, comprising:

[0059] Applying microwave radiation to animals to obtain an animal model of object-position association recognition memory impairment.

[0060] Wherein, the microwave radiation conditions are:

[0061] The microwave carrier frequency was 9-10 GHz, the average power density was 20-40 mW / cm2, the exposure time was 15 minutes per time, and there were 3 exposures in total, with an interval of 5 minutes between each exposure.

[0062] According to a preferred embodiment of the present invention, the conditions of the microwave radiation are:

[0063] The microwave carrier frequency was 9.375 GHz, the average power density was 30 mW / cm2, and the exposure time was 15 minutes per exposure, for a total of 3 exposures, with an interval of 5 minutes between each exposure.

[0064] According to a specific embodiment of the present invention, the animal can be a mouse, a dog, a rabbit or other mammals.

[0065] The scheme of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the examples below are only for illustration of the present disclosure and should not be considered as limiting the scope of the present disclosure. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained on the market.

[0066] Example 1: Microwave exposure of mice

[0067] 8-9-week-old C57BL / 6N male mice were raised and randomly divided into C and R groups. After the X-band high-power emission source was turned on, it was preheated for 30 minutes, the microwave carrier frequency was set to 9.375 GHz, and the average power density was 30 milliwatts per square centimeter. The R group was irradiated for 15 minutes per time, a total of 3 times, with an interval of 5 minutes each time, and the temperature rise was 0.9 degrees Celsius. The C group of mice was treated the same way, but without microwave exposure. The dose monitoring and temperature measurement results are shown in Figure 1 . Figure 1 The results show that the temperature rise of the mice after microwave exposure did not exceed 1 degree Celsius, indicating that the effect of microwave exposure on the mice under this condition is mainly non-thermal effect, and the thermal effect on the mice can be ignored.

[0068] Example 2: 9.375 GHz / 30 milliwatts per square centimeter exposure for 15 minutes 3 times, with an interval of 5 minutes each time, can cause object-location recognition memory impairment in mice

[0069] The object-location association recognition memory task adaptation stage lasted for 3 days, and the mice were placed in a 50x50x40 cm white open field with spatial cues, allowing the mice to freely explore for 10 minutes. The learning stage was separated from the test stage by 24 hours. During the learning stage, 4 different 3x4x4 cm blocks were fixed in the open field, and each mouse explored the open field for 10 minutes. Microwave exposure was performed 6 hours before the test stage, and during the test stage, two blocks from the learning stage were changed in position, and the remaining conditions were exactly the same as in the learning stage. Each mouse was allowed to freely explore the open field for 5 minutes, and the time and number of times each mouse explored each object were recorded (e.g. Figure 2 The object-location association recognition memory task discrimination index = (new position object exploration time - old position object exploration time) / total object exploration time x 100%.

[0070] Figure 2 The results in Figs. B and C show that the discrimination index of mice for new objects decreases after specific microwave radiation.

[0071] Example 3: 9.375 GHz / 30 milliwatts per square centimeter exposure for 15 minutes 3 times, with an interval of 5 minutes each time, causes object-location association recognition memory impairment closely related to PRH-mPFC

[0072] AAV-Retro-CaMKIIa-Cre (provided by Bicase (Wuhan) Biotechnology Co., Ltd.) was injected into the mPFC by brain stereotactic injection, and rAAV-EFla-DIO-taCasp3-EGFP (provided by Bicase (Wuhan) Biotechnology Co., Ltd.) or rAAV-EFla-DIO-taCasp3-T2A-TEVp-EGFP (provided by Bicase (Wuhan) Biotechnology Co., Ltd.) was injected into the PRH or vHPC. The coordinates of the mPFC brain region were AP: 1.7 mm, ML: ±0.3 mm, DV: -2.5 mm, the coordinates of the PRH were AP: -1.7 mm, ML: ±4.0 mm, DV: -4.0 mm, and the coordinates of the vHPC were AP: -3.16 mm, ML: ±3.0 mm, DV: -4.3 mm according to the mouse brain stereotactic atlas. After the completion of virus injection, the mouse was sutured with the scalp, and microwave irradiation and object-place recognition memory task were performed after 3 weeks of virus expression.

[0073] Figure 3 The effect of microwave irradiation on the object-place recognition memory ability of PRH-mPFC apoptotic mice was demonstrated, A is the schematic diagram of apoptotic virus injection, which shows the specific position of virus injection; B is the fluorescence protein expression imaging of control virus (rAAV-EFla-DIO-taCasp3-EGFP) and apoptotic virus (rAAV-EFla-DIO-taCasp3-T2A-TEVp-EGFP) in the PRH brain region; C is the discrimination index statistical analysis of PRH-mPFC apoptotic mice in the object-place recognition memory task, and the results show that:

[0074] In PRH-mPFC apoptotic mice, green fluorescent protein expression of control virus and apoptotic virus can be seen in the PRH brain region, and there is no significant difference in the discrimination index of PRH-mPFC apoptotic mice after microwave irradiation Figure 3 C).

[0075] Figure 4 The effect of microwave irradiation on the object-place recognition memory ability of vHPC-mPFC apoptotic mice was demonstrated, wherein A is the schematic diagram of apoptotic virus injection, which shows the specific position of virus injection; B is the fluorescence protein expression imaging of control virus and apoptotic virus in the PRH brain region; C is the discrimination index statistical analysis of vHPC-mPFC apoptotic mice in the object-place recognition memory task, and the results show that:

[0076] In vHPC-mPFC apoptotic mice, green fluorescent protein expression of control virus and apoptotic virus can be seen in the vHPC brain region, as shown in Figure 4 B, the discrimination index of vHPC-mPFC apoptotic mice significantly decreased after microwave irradiation (P<0.05)Figure 4 C).

[0077] Example 4: Activation of PRH-mPFC glutamatergic circuit alleviates object-location recognition memory impairment induced by microwave radiation

[0078] AAV-Retro-CaMKIIa-Cre was injected into mPFC and rAAV-CaMKIIa-DIO-hM3D(Gq)-EGFP (hM3D(Gq) protein is a human-made receptor from human muscarinic acetylcholine receptor M3, which only responds to CNO (clozapine-n-oxide), and after CNO injection, hM3D(Gq) enhances neuronal activity) was injected into PRH. The coordinates of mPFC brain region were determined according to the mouse brain stereotaxic atlas as AP: 1.7 mm, ML: ± 0.3 mm, DV: -2.5 mm, and the coordinates of PRH were AP: -1.7 mm, ML: ± 4.0 mm, DV: -4.0 mm. After the completion of virus injection, the mice were sutured on the scalp, and after three weeks of virus expression, microwave radiation and object-location association memory task were performed.

[0079] After microwave radiation, the chemical activation of virus mice activated the PRH-mPFC glutamatergic circuit, and the discrimination index of new objects in the object-location association recognition memory task increased (P < 0.05), as shown in Figure 5 .

[0080] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", "some implementations" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0081] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. Use of an agent or a pharmaceutical composition activating PRH-mPFC glutamate neural circuit in the preparation of a medicament for treating or alleviating impairment of object-place associative recognition memory ability in animals caused by microwave radiation.

2. Use according to claim 1, characterized in that, The conditions of the microwave radiation are: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, with an interval of 5 minutes each time.

3. Use of PRH-mPFC glutamate neural circuit in the screening of a medicament for treating or alleviating impairment of object-place associative recognition memory ability in animals caused by microwave radiation.

4. Use according to claim 3, characterized in that, The conditions of the microwave radiation are: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, with an interval of 5 minutes each time.

5. A method for screening a drug for treating or alleviating impairment of object-place associative recognition memory in an animal caused by microwave irradiation, characterized by, Comprising: administering a drug to be screened to an animal model of impairment of object-place associative recognition memory ability in animals caused by microwave radiation, determining the degree of activation of the PRH-mPFC glutamate neural circuit of the animal model under the action of different drugs to be screened, The drug to be screened can activate the PRH-mPFC glutamate neural circuit of the animal model, indicating that the drug to be screened is a medicament for treating or alleviating impairment of object-place associative recognition memory ability in animals caused by microwave radiation.

6. The method of claim 5, wherein, The conditions of the microwave radiation are: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, with an interval of 5 minutes each time.

7. A method for preparing an animal model of impairment of object-place associative recognition memory ability caused by microwave irradiation, characterized by, Comprising: applying microwave radiation to animals in order to obtain an animal model of impairment of object-place associative recognition memory ability, wherein the conditions of the microwave radiation are: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, with an interval of 5 minutes each time.

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