Systems, products, and applications for treating depression

Abstract

The present application relates to the technical field of depression treatment, and in particular to a system, product, and application for treating depression. The system for treating depression comprises: an expression vector implantation unit: used to implant a vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S into a subject for expression; an acoustic genetics regulation unit: used to perform ultrasonic stimulation on the subject implanted with the above-mentioned vector. The present application utilizes a mutant of the large-conductance mechanosensitive ion channel MscL-G22S as an acoustic genetics tool. By expressing it in a subject, the response of serotonergic neurons to ultrasound can be enhanced, and acoustic genetics regulation has a good antidepressant effect on the subject, thereby achieving the effect of treating depression.

Description

Technical Field

[0001] The present application belongs to the technical field of depression treatment, and in particular relates to a system, product, and application for treating depression. Background Art

[0002] Depression is a common mental disorder characterized by a significant and persistent low mood, accompanied by a loss of interest and pleasure in previously rewarding activities, and persistent hopelessness. It often interferes with work, study, and social functioning and is the second-leading contributor to the burden of chronic disease. Depression can be caused by a variety of factors, including genetic, biological, psychosocial, and environmental factors.

[0003] Clinically, many types of antidepressants are used to treat depression, including selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), monoamine oxidase inhibitors (MAOIs), and tricyclic antidepressants (TCAs). These drugs primarily relieve depressive symptoms by increasing the concentration of neurotransmitters, particularly serotonin (5-HT), norepinephrine, and dopamine, in the central nervous system (CNS). However, a review shows that the efficacy of these antidepressants is approximately 50%, and their therapeutic effects generally require more than several weeks.

[0004] Alternative non-drug neuromodulatory therapies are also used clinically to treat depression, including electroconvulsive therapy (ECT), vagus nerve stimulation (VNS) (only for adults), and transcranial magnetic stimulation (TMS). They provide valuable contributions to the treatment of depression, especially for patients who do not respond to antidepressants. However, they also have their own disadvantages, including the potential risk of cognitive impairment and even long-term memory loss, surgical invasiveness, and a long duration of antidepressant effect (usually more than 4 weeks) or even no response. Summary of the Invention

[0005] The purpose of this application is to provide a system, product, and application for treating depression, aiming to solve the technical problem of how to better treat depression.

[0006] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0007] In a first aspect, the present application provides a system for treating depression, comprising:

[0008] Expression vector implantation unit: used to implant a vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S into a subject for expression;

[0009] Acoustogenetic regulation unit: used for performing ultrasound stimulation on the subject implanted with the vector.

[0010] In some embodiments, the vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S comprises a viral vector having a Tph2 promoter.

[0011] In some embodiments, the vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S is implanted into the dorsal raphe nucleus of the subject for expression.

[0012] In some embodiments, the ultrasound stimulation includes: center frequency, 0.5-1.0 MHz; intensity, 0.2-0.3 MPa; pulse width, 450-550 μs; pulse repetition frequency, 0.5-1.5 kHz; stimulation duration, 250-350 ms; and stimulation interval, 2-4 s.

[0013] In some embodiments, the system further includes: a testing unit configured to test the effect of the ultrasonic stimulation.

[0014] In a second aspect, the present application provides a product for treating depression, comprising:

[0015] A vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S;

[0016] Ultrasonic collimator.

[0017] In some embodiments, the vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S comprises a viral vector having a Tph2 promoter.

[0018] In some embodiments, the ultrasonic collimator operating parameters include: center frequency, 0.5-1.0 MHz; intensity, 0.2-0.3 MPa; pulse width, 450-550 μs; pulse repetition frequency, 0.5-1.5 kHz; stimulation duration, 250-350 ms; and stimulation interval, 2-4 s.

[0019] In some embodiments, further comprising: optical fibers and data analysis software.

[0020] In a third aspect, the present application provides an application, namely, the application of ultrasound and a vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S in the preparation of a product for treating depression.

[0021] The first aspect of the present application provides a system for treating depression, comprising: (1) an expression vector implantation unit: a vector expressing the large-conductance mechanosensitive ion channel MscL-G22S can be implanted into a subject for expression; and (2) an acoustic genetics regulation unit: a subject implanted with the above-mentioned vector in the expression vector implantation unit can be subjected to ultrasonic stimulation. The present application can utilize the mutant MscL-G22S of the large-conductance mechanosensitive ion channel as an acoustic genetics tool, and its expression in a subject can enhance the response of serotonergic neurons to ultrasound, promote the secretion of serotonin through acoustic genetics regulation, produce a rapid and powerful antidepressant effect, and can quickly reverse despair-like behavior. Therefore, the system of the present application can effectively achieve the effect of treating depression.

[0022] The second aspect of this application provides a product for treating depression, comprising: (1) a vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S; and (2) an ultrasonic collimator. This application can utilize this vector to express in subjects with depression, thereby enhancing the response of serotonergic neurons to ultrasound. The ultrasonic collimator can then be used to stimulate ultrasound to promote serotonin secretion, thereby effectively treating depression.

[0023] The third aspect of this application provides an application for non-invasively activating specific neuronal populations by heterogeneously overexpressing the ultrasound-sensitive, high-conductance mechanosensitive ion channel MscL-G22S, thereby sensitizing neurons to ultrasound. Therefore, a product for treating depression can be prepared using ultrasound and a vector expressing the high-conductance mechanosensitive ion channel MscL-G22S. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 This is a result diagram of increasing neuronal activity and serotonin release in DRN using acoustic genetics regulation in an embodiment of the present application; wherein,

[0026] a is a schematic step of a method for virus injection and optical fiber implantation determination, the specific process is to co-inject and deliver the genetically encoded sensor AAV-hSyn-jRGECO1a and AAV-TPH2-MscL-EGFP (i.e., MscL group) or AAV-TPH2-EGFP (i.e., Ctrl group) into the DRN, and then implant the optical fiber into the DRN. Four weeks later, the change in red fluorescence intensity during ultrasound stimulation is measured in vivo; the ultrasound stimulation parameters are: center frequency = 1 MHz, intensity = 0.11 MPa or 0.25 MPa, pulse width = 500 μs, pulse interval = 1 ms, stimulation duration = 300 ms, and stimulation interval = 3 s. b is a representative image of DRN overexpressing jRGECO1a and MscL-EGFP (MscL group) or EGFP (Ctrl group) at low magnification (scale bar = 100 μm) and high magnification (scale bar = 50 μm). Green, red, and blue fluorescence indicate neurons expressing MscL or EGFP, jRGECO1a signals, and DAPI signals, respectively. The dotted rectangle indicates the position of the optical fiber. c and d are graphs showing increased neuronal activity in the DRN due to ultrasound stimulation, with blue and red representing the Ctrl group (n=6 mice) and the MscL group (n=4 mice), respectively. c is the jRGECO1a fluorescence trace of MscL- and EGFP-expressing mice when receiving multiple pulses of 0.25 MPa ultrasound stimulation (left panel of c) and the average jRGECO1a fluorescence signal from the left panel (right panel of c). d is the average peak change (peak ΔF / F) of jRGECO1a fluorescence in response to 0 MPa, 0.11 MPa, and 0.25 MPa ultrasound stimulation. Data are shown as mean ± sem, ***p < 0.0001.

[0027] e is a schematic step of another method of virus injection and optical fiber implantation measurement, the specific process is to co-inject the genetically encoded sensors AAV-hSyn-5-HT2.1 and AAV-TPH2-MscL-mCherry (i.e., MscL group) or AAV-TPH2-mCherry (i.e., Ctrl group) into the DRN, and then implant the optical fiber into the DRN. Four weeks later, the change in green fluorescence intensity during ultrasound stimulation is measured in vivo; the ultrasound stimulation parameters are: center frequency = 0.5 MHz, intensity = 0.11 or 0.25 MPa, pulse width = 500 μs, pulse interval = 1 ms, stimulation duration = 300 ms, and stimulation interval = 3 s. f is a representative image of DRN overexpressing 5-HT and MscL (MscL group) or mCherry (Ctrl group) at low magnification (scale bar = 100 μm) and high magnification (scale bar = 50 μm). Green, red, and blue fluorescence indicate 5-HT signals, neurons expressing MscL or mCherry, and DAPI signals, respectively. The dotted rectangle indicates the position of the optical fiber. g and h are graphs of serotonin release in DRN induced by ultrasound stimulation, with blue and red representing the Ctrl group (n=4 mice) and the MscL group (n=5 mice), respectively. g is the 5-HT fluorescence trace of MscL and mCherry-expressing mice when receiving multiple pulses of 0.25 MPa ultrasound stimulation (left panel of g) and the average 5-HT fluorescence signal from the left panel (right panel of g). h is the average peak change (peak ΔF / F) of 5-HT fluorescence in response to 0 MPa, 0.11 MPa, and 0.25 MPa ultrasound stimulation. Data are shown as mean ± sem, ***p < 0.0001.

[0028] Figure 2 For the present application, the embodiment utilizes acoustic genetics to regulate the selective activation of 5-HT DRN The results of neurons and rapid reversal of despair-like behavior;

[0029] a is a schematic diagram of the steps of viral injection and ultrasound stimulation. The specific process is to deliver the genetically encoded sensor AAV-TPH2-MscL-EGFP or AAV-TPH2-EGFP into the DRN. After the mice recovered, they received CRS treatment for 14 consecutive days. Then, an ultrasound collimator was installed above the DRN. One week later, a custom wearable transducer was installed in the ultrasound collimator, and behavioral tests related to depression were performed during ultrasound treatment. Ultrasound treatment was performed from 15 minutes before the test to the end of the test 6 minutes later (a total of 21 minutes); the ultrasound stimulation parameters were: center frequency = 0.8 MHz, intensity = 0.3 MPa, pulse width = 500 μs, pulse interval = 1 ms, stimulation duration = 300 ms, and stimulation interval = 3 s; b is a DAPI-MscL fluorescence image in the DRN (scale bar = 100 μm);

[0030] cg is 5-HT DRN Sonogenetic modulation of neurons reversed the despair-like behavioral effect data, and c is the immobility time of the TST test, n=6-7 mice (group), d is the struggling time of the TST test, n=6-7 mice (group), e is the immobility time of the FST test, n=6-8 mice (each group), f is the struggling time of the FST test, n=6-8 mice (each group), g is the velocity of the OFT test, n=6-8 mice (each group);

[0031] hj is 5-HT DRN Sonogenetic modulation of neurons improves neuronal activity. Data are shown in Figure 3. h is a representative image of c-Fos expression in the DRN at low magnification (scale bar = 100 μm) and high magnification (scale bar = 50 μm). Green, red, and blue fluorescence indicate neurons expressing MscL or EGFP, c-Fos-positive signals, and DAPI signals, respectively. i is the count of c-Fos-positive cells per slice imaged in the DRN. j is the ratio of c-Fos- and EGFP-positive cells to c-Fos-positive cells in the DRN. Data are shown as mean ± sem. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.

[0032] The MscL+US group indicated the group injected with AAV-TPH2-MscL-EGFP and underwent ultrasound stimulation, the MscL-US group indicated the group injected with AAV-TPH2-MscL-EGFP and without ultrasound stimulation, the Ctrl+US group indicated the group injected with AAV-TPH2-EGFP and underwent ultrasound stimulation, and the Ctrl-US group indicated the group injected with AAV-TPH2-EGFP and without ultrasound stimulation.

[0033] Figure 3 This is a diagram showing the results of the in vivo biosafety assessment of the embodiments of the present application; wherein,

[0034] a is a representative image of Iba1 in DRN (scale bar = 100 μm); b is a representative image of GFAP in DRN (scale bar = 100 μm); c is the count of Iba1-positive cells per section imaged in DRN, n = 4-5 mice (per group), d is the count of GFAP-positive cells per section imaged in DRN, n = 3-4 mice (per group), e is a low-magnification (scale bar = 100 μm) and high-magnification (scale bar = 20 μm) hematoxylin and eosin (H&E) staining in DRN, n = 1 mouse Mice (each group); data are shown as mean ± sem, and were statistically insignificant by two-way ANOVA and post hoc Tukey test; and the MscL+US group represents the group injected with AAV-TPH2-MscL-EGFP and subjected to ultrasound stimulation, the MscL-US group represents the group injected with AAV-TPH2-MscL-EGFP and without ultrasound stimulation, the Ctrl+US group represents the group injected with AAV-TPH2-EGFP and subjected to ultrasound stimulation, and the Ctrl-US group represents the group injected with AAV-TPH2-EGFP and without ultrasound stimulation. DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0036] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0037] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0038] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0039] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0040] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0041] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be understood to indicate or imply relative importance or to implicitly specify the quantity of the technical features being referred to. For example, without departing from the scope of the embodiments of this application, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0042] The efficacy of antidepressants on the market is low, and the currently commonly used non-drug neuromodulation therapies have the potential risk of cognitive impairment and even long-term memory loss. In addition, they are surgically invasive and may trigger antidepressant effects for a long time or even without response. Therefore, it is necessary to develop new neuromodulation methods with predictable and controllable therapeutic effects, tolerability and rapid effects.

[0043] Optogenetics, a neuromodulatory method, can be used to manipulate the activity of specific cell types through heterogeneous overexpression of light-sensitive proteins combined with precise spatiotemporal resolution to make neurons sensitive to light responses. This provides a high-fidelity neuromodulatory tool for dissecting abnormalities in neural circuits underlying diseases such as depression. In optogenetics, due to the opacity of brain tissue, optical fibers need to be invasively implanted into the target brain region to deliver light. Ultrasound neuromodulation, as an emerging neuromodulatory method, has attracted widespread attention due to its advantages such as non-invasiveness, deep penetration depth, and high spatiotemporal resolution. Acoustogenetics, the counterpart of optogenetics, uses low-intensity ultrasound to non-invasively activate specific neuronal populations by heterogeneously overexpressing ultrasound-sensitive ion channels, thereby making neurons sensitive to ultrasound.

[0044] In the examples of the present application, sonogenetic regulation is used to treat depression by enhancing specific depression-related neuronal populations. The subject in the examples of the present application refers to a patient with depression, which can be a human or an animal, such as a rodent or a large animal.

[0045] Specifically, the present invention uses a mutant of a large conductance mechanosensitive channel (MscL): MscL-G22S as an acoustic genetic tool to target serotoninergic neurons in the dorsal raphe nucleus (DRN), namely 5-HT DRN Neurons and enhance their response to ultrasound. The dorsal raphe nucleus is the main source of serotonergic neurons. This application found that sonogenetic modulation of serotonergic neurons in the dorsal raphe nucleus enhanced neuronal activity and serotonin release in the dorsal raphe nucleus. Therefore, the examples of this application combine genetic modification of MscL-G22S with non-invasive ultrasound stimulation to achieve enhancement of specific neuronal populations.

[0046] Importantly, the examples of the present application used acoustic genetics to induce a rapid antidepressant effect in mice, which was reflected in the tail suspension test and the forced swim test (a widely used measurement of despair-like behavioral states in rodents). They spent less time immobile and more time struggling. In addition, there was no abnormal activation of microglia and astrocytes or signs of tissue damage. Based on this, the examples of the present application used MscL-G22S as an acoustic genetics tool for the first time, attempting to treat depression by acoustic genetics regulation of specific neuronal populations in rodents, providing a new method for studying depression, including systems, products, and applications for treating depression. The specific scheme is as follows.

[0047] A first aspect of an embodiment of the present application provides a system for treating depression, comprising:

[0048] (1) Expression vector implantation unit: used to implant a vector capable of expressing the large conductance mechanosensitive ion channel MscL-G22S into a subject for expression. The vector can express the MscL-G22S modified gene in the subject.

[0049] (2) Acoustogenetic regulation unit: used to perform ultrasonic stimulation on the subject implanted with the above-mentioned vector.

[0050] In the embodiment of the present application, ultrasound stimulation can be performed on a subject in which the above-mentioned vector is implanted in an expression vector implantation unit, and the large-conductance mechanosensitive ion channel mutant MscL-G22S is used as an acoustic genetics tool. Its expression in the subject can enhance the response of serotonergic neurons to ultrasound, and has a good antidepressant effect through acoustic genetics regulation, thereby achieving the effect of treating depression.

[0051] In some embodiments, the expression vector used in the implantable unit to express the large-conductance mechanosensitive ion channel MscL-G22S includes a viral vector with a Tph2 promoter. This allows for better expression in specific neuronal types. For example, it can be AAV-TPH2-MscL-EGFP or AAV-TPH2-MscL-mCherry.

[0052] In some embodiments, a vector expressing the large-conductance mechanosensitive ion channel MscL-G22S in an expression vector implant unit is implanted and expressed in serotonergic neurons in the dorsal raphe nucleus of a subject. The dorsal raphe nucleus is the primary source of serotonergic neurons, and sonogenetic modulation of serotonergic neurons in the dorsal raphe nucleus enhances neuronal activity and serotonin release in the dorsal raphe nucleus.

[0053] In some embodiments, the ultrasound stimulation used by the acoustic genetic modulation unit includes: a center frequency of 0.5-1.0 MHz; an intensity of 0.2-0.3 MPa; a pulse width of 450-550 μs; a pulse repetition frequency of 0.5-1.5 kHz; a stimulation duration of 250-350 ms; and an interstimulus interval of 2-4 seconds. These conditions can effectively modulate the response of serotonergic neurons to ultrasound.

[0054] Ultrasound is safe, has high spatiotemporal resolution, and can be delivered noninvasively to brain regions of interest, even deep within the brain. Acoustogenetic modulation can target specific neuronal populations, making therapeutic effects more controllable and predictable.

[0055] In some embodiments, the system for treating depression further includes a testing unit for testing the effectiveness of ultrasound stimulation. For example, the testing unit may include fiber optic photometry recording, chronic restraint stress, tail suspension test, forced swim test, open field test, staining, and statistical analysis tools.

[0056] The second aspect of the embodiments of the present application provides a product for treating depression, including: (1) a vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S, which is used for implantation into a subject with depression, thereby enhancing the response of serotonergic neurons to ultrasound; and (2) an ultrasonic collimator, which can generate ultrasonic stimulation, thereby effectively achieving non-invasive treatment of depression in the subject.

[0057] The embodiments of the present application can utilize the above-mentioned vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S with an ultrasonic collimator to treat depression in a controllable, predictable, tolerable and fast-acting non-invasive manner.

[0058] In some embodiments, the vector capable of expressing the large conductance mechanosensitive ion channel MscL-G22S comprises a viral vector having a Tph2 promoter.

[0059] In some embodiments, the ultrasonic collimator operating parameters include: center frequency, 0.5-1.0 MHz; intensity, 0.2-0.3 MPa; pulse width, 450-550 μs; pulse repetition frequency, 0.5-1.5 kHz; stimulation duration, 250-350 ms; and stimulation interval, 2-4 s.

[0060] In some embodiments, the aforementioned product for treating depression further comprises: optical fibers and data analysis software. Optical fibers refer to artificial fibers used to conduct light, also known as optical fibers, and can transmit data. The data analysis software can analyze the experimental results of the subjects after the ultrasonic collimator is activated.

[0061] The third aspect of the embodiments of the present application provides an application, namely, the use of ultrasound and a vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S in the preparation of a product for treating depression.

[0062] In this example, serotonergic neurons are made sensitive to ultrasound by heterogeneously overexpressing the large-conductance mechanosensitive ion channel MscL-G22S in a neuronal population. Therefore, a product for treating depression can be prepared using ultrasound and a vector expressing the large-conductance mechanosensitive ion channel MscL-G22S.

[0063] Specifically, the present examples evaluate depression-related behaviors during sonogenetic modulation of serotonergic neurons in the dorsal raphe nucleus, using sonogenetic modulation to treat depression. The sonogenetic tool, MscL-G22S, can sensitize neurons to ultrasound. Therefore, genetic modification of MscL-G22S is combined with non-invasive ultrasound stimulation to enhance the serotonergic neuron population.

[0064] The following describes the details in conjunction with specific embodiments.

[0065] Example

[0066] In this application, all experiments were performed on male C57BL / 6J mice. These mice were housed in the Centralized Animal Facility (CAF) of the Hong Kong Polytechnic University. All animal experiments were approved by the Animal Ethics Committee (ASESC) of the Hong Kong Polytechnic University and performed in accordance with the guidelines of the Department of Health Animals (Experimental Control) of the Hong Kong SAR Government.

[0067] Stereotaxic surgery: Mice were anesthetized with a cocktail of ketamine and xylazine (100 mg / kg for ketamine and 10 mg / kg for xylazine) and then fixed in a stereotaxic apparatus (RWD Life Science Co., Ltd.). The scalp of the mouse was removed with a sterile surgical blade to expose the bregma point and lambda point. After cleaning and flattening the skull, the virus was injected, and the optical fiber was implanted or the ultrasound collimator was installed. For details, please refer to the subsequent experimental section. After all stereotactic surgeries, the mice were immediately placed on a heating pad until they woke up. 7 days after the surgery, the mice were subjected to fiber photometry or behavioral testing.

[0068] Virus Injection: After the mouse skull was cleaned and flattened on a stereotaxic apparatus, a small hole was made in the skull above the target brain area using a microdrill (RWD Life Science Co., Ltd). To target the serotonergic neurons of the dorsal raphe nucleus, an adeno-associated virus carrying MscL-G22S and under the control of the Tph2 promoter (e.g., AAV-TPH2-MscL-EGFP or AAV-TPH2-MscL-mCherry) was injected into the dorsal raphe nucleus at a rate of 0.1 μL / min via a glass pipette (1 μL; AP, -4.4 mm; ML, 0 mm; DV, -3.4 mm). The glass pipette was left in place for an additional 5 minutes for infusion before removal. To detect calcium activity or serotonin release in the dorsal raphe nucleus, for example, AAV-hSyn-jRGECO1a (a genetically encoded calcium sensor with red fluorescence) or AAV-hSyn-5-HT2.1 (a genetically encoded GPCR activation-based 5-HT sensor with green fluorescence) is injected into the dorsal raphe nucleus.

[0069] Optical fiber implantation: The mouse's head was fixed in a stereotaxic apparatus. A portion of the scalp was removed, and a small hole was created in the skull above the dorsal raphe nucleus using a microdrill. A black optical fiber cannula (model, FOC-B-1.25-200-0.37-3.5; Inper Co., Ltd., Zhejiang, China) was then implanted into the dorsal raphe nucleus (AP, -4.4 mm from the anterior body; ML, 0 mm; DV, -3.35 mm) and secured with dental cement.

[0070] Ultrasonic collimator installation: After being fixed to a stereotaxic apparatus, a portion of the mouse scalp above the dorsal raphe nucleus was removed. A custom-made wearable ultrasound collimator was placed on the intact skull aligned with the target brain region and fixed with dental cement.

[0071] Ultrasound system and stimulation: The output of the function generator (AFG251, Tektronix Inc., Beaverton, USA) was connected to the input of a power amplifier (A075, Electronics & Innovation Ltd., Rochester, USA) via a BNC cable. The output of the amplifier was connected to a custom-made wearable transducer. The custom-made wearable transducer was then mounted in a collimator coupled to ultrasound gel. The two-dimensional sound field distribution was measured using a hydrophone. Exemplarily, the parameters of ultrasound stimulation in some tests can be: center frequency, 0.8 MHz; intensity, 0.15 or 0.3 MPa; pulse width, 500 μs; pulse repetition frequency, 1 kHz; stimulation duration, 300 ms; and stimulation interval, 3 s.

[0072] Fiber photometry recording: Mice were anesthetized with 1–2% isoflurane. Eye ointment was applied to prevent corneal drying. A fiber optic patch cable was used to connect the implanted optical fiber to the recording system (Thinker TechNanjing Biotech Co., Ltd, Nanjing, China). The excitation wavelengths for the red calcium-sensitive protein jRGECO1a and serotonin 5-HT were 570 nm and 480 nm, respectively. Data were collected at 100 Hz and analyzed using a custom MATLAB script. The change in fluorescence (ΔF / F) was calculated as (F − F0) / F0, where F0 is the baseline fluorescence signal.

[0073] Chronic restraint stress (CRS): Mice were individually placed in a well-ventilated 50 ml conical tube for approximately 3 hours per day for 14 consecutive days, while control mice remained in their cages. After each day of CRS, the restrained mice were removed from the tube and immediately returned to their cages.

[0074] Tail suspension test (TST): Mice were suspended 25 cm above a horizontal floor for 6 minutes by tape-taping their tails. The test was videotaped and analyzed by an observer blinded to the animal's group assignment. "Immobility time" was defined as the time during which the body and limbs remained motionless. "Struggle time" was defined as the time during which vigorous body movements and struggles were observed.

[0075] Forced swim test (FST): Mice were placed in a plexiglass cylinder (12 cm in diameter, 25 cm in height) containing water (24±1°C) at a depth of 10 cm and allowed to swim freely for 6 minutes. After each test, the cylinder was cleaned and refilled with fresh water. Video recording and analysis were performed by an observer who was unaware of the animal grouping. "Immobility time" was defined as the time during which the mouse floated without any movement within the last 4 minutes. "Struggle time" refers to the time during which the forelimbs were observed to flap rapidly on the water surface, indicating a strong desire to survive, within the last 4 minutes.

[0076] Open field test (OFT): Mice were individually placed in the center of a cubic plastic chamber (40 cm × 40 cm × 40 cm) and allowed to move freely for 6 minutes. The mouse's behavior was videotaped, and the mouse's movement speed over the 6-minute period was measured using ToxTrac software.

[0077] Immunofluorescence staining:

[0078] Mice were anesthetized with a cocktail of ketamine and xylazine. After anesthesia, mice were perfused with phosphate-buffered saline (PBS) (Cat. no., 70011044; Thermo Fisher Scientific Inc., Waltham, USA) and then perfused with 4% paraformaldehyde solution (PFA solution) (Cat. No.: sc-281692; Santa Cruz Biotechnology Inc., California, USA). The brain was extracted and fixed overnight in a 4°C refrigerator with 4% paraformaldehyde solution. Coronal sections with a thickness of 40 μm were obtained from the fixed brain using a vibratome (VT1200S; Leica Biosystems GmbH, Wetzlar, Germany). The sections were blocked with blocking solution (10% normal goat serum + 1% BSA + 0.3% Triton in 1X PBS) at room temperature for 90 minutes and incubated with the prepared primary antibody solution against c-Fos (1:500; Cat. no., 2250; CST, Massachusetts, USA), Iba-1 (1:1000; Cat. no., 17198, CST, Massachusetts, USA), or GFAP (1:1000, Cat. no., 12389; CST, Massachusetts, USA) in a 4°C refrigerator overnight.

[0079] The sections were then washed with 1X PBS (5 minutes, 3 times). After washing, the sections were incubated with secondary antibodies against rabbit Alexa Fluor 488, 555, or 633 (1:1000; Thermo Fisher Scientific Inc., Waltham, USA) at room temperature for 90 minutes. The sections were then washed in PBS and mounted on slides with mounting medium having DAPI (Cat. no., ab104139; Abcam, Cambridge, UK). The staining was visualized by confocal microscopy (TCS SP8 MP; Leica Microsystems GmbH, Wetzlar, Germany) and analyzed using ImageJ software by an observer who was blinded to the animal treatment.

[0080] Hematoxylin and eosin staining (H&E): Mice were anesthetized with a cocktail of ketamine and xylazine. After anesthesia, the mice were perfused with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde solution (PFA solution). The brain tissue was extracted and fixed with 4% paraformaldehyde solution in a 4°C refrigerator for 24 hours, and then washed with phosphate-buffered saline. Next experiment: The brain tissue was dehydrated with higher concentrations of alcohol (70%, 80%, 90%, 95%, 100%), cleared with xylene, and embedded in paraffin. 5 μm thick coronal brain sections were obtained from the paraffin-embedded blocks using a microtome (Leica RM2235) and stained with a hematoxylin-eosin staining kit (Solarbio G1121). After staining, the sections were dehydrated again with graded alcohol, cleared again with xylene, and fixed with resin for microscopic observation.

[0081] Data analysis: Data were analyzed and figures were generated using GraphPad Prism 7.0. Schematic diagrams were created using CorelDRAW X8. Data were analyzed using two-tailed unpaired t-tests or two-way analysis of variance (ANOVA) with post hoc Tukey's test. All data are presented as the mean ± standard error of the mean (SEM). P values ​​< 0.05 were considered statistically significant.

[0082] Verification Experiment 1

[0083] 5-HT DRN Sonogenetic modulation of neurons increases neuronal activity and serotonin release in the DRN:

[0084] This example investigated whether sonogenetic modulation could directly manipulate neuronal activity in the DRN, a brain region deeper than the mPFC. The red calcium-sensing protein jRGECO1a and adeno-associated viruses (AAVs) expressing either MscL-EGFP or EGFP were co-injected into the DRN. An optical fiber was then implanted into the DRN. Four weeks later, changes in red fluorescence intensity were measured in vivo during sonication.

[0085] The experimental results are as follows Figure 1 As shown: Before ultrasound stimulation, both the MscL and Ctrl groups showed comparable baseline fluorescence levels (peak ΔF / F, MscL vs. EGFP: 0.093±0.022% vs. 0.075±0.015%, p=0.5142, see Figure 1d); When stimulated by 0.11 MPa ultrasound, the jRGECO1a fluorescence intensity of the MscL group and the Ctrl group did not change significantly (peak ΔF / F, MscL vs. EGFP: 0.115±0.019% vs. 0.082±0.012%, p=0.1577, see Figure 1 d); however, repeated ultrasound stimulation at 0.25 MPa induced a synchronous increase in the jRGECO1a fluorescence intensity in the MscL group, but not in the Ctrl group (see Figure 1 (c) When stimulated by 0.25 MPa ultrasound, the peak ΔF / F of mice expressing MscL was significantly higher than that of mice expressing EGFP alone (peak ΔF / F, MscL vs. EGFP: 0.365±0.029% vs. 0.072±0.020%, p=2.4×10 -5 ,See Figure 1 Together, these results demonstrate that sonogenetic stimulation can increase calcium activity in DRNs in real time.

[0086] Based on the well-known biological phenomenon that increased neuronal activity leads to enhanced neurotransmitter release, the present application further observed the release of serotonin in DRN due to ultrasound stimulation. The present application co-injected a 5-HT sensor and AAVs for expressing MscL or mCherry into the DRN, then implanted an optical fiber into the DRN, and then measured the change in green fluorescence intensity during ultrasound treatment in vivo. There was no significant difference in baseline fluorescence between the MscL group and the Ctrl group before ultrasound stimulation (peak ΔF / F, MscL vs. mCherry: 0.054±0.020% vs. 0.048±0.009%, p=0.7947, see Figure 1 In addition, when stimulated by 0.11 MPa ultrasound, there was no significant change in the 5-HT fluorescence intensity between the MscL group and the Ctrl group (peak ΔF / F, MscL vs. mCherry: 0.114±0.022% vs. 0.063±0.015, p=0.1085, see Figure 1 h); however, 0.25 MPa ultrasound stimulation synchronously induced an increase in 5-HT fluorescence intensity in the MscL group, but not in the Ctrl group (see Figure 1 g). When ultrasound stimulation was applied at 0.25 MPa, the 5-HT fluorescence intensity in the MscL group was significantly increased (peak ΔF / F, MscL vs. mCherry: 0.482±0.028% vs. 0.078±0.026%, p=1.7×10 -5 ,See Figure 1 These results indicate that 5-HT DRN Sonogenetic modulation of neurons increased neuronal activity and serotonin release in the DRN in a real-time manner.

[0087] Verification Experiment 2

[0088] 5-HT DRN Sonogenetic modulation of neurons selectively activates 5-HT DRN Neurons and Rapid Reversal of Despair-Like Behaviors:

[0089] Based on 5-HT DRN The acoustic genetic modulation of neurons increases neuronal activity and enhances serotonin release in the DRN in a real-time manner. The present invention further investigates whether their acoustic genetic modulation can rapidly improve depression-related behaviors. DRN In this example, AAV carrying either MscL-EGFP or EGFP under the control of the Tph2 promoter was infused into the DRN. After recovery, the mice were treated with CRS for 14 consecutive days. An ultrasound collimator was then installed above the DRN. One week later, a custom wearable transducer was installed into the ultrasound collimator, and depression-related behaviors were observed during ultrasound treatment.

[0090] The experimental results are as follows Figure 2 As shown: When comparing the MscL-US and Ctrl-US groups, no significant behavioral changes in the TST and FST were observed, indicating that the acoustic genetic tool MscL did not affect the baseline of the despair-like state in the application setting. However, when ultrasound stimulation was applied, the immobility time in the TST of mice expressing MscL was significantly reduced compared with the control mice expressing EGFP (MscL+US group vs. Ctrl+US group: 53.29±13.74 seconds vs. 134.60±14.51 seconds, p=0.0009, see Figure 2 c), the struggling time was longer (MscL+US group vs.Ctrl+US group: 141.40±12.11 seconds vs. 58.14±10.82 seconds, p=2.1×10 -5 ,See Figure 2 Mice in the MscL+US group also showed shorter immobility time in the FST than mice in the Ctrl+US group (MscL+US vs. Ctrl+US: 58.50±10.28 sec vs. 121.40±18.02 sec, p=0.0373, Figure 2 e) and more struggling time (MscL+US group vs.Ctrl+US group: 129.10±15.17 seconds vs. 76.00±13.23 seconds, p=0.0367, see Figure 2 f in ). However, there was no significant difference in OFT movement speed between these groups (see Figure 2 These observations suggest that 5-HT DRNSonogenetic modulation of neurons can rapidly reverse despair-like behavior in stressed mice.

[0091] To further confirm that 5-HT DRN To investigate the selective specificity of sonogenetic regulation of neurons, this study examined whether the activated neurons were MscL-expressing neurons. Figure 2 The results showed that acoustic genetics significantly increased the expression of c-Fos protein in DRN (MscL+US group vs. Ctrl+US group, MscL-US group, Ctrl-US group = 81.50±5.92 vs. 32.33±5.40, 28.20±5.21, 22.60±4.76, p = 1.6×10 -5 , 1.0×10 -5 and 2.6×10 -6 Importantly, the colocalization between c-Fos positive and green EGFP fluorescence signals in the MscL+US group (67.78±3.60%) was significantly higher than that in the Ctrl+US group (21.39±3.74%, p=2.1×10 -8 ), MscL-US group (17.60±2.85%, p=1.3×10 -8 ). This indicates that the neurons activated by sonogenetic modulation are mainly neurons expressing MscL.

[0092] Verification Experiment 3

[0093] Biosafety Assessment Following DRN Sonogenetic Modulation:

[0094] Ionized calcium binding adaptor 1 (Iba1) and glial fibrillary acidic protein (GFAP) are typical biomarkers of activated microglia and astrocytes, respectively, and their activation has been identified as an early indicator of adverse tissue reactions. Therefore, immunofluorescence staining and hematoxylin and eosin (H&E) staining were performed in the present examples.

[0095] The experimental results are as follows Figure 3 As shown: No difference in the expression of Iba1 or GFAP was observed among the MscL+US, Ctrl+US, MscL-US and Ctrl-US groups (see Figure 3 Furthermore, no neuronal loss or changes in tissue responses were detected in the DRN following sonogenetic modulation (see Figure 3 Thus, these results suggest that sonogenetic modulation can selectively activate 5-HT DRN neurons and rapidly reversed despair-like behaviors with no signs of tissue damage.

[0096] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A system for treating depression, characterized in that include: An expression vector implantation unit: used to implant a vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S into a subject for expression, wherein the vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S comprises a viral vector having a Tph2 promoter and is implanted into the dorsal raphe nucleus of the subject for expression; Acoustogenetic regulation unit: used to perform ultrasonic stimulation on a subject implanted with the carrier, wherein the ultrasonic stimulation includes: center frequency, 0.5-1.0 MHz; intensity, 0.2-0.3 MPa; pulse width, 450-550 μs; pulse repetition frequency, 0.5-1.5 kHz; stimulation duration, 250-350 ms; and stimulation interval, 2-4 s.

2. The system for treating depression according to claim 1, wherein Also includes: Testing unit: used to test the effect of the ultrasonic stimulation.

3. A product for treating depression, characterized in that: include: A vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S, wherein the vector capable of expressing the large-conductance mechanosensitive ion channel MscL-G22S comprises a viral vector having a Tph2 promoter and is implanted into the dorsal raphe nucleus of a subject for expression; An ultrasonic collimator, wherein the operating parameters of the ultrasonic collimator include: center frequency, 0.5-1.0 MHz; intensity, 0.2-0.3 MPa; pulse width, 450-550 μs; pulse repetition frequency, 0.5-1.5 kHz; stimulation duration, 250-350 ms; and stimulation interval, 2-4 s.

4. The product for treating depression according to claim 3, characterized in that Also includes: Optical fibers and data analysis software.

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