A gene-environment interaction in vitro model based on reward stress stimulation and construction method and use thereof

Abstract

The application provides a gene-environment interaction in vitro model based on reward stress stimulation and a construction method and use thereof. The method places human midbrain organoids rich in dopaminergic neurons on a microelectrode array, applies electrical stimulation corresponding to the burst firing frequency of dopaminergic neurons, simulates persistent reward stress load, and thus reconstructs chronic environmental stress conditions. The electrical stimulation can selectively induce patient-derived organoids of gene-environment interaction related diseases to produce disease-related electrophysiological and molecular phenotype changes, reflecting the neuroplasticity defects under the synergistic action of genetic susceptibility and environmental stress. Compared with traditional low-frequency stimulation-induced long-term depression or non-stimulation conditions, the method has unique effectiveness in inducing depression-related pathological characteristics. The method can be used to construct an in vitro model of depression and other gene-environment interaction related diseases, and is suitable for drug screening and the development and evaluation of diagnosis and treatment strategies.

Description

Technical Field

[0001] This invention belongs to the fields of neurobiology, stem cell engineering, and in vitro disease model technology. Specifically, it relates to a reward stress simulation method based on human brain organoids, which is used to induce gene-environment (G×E) interaction effects in depression and to construct an in vitro model that can be used for disease mechanism and diagnosis research, drug screening, and neuromodulation assessment. Background Technology

[0002] Depression is a common mental illness with complex etiologies, and its occurrence is generally believed to be driven by both genetic susceptibility and environmental stressors. Clinically, one of the most core and diagnostically significant symptoms of depression is anhedonia, i.e., a diminished response to natural rewards or positive stimuli. Numerous studies have shown that anhedonia is closely related to dysfunction of the reward system, which is influenced by the interaction of genetic susceptibility and environmental stress. Therefore, understanding the role of "reward stress" on the dopaminergic (DA) neuron system and related neural networks is key to revealing the G×E interaction mechanism in depression.

[0003] However, due to significant limitations in in situ studies of the human brain's reward circuitry, the mechanisms by which environmental stress disrupts the reward system's function and further induces depressive-like pathological changes such as anhedonia remain poorly understood.

[0004] In recent years, the development of brain organoid technology has provided a new in vitro platform for studying human neuropsychiatric diseases. Brain organoids, formed from human pluripotent stem cells (iPSCs), possess a three-dimensional structure, multiple neuronal types, and spontaneous neural network activity. By introducing environmental stress factors into organoids, it is hoped that the developmental regulation of the human brain's reward system and its dysregulation under chronic stress can be simulated, thus providing a new experimental system for studying the formation mechanisms of core symptoms of depression, such as anhedonia. Therefore, constructing organoid models capable of reproducing the G×E interaction effects of depression is of great significance.

[0005] Although some studies have attempted to construct models of mental illness using organoids, such as the publicly available technical solution (CN120366218A) which provides a method for constructing a model of chronic unpredictable negative stress by introducing negative emotional electrical stimulation into a microelectrode array system, this model, while providing a platform for mental illness research, primarily simulates the continuous input of chronic negative stimulation to the overall neural network, lacking specific attention to the reward system. Therefore, this model cannot reflect the defects in neural plasticity under the synergistic effect of genetic susceptibility and environmental stress, and it is also difficult to reproduce the reward-related pathological phenotype in depression, which is centered on anhedonia.

[0006] Furthermore, existing in vitro stress simulation methods still have significant limitations. Traditional methods often use stress hormones or chemical stimulation to regulate neuronal activity, but these stimuli are usually broad-based and lack targeting, making it difficult to simulate the dysfunction of the reward system under complex backgrounds of chronic stress, and also difficult to induce neural network disorders or molecular abnormalities related to anhedonia in organoids. In addition, low-frequency electrical stimulation of physical stimuli is mainly used to induce widespread long-term inhibition (LTD) in neurons, and cannot specifically simulate the dynamic neural activity patterns specific to the reward pathway, thus making it difficult to reproduce the reward-related pathological phenotypes in depression centered on anhedonia.

[0007] Neuroscience research shows that midbrain dopaminergic neurons are a key cell population encoding reward, exhibiting brief, high-frequency phasic burst firing during natural reward or reward anticipation. This firing pattern directly participates in reward prediction errors, motivational drives, and the formation of positive experiences, and is also a key neural basis for "positive event sensitivity." Chronic stress, negative emotional experiences, or long-term lack of reward can disrupt this burst firing pattern, further leading to decreased neural network synchronicity, impaired synaptic plasticity, and weakened reward response capacity. This process is considered an important biological basis for anhedonia.

[0008] However, existing in vitro models struggle to reconstruct the characteristic activity patterns of DA, and there is a lack of technical approaches to simulate "reward stress overload" or "reward deprivation" based on these patterns. Traditional low-frequency stimulation fails to capture this neurodynamic feature closely related to anhedonia, making it difficult for current organoid models to induce pathological changes consistent with the core symptoms of depression.

[0009] Meanwhile, numerous animal models and clinical studies have revealed that depression involves multi-level pathologies related to neuroplasticity defects, including reduced neural network activity, impaired synchronicity, extracellular matrix remodeling, and abnormalities in various synaptic-related proteins and neurotransmitter systems. These phenomena are all closely related to anhedonia and decreased reward circuit function. However, current organoid models cannot systematically reproduce these comprehensive pathological effects caused by G×E interactions.

[0010] In summary, there is an urgent need for a new technical solution that can introduce characteristic neural activity patterns related to reward stress and anhedonia into organoids, thereby inducing specific electrophysiological and molecular changes in depression, achieving in vitro modeling that more closely resembles the actual mechanism of the disease, and providing a reliable experimental platform for research on the pathogenesis of depression, drug screening, and intervention evaluation. Summary of the Invention

[0011] This invention aims to overcome the shortcomings of existing in vitro models of depression in simulating gene-environment (G×E) interactions and core pathologies of reward pathways, particularly their inability to specifically reproduce neuroplasticity defects associated with anhedonia, lack of genetically dependent stress responses, and difficulty in inducing systemic, multi-level pathological phenotypes. Therefore, this invention provides a method for constructing an organoid model based on reward-based electrical stress stimulation that can highly simulate G×E interactions and anhedonia pathologies in depression in vitro.

[0012] This invention utilizes midbrain organoids obtained through three-dimensional induced differentiation of induced pluripotent stem cells (iPSCs) from healthy controls (HC) and patients with depression. These organoids are rich in dopaminergic neurons (DA), possessing both excitatory and inhibitory neuronal composition, and have formed a three-dimensional neural network structure with synchronized firing and functional connectivity. The mature organoids are loaded into a microelectrode array (MEA) system to ensure simultaneous electrical stimulation and network activity recording. The cellular composition and network basis of the brain organoids enable them to respond to physiologically significant reward-related rhythmic stimuli.

[0013] In a key step of this invention, a biphasic square wave electrical stimulation, preferably at 20 Hz, is applied to the midbrain organoid via MEA. This stimulation simulates the high-frequency burst firing pattern of the midbrain DA when natural reward expectation or reward prediction error signals appear. This rhythm can be considered a "reward stress" load, capable of reconstructing the plasticity challenge caused by disease-induced reward dysfunction in vitro. This invention is not limited to specific pulse amplitude, pulse width, or stimulation duration; any method that produces a neuromodulation effect equivalent to a 20 Hz reward rhythm can be used. 20 Hz is a preferred condition, but similar stress simulation effects can also be produced in relevant frequency bands in the 10-40 Hz range. To compare the specificity of this method, this invention introduces an unstimulated group as a baseline control and uses a 1 Hz low-frequency stimulation group as a representative of traditional long-term inhibition (LTD) conditions to distinguish the differences between "reward stress stimulation" and "stimulation that inhibits synaptic plasticity" in inducing pathological phenotypes.

[0014] The specific technical solution of the present invention is as follows:

[0015] This invention provides a method for constructing an in vitro model of gene-environment interaction based on reward stress stimulation, the method comprising the following steps:

[0016] (1) Obtain midbrain organoids rich in dopaminergic neurons;

[0017] (2) The midbrain organoid is loaded into a microelectrode array system;

[0018] (3) Apply exogenous electrical stimulation to the midbrain organoid through the microelectrode array system to simulate the reward-related burst firing rhythm of midbrain dopaminergic neurons in order to simulate reward stress load.

[0019] Furthermore, the induced pluripotent stem cells are obtained from the patient's peripheral blood mononuclear cells through a non-integrative reprogramming technique.

[0020] Furthermore, the induced differentiation includes:

[0021] Neuroectoderm induction was performed using a combination of small molecule inhibitors including SB431542, Noggin, and CHIR99021.

[0022] Midbrain ventral directional differentiation was performed using a midbrain patterning factor induction system containing SHH-C25II and FGF8.

[0023] During the mature culture stage, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, ascorbic acid, and cyclic adenosine monophosphate analogues are supplemented, and the culture time is 70-100 days.

[0024] Furthermore, immunofluorescence staining confirmed that the midbrain organoids, during their differentiation and maturation period (≥35 days), contained a wide range of TH-positive dopaminergic neurons and MAP2-positive mature neurons.

[0025] Furthermore, the midbrain organoids are obtained by inducing differentiation to maturity from the patient's induced pluripotent stem cells, and the midbrain organoids possess a midbrain network with spontaneous firing activity.

[0026] Furthermore, the patient suffers from a gene-environment interaction-related disease.

[0027] Furthermore, the gene-environment interaction-related diseases include depression, bipolar disorder, schizophrenia, post-traumatic stress disorder, autism spectrum disorder, anxiety disorder, or attention deficit hyperactivity disorder.

[0028] Furthermore, the gene-environment interaction-related disease is depression, and the model is a depression model.

[0029] Furthermore, the frequency of the exogenous electrical stimulation is 10 Hz to 40 Hz.

[0030] Furthermore, the frequency of the exogenous electrical stimulation is 20 Hz.

[0031] Furthermore, the pulse amplitude of the exogenous electrical stimulation is 300mV, and the duration of each phase of the biphasic square wave is 100μs.

[0032] Furthermore, the exogenous electrical stimulation is an intermittent burst stimulation mode, which includes alternating high-frequency pulse firing phases and resting phases.

[0033] Furthermore, within a stimulation cycle, there are multiple high-frequency pulse firing phases, with a resting phase between each firing phase.

[0034] Furthermore, within a stimulation cycle, there are three high-frequency pulse firing phases, with each phase firing 200, 300, and 500 pulses respectively, and a resting phase of 475 seconds between each two firing phases.

[0035] Furthermore, the exogenous electrical stimulation uses biphasic square wave pulses, applied for 1 to 3 stimulation cycles per day, with each stimulation cycle lasting 900 to 1100 seconds and the total stimulation duration being 14 to 28 days.

[0036] Furthermore, the total duration of each stimulation cycle is 1000 seconds, and the total stimulation duration is 21 days.

[0037] The present invention also provides an in vitro model of gene-environment interaction, which is obtained by the above-described construction method.

[0038] The present invention also provides the use of the above-mentioned in vitro gene-environment interaction model in screening or evaluating drugs for treating diseases caused by gene-environment interactions, studying the pathogenesis of diseases caused by gene-environment interactions, or evaluating therapies for diseases caused by gene-environment interactions.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. Precise induction of neuroplasticity damage associated with anhedonia: By applying electrical stimulation (preferably 20 Hz) that simulates the DA reward burst rhythm, the present invention successfully induced neuroplasticity damage characteristics in brain organoids in vitro that are highly associated with anhedonia and reward pathway dysregulation in depression. Specifically, these characteristics include reduced spontaneous firing frequency, disruption of neural network synchronization, and weakened functional connectivity.

[0041] 2. Triggering multi-level molecular pathological changes and systematically mimicking disease states: Further high-throughput proteomics analysis revealed that this reward stress stimulus induced widespread molecular expression abnormalities in organoids, including reduced synaptic structural proteins, enhanced neuroinflammatory pathways, decreased extracellular matrix components, and dysregulation of plasticity regulators. These changes systematically reproduced the multi-system pathological changes observed in the brains of patients with depression.

[0042] 3. Achieving Depression-Specific Gene-Environment Interactions in In Vitro: The most significant breakthrough of this invention lies in the clear gene background dependence of the 20Hz reward stress stimulus: this stimulus selectively induces significant neural network inhibition and molecular pathological changes in organoids derived from patients with depression, while producing no or only slight effects on organoids derived from healthy controls. In contrast, the 1Hz low-frequency stimulus produces broad non-specific inhibition in both groups of organoids, lacking this selectivity. This result successfully reconstructs the classic G×E effect in vitro, i.e., environmental stress must synergize with genetic susceptibility to trigger disease-related phenotypes.

[0043] 4. Providing a highly controllable, physiologically relevant, and translational research platform: Based on the above characteristics, this invention establishes an organoid model system that can reconstruct reward system dysfunction, simulate anhedonia, and reflect the G×E interaction mechanism unique to depression in vitro. This model not only provides a novel tool for revealing neural circuit defects and molecular mechanisms in depression, but also serves as a highly efficient experimental platform for disease diagnosis, drug screening, evaluation of antidepressant therapies, and optimization of neuromodulation strategies, possessing significant scientific research and clinical translational value.

[0044] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0045] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the overall process of the method of the present invention, showing the overall steps of obtaining PBMCs from healthy controls and patients with depression, reprogramming to obtain iPSCs, inducing the generation of midbrain organoids rich in dopaminergic neurons, loading the organoids onto the MEA platform and applying different electrical stimulation modes.

[0047] Figure 2 The immunofluorescence images show the development of midbrain organoids, demonstrating that the organoids express early midbrain markers such as LMX1B, OTX2, and SOX2 on day 14 (14DIV) of differentiation, and mature neuronal markers such as Tubulin, Ki67, TH (tyrosine hydroxylase), and MAP2 on day 35 (35DIV), proving that the organoids have the structural basis to carry out electrical stimulation experiments.

[0048] Figure 3This is a schematic diagram of a midbrain organoid placed on an MEA chip, showing the attachment interface between the organoid and the electrodes, as well as the layout of the recording and stimulation electrodes.

[0049] Figure 4 The diagram below shows the waveform of the electrical stimulation protocol used in this invention. The top diagram shows the 1Hz continuous stimulation mode (1000 pulses applied continuously within 1000 seconds); the bottom diagram shows the core 20Hz intermittent reward pressure stimulation mode of this invention, which includes a "stimulation-rest-stimulation" cyclic structure to simulate the burst firing characteristics of midbrain dopaminergic neurons.

[0050] Figure 5 The plot shows the population mean firing frequency over time of healthy controls and organoids derived from depression under blank stimulation, low-frequency stimulation (1 Hz), and reward stress stimulation (20 Hz), demonstrating the broad inhibitory effect of 1 Hz stimulation and the specific inhibitory effect of 20 Hz stimulation on MDD organoids.

[0051] Figure 6 This is a statistical comparison of the synchronization and topological properties of organoid neural networks under different stimulus conditions, showing four indicators: average participation coefficient, average node degree, average local efficiency, and network density. The results show that reward stress stimulation (20Hz) significantly reduced the network complexity and synchronization of depression organoids; asterisks indicate statistically significant differences. This indicates that p < 0.05. This indicates that p < 0.001.

[0052] Figure 7 Volcano plots were used to model the differences in organoid protein expression among groups 21 days after stimulation, comparing the distribution of differentially expressed proteins between the depression group and the healthy control group under blank, low-frequency (1Hz), and reward stress stimulation (20Hz). The results showed that 20Hz reward stress stimulation resulted in the largest differentially expressed protein between the two groups, while low-frequency stimulation led to a decrease in differentially expressed proteins between the two groups (upregulation, downregulation in blue).

[0053] Figure 8 The image shows a volcano plot illustrating the longitudinal changes in organoid proteomes before and after 20Hz reward stress stimulation. The left plot shows a large number of protein expression changes (137 upregulated and 325 downregulated) in the depression group after stimulation, while the right plot shows smaller changes in the healthy control group, further verifying that the stimulation has a selective effect on the genetic background of depression.

[0054] Figure 9This is a volcano plot showing the longitudinal changes in the organoid proteome before and after 1Hz low-frequency stimulation. The left plot shows that the normal control group showed some degree of protein changes after 1Hz low-frequency stimulation (161 proteins upregulated and 53 downregulated); the right plot shows that the changes in the depression group were relatively smaller (64 proteins upregulated and 45 downregulated). Figure 8 In contrast, 1Hz stimulation did not induce the characteristic large-scale protein downregulation in the depression group, demonstrating the nonspecificity of low-frequency stimulation. Detailed Implementation

[0055] The cells, culture system, multi-electrode array, and proteomics detection platform used in this invention are all commercially available products and readily accessible within the technical field. The embodiments of this invention are described in detail below with reference to the accompanying drawings, but should not be construed as limiting the scope of protection of this invention.

[0056] Example 1: Construction of midbrain organoids rich in dopaminergic neurons, MEA loading, and reward-pressure simulation stimulation

[0057] I. Obtaining PBMCs from healthy controls and patients with depression and constructing iPSC cell lines

[0058] To construct human midbrain organoids that can simulate neural plasticity under reward stress, this embodiment first collected peripheral blood samples from healthy controls (HC) and clinically diagnosed patients with depression, obtaining peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation. After obtaining informed consent from the donors and passing ethical approval, the PBMCs were reprogrammed into iPSCs using non-integrative reprogramming technology. The resulting iPSCs were verified by pluripotency marker staining and karyotype analysis, exhibiting typical pluripotency and genomic stability. This embodiment exemplarily used three HC-derived and three depression-derived iPSC cell lines, each independently used for subsequent organoid construction. Subsequent experiments independently constructed organoids based on each iPSC line, generating no fewer than three organoids from each line for data analysis and statistical testing to ensure experimental repeatability and statistical stability. The relevant procedures are as follows: Figure 1 As shown.

[0059] II. Construction and maturation culture of midbrain organoids rich in dopaminergic neurons

[0060] After obtaining a stable iPSC system, according to Figure 1The strategy described guides iPSCs into a midbrain-directed differentiation process. The specific steps are as follows: First, after iPSCs form embryoid bodies, they are induced to develop neuroectoderm by adding a combination of small molecule inhibitors containing 10 μM SB431542, 100 ng / ml Noggin, and 1 μM CHIR99021. Subsequently, the cell clumps are transferred to a midbrain modeling factor induction system containing 300 ng / ml SHH-C25II and 100 ng / ml FGF8 to direct their development towards the midbrain dopaminergic spectrum. Eight days later, the cell clumps are placed in a rotating flask or gently shaken three-dimensional culture system for further maturation. During this stage, the culture medium used is Neurobasa1 medium supplemented with 10 ng / ml brain-derived neurotrophic factor, 10 ng / ml glial cell-derived neurotrophic factor, 200 μM ascorbic acid, and 500 μM db-cAMP to promote the generation of TH-positive dopaminergic neurons, axonal growth, and synapse formation. During 70-100 days of culture, organoids gradually develop a midbrain network capable of spontaneous electrical activity. Immunofluorescence staining ( Figure 2 This study confirmed that early and mid-differentiation (14 DIV) organoids express midbrain progenitor cell markers such as LMX1B and OTX2, and mature (35 DIV and later) organoids widely contain TH-positive dopaminergic neurons and MAP2-positive mature neurons, indicating that they have the structural and molecular basis to carry out electrical stimulation experiments.

[0061] III. MEA Loading and Stabilization of Midbrain Organoids

[0062] The mature midbrain organoids were then gently loaded into the MEA system, in a manner as follows: Figure 3 As shown. The electrode area was pre-coated with poly-L-lysine, Matrigel, or Laminin to enhance adhesion, and diluted ECM gel or micromesh was used to assist in fixation according to the organoid volume, ensuring that its bottom was in close contact with the MEA electrode. The loaded organoids were equilibrated in a constant temperature environment of 37°C and 5% CO2 for about one week, during which a stable baseline firing pattern gradually formed, laying the foundation for subsequent stimulation experiments.

[0063] IV. Reward Stress Simulation and Control Stimulus Conditions

[0064] After MEA equilibration, the organoids were subjected to the following electrical stimulation conditions: pulse amplitude 300mV, biphasic square wave, each phase duration 100μs:

[0065] 1. Unstimulated group: Remained in a natural state, only spontaneous discharge activity was recorded;

[0066] 2. Low-frequency stimulation group (1Hz): Uses classic low-frequency pulses in a continuous stimulation mode, such as... Figure 4(Above) 1000 biphasic square wave pulses were applied over 1000 seconds for 21 days to simulate low-frequency modulation known to induce a wide range of LTD effects;

[0067] 3. Reward stress simulation group (20Hz): Employing the specific "intermittent burst stimulation" protocol designed in this invention. Specific parameters are as follows... Figure 4 As shown below: Within a 1000-second stimulation cycle, three phases of 20Hz pulse firing (200 pulses, 300 pulses, and 500 pulses respectively) are conducted, with a 475-second rest period between each firing phase. This high-frequency burst pattern, incorporating long resting intervals, more accurately simulates the intermittent response characteristics of midbrain dopaminergic neurons to reward prediction under physiological conditions. This stimulation is performed once daily for 21 days. This 20Hz rhythm corresponds to the burst firing pattern of midbrain dopaminergic neurons under natural reward situations, thus enabling the simulation of long-term reward deprivation stress in vitro.

[0068] All organoids were derived from the above six independent iPSC cell lines. Each cell line contained multiple organoid duplicates. Throughout the experiment, consistent culture conditions and replacement frequency were maintained to ensure that the differences between groups mainly stemmed from differences in stimulation frequency and donor class (normal control vs. depression) genetic background.

[0069] Example 2: Reward stress simulation of neural network effects, molecular pathological changes, and reversibility verification on midbrain organoids

[0070] I. Analysis of neural network electrical activity in donor-sourced brain organoids

[0071] After completing the different stimulation conditions in Example 1, the spontaneous electrical activity of organoids from various sources was recorded and analyzed over a long period using the MEA system. The core finding of this invention comes from comparing the differences in effects of different stimulation frequencies under different gene backgrounds, with the following results:

[0072] 1. Non-specific inhibitory effect of low-frequency stimulation (1Hz group): such as Figure 5 The population discharge frequency trajectory shown and Figure 6 Network topology analysis showed that 1Hz stimulation induced a broad inhibitory effect in all healthy controls and organoids derived from depression. The mean firing frequency of both groups of organoids decreased to extremely low levels after 1Hz stimulation. Figure 5 (Light blue and pink lines), at the same time Figure 6 The network density and average node degree of the group were significantly lower than those of the unstimulated group. This phenomenon is consistent with the characteristics of classic LTD stimulation, indicating that the inhibitory effect of low-frequency stimulation on midbrain organoids is a universal, non-selective effect independent of genetic background.

[0073] 2. Gene background-dependent effect of reward stress stimulation (20Hz group): In contrast, 20Hz reward stress stimulation exhibited a highly specific gene-environment interaction (G×E) effect:

[0074] In organoids derived from depression: 20Hz stimulation led to severe network function breakdown. Figure 5 This shows that its discharge frequency (dark blue line) is significantly suppressed, approaching the level of the 1Hz group. Figure 6 Further analysis of network synchronization revealed that the depression group showed a highly significant decrease in mean participation coefficient, mean node degree, mean local efficiency, and network density after 20Hz stimulation. This indicates that simulated reward pressure disrupts neural network connectivity in the context of susceptibility genes.

[0075] However, in organoids derived from healthy controls, 20Hz stimulation did not cause significant network damage. Figure 5 The discharge frequency (red line) remained active, even exceeding that of the unstimulated group at certain times. Figure 6 The data showed that the HC group maintained high levels of various network topology indicators (red bars) under 20Hz stimulation, with no statistically significant difference from the unstimulated group, and significantly higher than the depression group under the same conditions.

[0076] This selective response indicates that the 20Hz reward stress simulation only induces a decrease in pathological neural plasticity when the organoids have a genetic susceptibility background associated with depression. Organoids derived from HC patients exhibit a significant ability to maintain homeostasis under this stimulus, thus reproducing the typical G×E effect. These results collectively demonstrate that the method of this invention can reconstruct network impairment phenotypes closely related to depression in a human genetic background, exhibiting good reproducibility, robustness, and relevance to human samples.

[0077] II. Proteomic Pathological Changes Analysis

[0078] To further explore the molecular mechanism of this selective effect and verify the pathological specificity of 20Hz reward stress stimulation, this invention utilizes a proteomics platform to perform high-throughput detection of culture medium-secreted proteins in two groups of organoids under different stimulation conditions. Through comparison... Figure 7 (Intergroup comparison) Figure 8 (20Hz longitudinal comparison) and Figure 9 (1Hz longitudinal comparison) revealed significant differences:

[0079] 1. 20Hz reward stress stimulation induces specific "molecular collapse" such as Figure 8As shown, after 21 days of 20Hz reward stress stimulation, organoids derived from depression (MDD) exhibited dramatic proteomic remodeling, with 325 proteins significantly downregulated and 137 proteins significantly upregulated. This large-scale downregulation involved extracellular matrix (ECM) degradation, loss of synaptic functional proteins, and reduction of neurotrophic factors, constituting a "molecular collapse" phenotype. In contrast, organoids derived from healthy controls (HC) showed extremely strong homeostasis under the same 20Hz stimulation, with only 39 proteins downregulated and 75 proteins upregulated, without any pathological changes.

[0080] 2. 1Hz low-frequency stimulation only produces non-specific modulation. To eliminate the non-specific interference of electrical stimulation itself, this invention introduces 1Hz stimulation as a control. Figure 9 Unlike 20Hz, 1Hz stimulation did not induce the aforementioned large-scale downregulation pathology in MDD organoids; the changes were limited to downregulation of 45 and upregulation of 64. Figure 9 (Data on the right). Notably, the HC group responded even slightly better to 1Hz stimulation than the MDD group (down-regulation of 53 and up-regulation of 161). Figure 9 (Data on the left).

[0081] The above comparison confirms that 1Hz stimulation only induces non-specific basal metabolic regulation and has no selectivity for gene background; while the 20Hz reward stress stimulation identified in this invention is the only key condition that can synergize with depression susceptibility genes (G×E effect) to directionally induce specific pathological changes in organoids characterized by "large-scale protein expression inhibition". This is highly consistent with the aforementioned results of electrophysiological network functional decline.

[0082] III. Verification of the reversibility of neural network damage in donor-derived organoids

[0083] While verifying pathological damage, this invention also tested the reversibility of neural network damage. By applying γ-θ rhythmic electrical stimulation to MDD-derived organoids subjected to 20Hz stimulation, partial recovery of organoid network activity, synchronicity, and functional connectivity was observed, with the recovery trend as follows: Figure 9 As shown, antidepressants (such as fluoxetine) can also promote the recovery of synaptic function and network activity to some extent, while HC organoids, being undamaged, have no significant room for recovery. Intervention experiments demonstrate that the organoid model established in this invention can not only simulate the neural network damage phenotype associated with depression, but also serve as an in vitro platform for evaluating treatment strategies, drug intervention effects, and neuromodulation parameters.

[0084] In summary, the reward-pressure simulation system based on human brain organoids constructed in this invention successfully reconstructs the characteristic mechanism of gene-environment interaction in depression: low-frequency stimulation, as a broad-spectrum inhibitory mechanism, exerts a non-selective inhibitory effect on neurons in all brain organoids, while reward-pressure simulation induces neuroplasticity damage only in organoids with a background of depression genes. This selective damage effect of depression induced by reward-pressure simulation is an inventive discovery that cannot be expected or replaced by existing technologies, making the model of this invention have significant theoretical value and application prospects in the study of the mechanisms of depression, drug screening, and the development of neuromodulation strategies.

Claims

1. A method for constructing an in vitro model of gene-environment interaction based on reward-pressure stimuli, characterized in that, The method includes the following steps: (1) Obtain midbrain organoids rich in dopaminergic neurons; (2) The midbrain organoid is loaded into a microelectrode array system; (3) Apply exogenous electrical stimulation to the midbrain organoid through the microelectrode array system to simulate the reward-related burst firing rhythm of midbrain dopaminergic neurons in order to simulate reward stress load; The midbrain organoids were obtained by inducing pluripotent stem cells from the patient to mature, and the midbrain organoids possess a midbrain network with spontaneous firing activity. The patient suffers from a gene-environment interaction-related disease. The gene-environment interaction-related disease is depression; The frequency of the exogenous electrical stimulation is 20 Hz; The exogenous electrical stimulation is an intermittent burst stimulation mode, which includes alternating high-frequency pulse firing phases and resting phases; One stimulation cycle of exogenous electrical stimulation includes three high-frequency pulse firing phases, with 200, 300 and 500 pulses fired in each phase, respectively, and a resting phase of 475 seconds between each two firing phases.

2. The construction method according to claim 1, characterized in that, The exogenous electrical stimulation uses biphasic square wave pulses, applied for 1 to 3 stimulation cycles per day, with each stimulation cycle lasting 900 to 1100 seconds, and the total stimulation duration being 14 to 28 days.

3. An in vitro model of gene-environment interaction, characterized in that, The gene-environment interaction in vitro model is obtained by the construction method described in any one of claims 1 to 2.

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