An olfactory training system for activating motor-olfactory-hippocampal neural network and application

Abstract

The application discloses an olfactory training system for activating a motor-olfactory-hippocampal neural network and application, the system comprises a plurality of different odor sources, the odor source is used for alternating olfactory stimulation to stroke patients, so as to improve the cognitive function and / or olfactory function of the patient. The application firstly discloses that the function connection of the motor-olfactory-cognitive brain area is disordered after stroke, the function of the VIP neuron of the motor cortex (MO) around the infarction is inhibited; the VIP neuron of the MO region dominates the GABA neuron of the dorsal peripeduncular area (DP) through an inhibitory pathway, activating the pathway can remove the inhibition of the glutamatergic pyramidal neuron of the DP, enhance the excitatory input to the hippocampal CA3 region, and restore the olfaction and spatial memory. The application proves that the structured olfactory training can activate the loop, and significantly improve the cognitive function of the stroke patient. The application provides a simple and non-invasive stroke rehabilitation strategy.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to an olfactory training system for activating the motor-olfactory-hippocampal neural network and its application. Background Technology

[0002] As one of the oldest sensory modules, olfaction provides a crucial entry point for cognitive processes through its neural connections with cortical and limbic system circuits. The synergistic effect of motor control, olfactory perception, and cognitive processing reflects the integrative nature of brain organization, where seemingly disparate brain regions work collaboratively to support complex behaviors essential for survival. In this neural network composed of three major functional systems, damage to any link can trigger cascading functional impairments throughout the system, but the mechanisms of their interdependence and dynamic regulation remain poorly understood. Stroke is one of the leading causes of death and disability worldwide, and stroke patients have a very high probability of experiencing impairment in motor, olfactory, and cognitive functions after stroke. This provides an ideal and clinically valuable research model for exploring the interaction between motor, olfactory, and cognitive functions. Elucidating how pathological damage disrupts this network is crucial for advancing interventions and treatments for related neurological diseases.

[0003] Post-stroke olfactory dysfunction has long been underestimated and neglected in clinical practice, primarily due to the unreliability of patient-reported olfactory assessments and the difficulty in early identification of abnormal changes in olfactory function. Recent standardized assessments indicate that up to 43% of stroke patients experience olfactory impairment, clinically manifesting as decreased or even complete loss of smell. Approximately 46% of stroke patients develop post-stroke cognitive impairment (PSCI), a serious long-term complication whose severity directly determines a patient's functional independence and quality of life. However, individualized prevention and precise treatment of PSCI remain challenging in current clinical practice. Notably, olfactory dysfunction often occurs concurrently with, or even earlier than, cognitive decline. Despite their strong correlation, the neural circuits connecting post-stroke olfactory dysfunction and PSCI remain largely unknown. Summary of the Invention

[0004] The purpose of this invention is to provide an olfactory training system and its application for activating the motor-olfactory-hippocampal neural network, aiming to solve the technical problem that motor, olfactory and cognitive dysfunctions coexist and influence each other after stroke, but their coupling mechanism is not yet clear, resulting in a lack of mechanism-based non-invasive intervention strategies in clinical practice.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An olfactory training system for activating motor-olfactory-hippocampal neural networks, the system comprising multiple different odor sources for alternating olfactory stimulation of stroke patients to improve their cognitive and / or olfactory functions.

[0007] Furthermore, the motor-olfactory-hippocampal neural network includes vasoactive intestinal peptide (VIP) neurons in the motor cortex (MO), gamma-aminobutyric acid (GABAergic) neurons in the dorsal tegmental area (DP), glutamatergic pyramidal neurons in the dorsal tegmental area (DP), and neurons in the CA3 region of the hippocampus. The activation is achieved by activating VIP neurons in the motor cortex (MO) around the infarct through olfactory training, thereby inhibiting GABAergic neurons in the dorsal tegmental area (DP) through the inhibitory pathway from VIP neurons in the motor cortex (MO) to GABAergic neurons in the dorsal tegmental area (DP), thereby inhibiting GABAergic neurons in the dorsal tegmental area (DP) and enhancing their excitatory input to the CA3 region of the hippocampus.

[0008] Furthermore, the various odor sources include at least two odors selected from the group consisting of clove, lemon, rose, and eucalyptus.

[0009] Furthermore, the odor source is used to alternately provide the patient with different odors each day.

[0010] Furthermore, the system also includes a training program specification documenting an olfactory training program lasting at least 21 days.

[0011] Furthermore, the cognitive functions include the ability to recognize new objects and / or the ability to learn and retain spatial memories; the olfactory functions include the ability to recognize odor preferences and / or the ability to recognize odor aversions.

[0012] The present invention also provides applications of the aforementioned olfactory training system.

[0013] The olfactory training system is used in the preparation of devices or drugs for activating motor-olfactory-hippocampal neural networks and / or improving post-stroke cognitive and / or olfactory functions.

[0014] Furthermore, the activation of the motor-olfactory-hippocampal neural network and / or improvement of post-stroke cognitive and / or olfactory function is achieved by restoring the activity of vasoactive intestinal peptide (VIP) neurons in the peri-infarct motor cortex (MO), enhancing the intrinsic excitability of glutamatergic pyramidal neurons in the dorsal tegmental area (DP), and / or increasing the number of c-Fos positive neurons in the dorsal tegmental area (DP) and the CA3 region of the hippocampus.

[0015] Furthermore, the improvement in cognitive function was assessed using the Montreal Cognitive Assessment (MoCA) score, and the improvement in olfactory function was assessed using the olfactory function score, with the olfactory score being positively correlated with the Montreal Cognitive Assessment (MoCA) score.

[0016] The present invention also provides a method for screening candidate substances for activating the motor-olfactory-hippocampal neural network and / or improving post-stroke cognitive function and / or olfactory function, the method comprising: detecting the effect of the candidate substance on the activity of vasoactive intestinal peptide (VIP) neurons in the motor cortex (MO), the activity of γ-aminobutyric acid (GABAergic) neurons in the dorsal tegmental area (DP), the activity of glutamatergic pyramidal neurons in the dorsal tegmental area (DP), or the activity of neurons in the CA3 region of the hippocampus; if the candidate substance can activate vasoactive intestinal peptide (VIP) neurons, inhibit γ-aminobutyric acid (GABAergic) neurons, de-inhibit glutamatergic pyramidal neurons, or enhance the activity of neurons in the CA3 region of the hippocampus, then the candidate substance is indicated to be suitable for activating the motor-olfactory-hippocampal neural network and / or improving post-stroke cognitive function and / or olfactory function.

[0017] Furthermore, the detection includes detecting c-Fos expression levels in the animal brain, changes in neuronal calcium signaling, and / or changes in intrinsic neuronal excitability.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) This invention discovers that abnormal changes occur in the motor-olfactory-cognitive neural network in the brain of patients after ischemic stroke. This result has been verified in animal models, and the stroke animal models show significant olfactory and cognitive dysfunction. This invention reveals for the first time that neurons expressing vasoactive intestinal peptide (VIP) in the peri-infarct motor cortex (MO) exhibit structural resilience after injury, but functionally exhibit low activity. These findings elucidate the neural basis for the coexistence and mutual influence of motor, olfactory, and cognitive dysfunction after stroke.

[0020] (2) This invention discovers that VIP neurons in the peri-infarct motor cortex (MO) establish direct inhibitory synapses in the dorsal tegmental area (DP), a key olfactory center. Activation of this MO... VIP -DP GABA The circuit unblocks the inhibition of DP pyramidal neurons, enhances the excitatory drive of the CA3 region of the hippocampus, thereby restoring olfactory discrimination and spatial memory. This is achieved through optogenetic activation of MO. VIP -DP GABA The circuit can enhance signal transmission in the DP-CA3 region, significantly improving olfactory discrimination impairment and post-stroke cognitive impairment.

[0021] (3) This invention further discovers that structured olfactory training can activate this circuit and significantly improve cognitive function in rodents and clinical stroke patients. Crucially, olfactory training can bring significant cognitive improvement to stroke patients. Clinical data show that patients in the olfactory training group showed significant improvements in both olfactory scores and MoCA scores, and that olfactory scores were positively correlated with MoCA scores.

[0022] (4) These findings reveal that the olfactory system is a key node in post-stroke network dysfunction and establish sensory-based olfactory training as a promising strategy for comprehensive stroke rehabilitation. The olfactory training system provided by this invention is a simple, non-invasive stroke treatment strategy with good clinical translation prospects and broad industrial application value. (See attached figures)

[0023] Figure 1 To map the changes in whole-brain functional connectivity in AIS patients and PT mice based on rs-fMRI. (A) Schematic diagram of rs-fMRI studies in non-stroke control groups and acute ischemic stroke (AIS) patients. (B and C) Heatmap (B) and bar chart (C) showing the differences in resting-state functional connectivity between the infarct-side olfactory cortex (OLF) and the healthy parahippocampal gyrus (PHG) and bilateral motor cortex (SMA), using the OLF as the seed point. (D) Flowchart of the timeline for mouse PT model construction, rs-fMRI detection, and olfactory and cognitive behavioral experiments. (E) Definition of peri-infarct regions: infarct core region (red), motor cortex-side peri-infarct region (MO, green), and somatosensory cortex-side peri-infarct region (SS, blue). (F) Voxel-based analysis using the MO-side peri-infarct region as the seed point; red-orange areas indicate significant changes in functional connectivity in PT mice; the right side is an enlarged view of the DP brain region shown in the white dashed box on the left. (G and H) On day 7 after PT, the olfactory preference test (L) and aversion test (M) were used to detect the time mice spent exploring between preferred / averse odors and control odors; (I) The buried food search test: the total time mice spent finding buried food on day 7 after PT. (J) The new object recognition test: the discrimination index of mice for new objects on day 21 after PT. (KM) Morris water maze test on days 22-28 after PT: learning curve (K), trajectory map (L), time spent in the target quadrant, number of times crossing the target, percentage of time spent in the target quadrant, and average swimming speed (M).

[0024] Figure 2Molecular mapping analysis of the infarct-side cortex after stroke. (A) Schematic diagram of snRNA-seq and ST detection process in PT model mice. (B) t-SNE plot based on snRNA-seq data, showing the distribution of different neuronal types in the infarct-side cortex. (C) Violin plot showing the expression levels of cell type-specific marker genes in different cell clusters. (D) Proportional distribution of cortical cells in the capture area after stroke; the SPOTlight tool can accurately capture cortical structures and distinguish highly similar neuronal subtypes. (E) Cell proportion of VIP neurons in the peri-infarct area of ​​the sham-operated group and the PT group. (F) Experimental procedure for verifying the distribution of VIP neurons through microinjection of virus into the brain of VIP-Cre mice. (G) Left: Representative images of virus-labeled VIP neurons and RNAscope detection; Right: Quantitative results of RNAscope verification of the proportion of virus-labeled VIP neurons. (H) Left: Representative image of VIP neurons in the peri-infarct area; Right: Quantitative analysis of VIP neuron density in the peri-infarct area.

[0025] Figure 3 This study describes the detection and optogenetic manipulation of VIP neuron activity in the peri-infarct region. (A) Schematic diagram of continuous calcium imaging tracking of VIP neuron calcium signaling events in the peri-infarct region before and after stroke; top left corner: recording site in the peri-infarct region. (B) Representative calcium imaging images before and after PT modeling; the same neuron location is circled in white. (C) Average peak value of calcium signal in VIP neurons before and after stroke. (D) Left: Schematic diagram of optogenetic manipulation of VIP neurons in the MO brain region; right: representative image of fiber optic implantation site. (E) c-Fos after photoactivation of VIP neurons in Sham and PT mice. + Schematic diagram and quantitative results of VIP neuron count. (F) Time axis of optogenetic activation of VIP neurons experiment design. (GI) Repair effect of optogenetic activation of VIP neurons in the MO brain region on post-stroke motor dysfunction: grid walking test (G), cylinder test (H), adhesive removal test (I). (J and K) Repair effect of optogenetic activation of VIP neurons in the MO region on post-stroke olfactory dysfunction: olfactory preference test (J) and aversion test (K) on day 22 after PT modeling to detect the exploration time of mice in preferred / averse odors and control odors. (L and M) Food burial test on day 22 after PT modeling: mouse food-finding latency (L) and average movement speed (M). (N) New object recognition test: discrimination index of mice in each group. (O) Learning curve of escape latency in Morris water maze test on days 23–27 after modeling. (P) Schematic diagram of representative water maze movement trajectories. (QT)PT probe experiment on the 28th day after platform removal: total time spent in the target quadrant (Q), number of times the target was crossed (R), percentage of time spent in the target quadrant (S), and average swimming speed (T).

[0026] Figure 4 This diagram illustrates the efferent projection pathway of VIP neurons in the MO brain region. (A) Schematic diagram of anterograde tracing experiments of VIP neurons in the MO brain region. (B and C) Representative images of EGFP signals at the injection site (B) and downstream brain region (C). (D) Schematic diagram of retrograde tracing using VIP-Cre mice and identification of upstream neurons using RNAscope. (E–F) Representative images of mCherry or Vip signals at the injection site (E) and upstream brain region (F). (G) Patch-clamp experimental design for monosynaptic GABAergic transmission from VIP neurons in the MO brain region to the DP brain region, resolving their projection function; the right side shows an example of a sealed DP neuron. (H) Photoactivation of ChR2-expressing VIP neuron terminals from the MO brain region induced inhibitory postsynaptic currents (IPSCs) on DP neurons, and observation of the blocking effect of a GABA receptor antagonist (picristol) on this current. (I) The initial latency of optogenetic stimulation-induced IPSC in DP neurons; n=4 mice (10 cells, 2-3 cells per mouse patch-clamp recording). (J) Tetrandrolone completely blocks optogenetic-induced IPSC. (K and L) Optogenetic-induced IPSC can be blocked by tetrodotoxin (TTX) and recovered after combined administration of TTX and 4-aminopyridine (4-AP). (M and N) Representative images of biotin-labeled cells induced by photoactivation of ChR2-expressing VIP neuronal terminals from MO for cell type identification. Biotin-labeled DP neurons are co-labeled with GABA (M) but not with glutamate (N).

[0027] Figure 5 This section describes the optogenetic stimulation of VIP neurons projected to the DP and its mechanism of action. (A) Left: Schematic diagram of the position of the end of the optogenetic stimulation fiber; Right: Experimental design timeline. In the study of MO... VIP Behavioral tests were conducted after photoactivation of neuronal projections to the DP. (B–C) On day 22 after photothrombosis (PT) modeling, the time mice spent in preferred / averse odors and control odors was measured using odor preference test (B) and odor aversion test (C). (D–E) On day 22 after PT modeling, the sham-hunting test was used to measure sham-hunting behavior. VIP-mCherry PT VIP-mCherry With PT VIP-ChR2 (D) Foraging latency and average movement speed (E) of mice. (F) Discrimination index of mice in each group as tested in the novel object recognition test. (G) Learning curve of foraging latency in the Morris water maze test on days 22-27 after PT modeling. (H) Representative route maps of swimming trajectories of mice in each group on day 28. (I–L) Probe test after platform removal on day 28 to detect sham... VIP-mCherry PT VIP-mCherry With PT VIP-ChR2The total time spent in the target quadrant (I), the number of times the mouse crossed the platform (J), the percentage of time spent in the target quadrant (K), and the average swimming speed (L) were measured. (M) Validation of MO VIP Schematic diagram of the experiment demonstrating the disinhibition effect of neurons on DP projection neurons via local GABAergic interneurons. (N) Inhibitory postsynaptic currents (IPSCs) recorded on DP projection neurons during 30 seconds of continuous blue light stimulation. (O) Bar graph showing the amplitude and frequency of IPSCs before and after optogenetic stimulation of the DP region; n=4 mice (11 cells in total, 2-3 cells recorded per mouse). (P) Schematic diagram of anterograde tracing of DP projection neurons. (Q) Representative images of DP region initiating cells and downstream nerve fibers of the CA3 region in the dorsal hippocampus. (R) Schematic diagram of retrograde tracing from CA3 pyramidal neurons. (S) Representative images of CA3 region initiating cells and upstream cells of the DP region.

[0028] Figure 6 Olfactory training was used to activate DP (distracted sensory organs) and improve olfactory and cognitive dysfunction after stroke. The study included: (A) a flowchart of the olfactory enrichment stimulation experiment; and (B) sham surgery group, photothrombosis group (PT), and photothrombosis + olfactory training group (PT). OT (C) Waveforms of representative action potentials of intrinsic excitability in projection neurons of the mouse DP region. OT Intrinsic excitability of projection neurons in the DP region of mice. (D–E)sham, PT, PT OT c-Fos expression levels in DP projection neurons (D) and CA3 pyramidal neurons (E) of mice in the (F–G) group were measured. On day 28 after PT modeling, odor preference (F) and odor aversion (G) tests were performed to determine the time mice spent in preferred / averse odors and control odors. On day 28 after PT modeling, a foraging test was performed to measure sham, PT, and PT levels. OT The foraging latency (H) and average movement speed (I) of the group of mice. (J) New object recognition experiment to detect sham, PT, PT OT Group discrimination index of mice. (K) Learning curve of Morris water maze latency for mice on days 31-36 after PT modeling. (L) Representative route map of swimming trajectory of mice in each group on day 37. (M–P) Probe test to detect sham, PT, and PT after platform removal on day 37. OTThe total time spent in the target quadrant (M), the number of times the platform was crossed (N), the percentage of time spent in the target quadrant (O), and the average swimming speed (P) of the mice were measured. (Q) Flowchart of the experiment dividing patients into a simple cognitive function training group and a cognitive + olfactory combined function training group. Cognitive and olfactory functions were tested before and after training. (R–S) Olfactory scores (R) and MoCA scores (S) before and after training in the control group and the olfactory training group (OT group). (T) Correlation analysis between olfactory scores and MoCA scores. Detailed Implementation

[0029] The present invention will be described in detail below with reference to embodiments, but the present invention is not limited to these embodiments. Any equivalent transformations or modifications made by those skilled in the art based on the core ideas of the present invention fall within the protection scope of the present invention.

[0030] This invention provides an olfactory training system for activating the motor-olfactory-hippocampal neural network, comprising multiple different odor sources configured to provide alternating olfactory stimulation to stroke patients. By alternately providing different odors, the motor-olfactory-hippocampal neural network in the patient's brain can be activated, specifically including vasoactive intestinal peptide (VIP) neurons in the motor cortex (MO), γ-aminobutyric acid (GABAergic) neurons in the dorsal tegmental area (DP), glutamatergic pyramidal neurons in the DP, and neurons in the CA3 region of the hippocampus. Its mechanism of action is as follows: olfactory training activates VIP neurons in the peri-infarct motor cortex (MO), which in turn inhibits GABAergic neurons in the DP region through an inhibitory pathway from VIP neurons in the MO region to GABAergic neurons in the DP region, thereby inhibiting glutamatergic pyramidal neurons in the DP region, enhancing their excitatory input to the CA3 region of the hippocampus, and ultimately improving the cognitive and olfactory functions of stroke patients.

[0031] The preferred odor sources include at least two scents selected from clove, lemon, rose, and eucalyptus, with patients receiving different scents alternately each day. The training program is preferably sustained for at least 21 days. Improvements in cognitive function can be evaluated using indicators such as the ability to recognize new objects, spatial learning, and memory retention; improvements in olfactory function can be evaluated using indicators such as the ability to recognize odor preferences and odor aversions.

[0032] The present invention also provides the application of the olfactory training system in the preparation of devices or drugs for activating motor-olfactory-hippocampal neural networks and / or improving post-stroke cognitive function and / or olfactory function, as well as a method for screening candidate substances based on the neural pathway.

[0033] The present invention will be further illustrated below through specific embodiments.

[0034] Example 1: Mapping the changes in whole-brain functional connectivity in AIS patients and PT mice based on rs-fMRI

[0035] To investigate the mechanism of brain functional network deficits after stroke, this invention first analyzed resting-state functional magnetic resonance imaging (rs-fMRI) data of patients with acute ischemic stroke (AIS) within 2 weeks of onset. Figure 1 (A). Compared with the control group, the functional connectivity between the olfactory brain region and the parahippocampal gyrus on the affected side was significantly reduced in AIS patients, while the functional connectivity with the motor cortex on the affected side was abnormally enhanced. Figure 1 (B and C). The above changes were unrelated to the infarct location, suggesting widespread damage to the motor-olfactory-cognitive neural circuit.

[0036] To further explore the underlying mechanisms of abnormalities in the motor-olfactory-cognitive circuitry after stroke, this invention used photochemical embolization (PT) to construct a mouse model of sensorimotor cortical infarction. Three days later, rs-fMRI was performed. Figure 1 The results showed that functional connectivity between the periinfarct motor cortex (MO) and the ipsilateral dorsal tegmental area (DP) was significantly reduced in PT mice, suggesting disruption of neural communication between these key cortical regions after stroke. Figure 1 (E and F in the middle).

[0037] Behavioral verification experiments further corroborated the above imaging findings, showing that the PT model mice exhibited significant olfactory and cognitive deficits. Figure 1 (Middle GM). In summary, the above data confirm that stroke can induce abnormalities in the coordinated function of the motor-olfactory-cognitive network, among which olfactory circuit damage is a key node driving post-stroke neurological functional decline, providing direct experimental evidence for subsequent mechanism research and intervention strategy design.

[0038] The inclusion and exclusion criteria for patients with acute ischemic stroke (AIS) used in this invention are as follows:

[0039] Inclusion criteria: 1) Acute neurological deficit symptoms, clinical and imaging examinations confirming ischemic stroke, or meeting the diagnosis of TIA based on medical history; 2) Ability to cooperate in completing scale assessments, such as NIHSS, modified Rankin Scale, BI index, etc.

[0040] Exclusion criteria: 1) Brain MRI showing intracranial space-occupying lesions; 2) Unstable vital signs, severe impairment of consciousness, or multiple organ dysfunction during admission; 3) History of other neurological or psychiatric diseases; history of tumors; 4) History of surgery within the past three months; 5) Use of unfractionated heparin or low molecular weight heparin within the past month; recurrence of cerebral infarction or other neurological diseases during follow-up.

[0041] Example 2: VIP neurons have a survival advantage in the peri-infarct area.

[0042] To locate the core neuronal subpopulations of the motor-olfactory-cognitive neural circuit after stroke, this invention performed single-cell nuclear RNA sequencing (snRNA-seq) on the ischemic cortex of PT model mice, successfully resolving the transcriptome features of 19,926 high-quality cell nuclei from 20 mice. Figure 2 (A). Unsupervised clustering analysis was performed on all sequencing data. To further refine neuronal characteristics, secondary clustering was conducted on the nuclei of 16,264 neurons, identifying 16 neuronal subpopulations with significant differences at the transcriptome level based on classic marker genes. Figure 2 (B and C). To clarify the spatial heterogeneity of neuronal populations in the ischemic cortex, this invention employs spatial transcriptomics (ST) technology, combined with snRNA-seq data, to map the distribution of 16 neuronal subtypes in the ischemic cortex, providing important evidence for revealing region-specific transcriptomic abnormalities in the cortex after stroke. Figure 2 (D).

[0043] The classic cortical layering structure in the peri-infarct area was disrupted; quantitative analysis of the proportion of neuronal subsets in the peri-infarct area revealed a significant increase in the relative abundance of VIP neurons in the peri-infarct area. Figure 2 To quantify the number of VIP neurons, we injected VIP-Cre mice with AAV-DIO-EGFP virus to achieve Cre-dependent fluorescent labeling of VIP neurons (E). Figure 2 (F, G). Post-stroke testing results showed a significant reduction in the absolute number of VIP neurons (F, G). Figure 2 The H-value suggests that the increased relative abundance of VIP neurons is due to the greater loss of other neuronal subtypes, rather than the selective retention of VIP neurons.

[0044] Example 3: Decreased activity of VIP neurons in the peri-infarct motor cortex participates in mediating post-stroke olfactory and cognitive dysfunction.

[0045] The findings of Example 2 raise a key scientific question: Is the activity of VIP neurons similarly impaired after stroke, and can restoring their activity improve post-stroke brain function? Therefore, we used VIP-Cre mice to specifically express the calcium indicator GCaMP6f in VIP neurons of the MO region. Figure 3 (A). Quantitative analysis using two-photon calcium imaging showed that the PT model significantly reduced the peak spontaneous calcium transient in VIP neurons of the MO region ( Figure 3 (B and C) suggests that the activity of VIP neurons in the peri-infarct area is significantly inhibited.

[0046] To clarify the causal relationship between decreased VIP neuronal activity and post-stroke functional deficits, we microinjected AAV-DIO-ChR2-mCherry or the control virus AAV-DIO-mCherry into the MO region of VIP-Cre mice. Figure 3 (Middle D). Consistent with calcium imaging results, c-Fos expression in peri-infarct VIP neurons labeled with mCherry in PT mice was significantly lower than in the sham-operated group ( Figure 3 (E); while optogenetic stimulation (20Hz, 10ms pulse, 5mW / mm²) can effectively restore the activity of VIP neurons around the infarct, as evidenced by the restoration of c-Fos expression to normal levels ( Figure 3 (E).

[0047] We then conducted a comprehensive behavioral assessment of the functional recovery following VIP neuron activation. Figure 3 (F). Activation of VIP neurons around the infarct using wireless optogenetics significantly reduced the number of foot errors in the grid walking experiment. Figure 3 In the middle G), the bilateral limb symmetry was significantly restored in the cylinder test and adhesive removal test. Figure 3 The presence of H and I neurons suggests improved motor function. Notably, specific activation of VIP neurons surrounding the infarct can also significantly restore olfactory function. Figure 3 JM). Cognitive function assessment showed a more significant reparative effect: recovery of new object recognition ability (JM). Figure 3 (N), spatial learning ability is enhanced (escape latency during training period is shortened, Figure 3 (O), improved memory retention (shortened escape latency, increased number of target crossings, and increased time spent in the target quadrant in probe experiments), Figure 3 (P–S), and has no significant effect on swimming speed ( Figure 3 (China T).

[0048] In conclusion, restoring the activity of VIP neurons damaged in the peri-infarct motor cortex after stroke can simultaneously improve motor function recovery, olfactory discrimination ability, and cognitive function in stroke mice.

[0049] Example 4: Monosynaptic inhibitory projections exist between VIP neurons in the MO brain region and GABAergic neurons in the DP brain region.

[0050] To further investigate whether VIP neurons in the MO brain region directly project to olfactory or cognitive-related brain regions, we injected AAV-DIO-EGFP into the MO region of VIP-Cre mice for anterograde tracing. Figure 4 (A). The results showed that EGFP-positive nerve fibers were mainly distributed in the DP region of the olfactory cortex (A). Figure 4(B and C). Further retrograde tracing was performed by injecting AAV-Retro-DIO-mCherry into the DP region of VIP-Cre mice, combined with RNAscope labeling of VIP neurons. The results showed that mCherry-positive VIP neurons were detected in the MO region, confirming a direct projection of VIP neurons in the MO region to the DP region. Figure 4 (Middle D–F).

[0051] To clarify the synaptic characteristics of VIP neurons projecting from the MO region to the DP region, we injected AAV-DIO-ChR2-mCherry into the MO region of VIP-Cre mice and conducted photoelectrophysiological experiments using acute brain slices. Figure 4 (G). Photoactivation of ChR2-positive nerve endings can induce stable inhibitory postsynaptic currents (IPSCs) in DP neurons, with a mean latency of 4.75 ms. Figure 4 (H and I). This current can be completely blocked by picrotoxin, suggesting GABAergic inhibitory synaptic transmission ( Figure 4 (H and J). Furthermore, optogenetically induced IPSCs can be blocked by tetrodotoxin (TTX) and restored by 4-aminopyridine (4-AP), confirming monosynaptic connections (H and J). Figure 4 K and L). Notably, all DP neurons that recorded light-induced IPSCs were GABA-immunopositive and glutamate-immunonegative (K and L). Figure 4 The presence of M and N in the MO region indicates that VIP neurons in the MO region specifically form functional monosynaptic inhibitory connections with GABAergic neurons in the olfactory-related DP region.

[0052] In summary, the above results confirm that VIP neurons in the MO region selectively innervate GABAergic neurons in the DP region through monosynaptic inhibitory connections.

[0053] Example 5: Activating MO VIP -DP GABA The pathway improves post-stroke olfactory and cognitive dysfunction through deinhibition mechanisms.

[0054] We then investigated selective activation of MO. VIP -DP GABA Whether the pathway can alleviate post-stroke olfactory dysfunction (PSOD) and post-stroke cognitive impairment (PSCI) in photothrombotic (PT) mice. AAV-DIO-ChR2-mCherry was injected into the MO brain region of VIP-Cre mice, and an optical fiber was implanted above the DP region. Figure 5 (A). Using a wireless device to control the MO within the DP. VIP Photogenetic activation of the terminal buds can significantly improve the peanut butter aroma preference index. Figure 5 (B), enhances the avoidance reaction to 2-methylbutyric acid ( Figure 5(C), and shortens foraging time without changing average movement speed ( Figure 5 D, E), prompt MO VIP -DP loop can specifically mediate the recovery of olfactory function.

[0055] In cognitive behavioral tests, PT VIP-ChR2 The mice's ability to prefer new objects was restored. Figure 5 (Middle F), Morris water maze spatial learning ability significantly improved ( Figure 5 (G), and the memory retention ability in the probe experiment was significantly improved ( Figure 5 (H–L). The above results indicate that activating MO VIP-DP The pathway can simultaneously alleviate post-stroke olfactory dysfunction and cognitive impairment. Activation of MO VIP -DP GABA The functional improvement in both olfactory and cognitive deficits following the pathway intervention prompted us to further investigate whether this olfactory circuit projects to cognitively related brain regions. Given that glutamatergic pyramidal neurons are the main projection neurons within the DP (digestive tract), we first evaluated the MO (molarity disorder). VIP Whether activation modulates these pyramidal neurons through local GABAergic interneurons was investigated. AAV-DIO-ChR2-mCherry was injected into MOs of VIP-Cre mice, and AAV-CaMKIIα-EGFP was injected into DPs to label glutamatergic pyramidal neurons. Figure 5 (M).

[0056] on MO VIP Whole-cell patch-clamp recordings of DP-glutamatergic pyramidal neurons during peripheral light stimulation showed a significant decrease in the frequency of inhibitory postsynaptic currents (IPSCs). Figure 5 (medium N, O), prompt MO VIP Activation can induce deinhibition of DP pyramidal neurons through local GABAergic interneurons.

[0057] To identify the downstream target region of DP, we used anterograde nerve tracing technology to inject AAV-CaMKIIα-tdTomato into DP ( Figure 5 (P). Four weeks later, mCherry-positive nerve fibers were observed in the CA3 region of the hippocampus. Figure 5 The presence of β-D-type glutamatergic neurons (DP-D) suggests that DP-D-D neurons can project directly to CA3. Retrograde transsynaptic tracing using rabies virus (RV) further validated this connection. Figure 5 (R): Four days after RV was injected into the CA3 region, tdTomato-positive neurons were detected in the DP region. Figure 5 (S).

[0058] In summary, this invention confirms the existence of a direct excitatory pathway from DP glutamatergic neurons to the CA3 region of the hippocampus, and suggests that activation of MO... VIP Neurons can promote this downstream projection through disinhibition mechanisms.

[0059] Example 6: Olfactory training improves olfactory and cognitive impairment after stroke in PT mice by activating the DP-CA3 pathway.

[0060] Given the activation of MO VIP The olfactory circuit of the DP (diastolic disc) can alleviate olfactory and cognitive deficits after stroke by deinhibiting DP pyramidal neurons. We further investigated whether enriched olfactory stimuli could directly activate the DP and its downstream target area CA3 to promote functional recovery. Mice were subjected to olfactory training (OT) for 21 consecutive days using alternating scents of clove, lemon, rose, and eucalyptus. Figure 6 (A)

[0061] After training, we examined the intrinsic excitability of DP-glutamatergic pyramidal neurons. The results showed that olfactory training significantly restored the intrinsic excitability of damaged DP-glutamatergic pyramidal neurons in PT mice. Figure 6 (B, C). Furthermore, by quantitatively analyzing the levels of c-Fos-positive glutamatergic neurons in the DP and CA3 regions to assess neuronal activation status, it was found that stroke significantly reduces their number, and olfactory training can reverse this effect. Figure 6 D, E).

[0062] Behavioral results showed that PT mice had significant olfactory dysfunction. Figure 6 FG), while PT trained with olfactory sense OT The group of mice showed a significantly enhanced preference for the aroma of peanut butter. Figure 6 The avoidance response to 2-methylbutyric acid (2-MB) was significant (F). Figure 6 (Medium G) Foraging time is shortened, but exploration speed is not affected. Figure 6 The results (H, I) suggest that olfactory training can improve olfactory dysfunction after stroke.

[0063] In terms of cognition, olfactory training can restore the ability to prefer new objects. Figure 6 (J), to improve the spatial learning efficiency of Morris water maze ( Figure 6 (K), and improved memory retention in probe experiments; compared with untreated PT mice, PT OT The mice in the group traversed the platform more times and stayed in the target quadrant for a longer period of time. Figure 6 (Middle L–P).

[0064] The above results indicate that olfactory training can enhance the activation of DP glutamatergic neurons and regulate the DP-CA3 pathway, thereby promoting the repair of olfactory and cognitive dysfunction after stroke.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An olfactory training system for activating the motor-olfactory-hippocampal neural network, characterized in that, The system includes multiple different odor sources used to provide alternating olfactory stimulation to stroke patients in order to improve their cognitive and / or olfactory functions.

2. The system according to claim 1, characterized in that, The motor-olfactory-hippocampal neural network includes vasoactive intestinal peptide neurons in the motor cortex, γ-aminobutyric acid (GABA) neurons in the dorsal tegmental area (DTA), glutamatergic pyramidal neurons in the DTA, and neurons in the CA3 region of the hippocampus. The activation is achieved by activating vasoactive intestinal peptide neurons in the motor cortex surrounding the infarct through olfactory training. This, in turn, inhibits GABA neurons in the DTA via the inhibitory pathway from vasoactive intestinal peptide neurons in the motor cortex to GABA neurons in the DTA, thereby inhibiting glutamatergic pyramidal neurons in the DTA and enhancing their excitatory input to the CA3 region of the hippocampus.

3. The olfactory training system according to claim 1, characterized in that, The various scent sources include at least two scents selected from the group consisting of clove, lemon, rose, and eucalyptus; the scent sources are used to alternately provide the patient with different scents each day.

4. The olfactory training system according to claim 1, characterized in that, The system also includes a training program manual that describes an olfactory training program lasting at least 21 days.

5. The olfactory training system according to claim 1, characterized in that, The cognitive functions include the ability to recognize new objects and / or the ability to learn and retain spatial memories; the olfactory functions include the ability to recognize odor preferences and / or the ability to recognize odor aversions.

6. The use of the olfactory training system according to any one of claims 1-5 in the preparation of a device or medicament for activating the motor-olfactory-hippocampal neural network and / or improving post-stroke cognitive function and / or olfactory function.

7. The application according to claim 6, characterized in that, The activation of the motor-olfactory-hippocampal neural network and / or improvement of post-stroke cognitive and / or olfactory function are achieved by restoring the activity of vasoactive intestinal peptide neurons in the motor cortex surrounding the infarct, enhancing the intrinsic excitability of glutamatergic pyramidal neurons in the dorsal tegmental area, and / or increasing the number of c-Fos-positive neurons in the dorsal tegmental area and the CA3 region of the hippocampus.

8. The application according to claim 6, characterized in that, The improvement in cognitive function was assessed using the Montreal Cognitive Assessment score, and the improvement in olfactory function was assessed using the olfactory function score, with the olfactory score being positively correlated with the Montreal Cognitive Assessment score.

9. A method for screening candidate substances for activating motor-olfactory-hippocampal neural networks and / or improving post-stroke cognitive function and / or olfactory function, characterized in that, The method includes: detecting the effect of a candidate substance on the activity of vasoactive intestinal peptide neurons in the motor cortex, the activity of γ-aminobutyric acid (GABA) neurons in the dorsal tegmental area, the activity of glutamatergic pyramidal neurons in the dorsal tegmental area, or the activity of neurons in the CA3 region of the hippocampus; if the candidate substance can activate vasoactive intestinal peptide neurons, inhibit GABA neurons, de-inhibit glutamatergic pyramidal neurons, or enhance the activity of neurons in the CA3 region of the hippocampus, it indicates that the candidate substance can be used to activate the motor-olfactory-hippocampal neural network and / or improve post-stroke cognitive function and / or olfactory function.

10. The method according to claim 9, characterized in that, The detection includes: detecting c-Fos expression levels in the animal brain, changes in neuronal calcium signaling, and / or changes in intrinsic neuronal excitability.

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