Application of anterior piriform cortex gamma-aminobutyric acid neurons as analgesic target

Abstract

The application discloses application of anterior piriform cortex GABAergic neurons as an analgesic target point, and relates to the technical field of neuroscience and pain. GABA The application discloses that the neural loop (APC GABA →MD) from the anterior piriform cortex GABAergic neurons to the thalamic dorsal medial nucleus plays a key role in pain regulation. Experiments prove that the loop can not only relieve acute pain, but also has a significant analgesic effect on the clinical treatment of intractable chronic inflammatory pain and neuropathic pain models, and shows a wide treatment application prospect.

Description

Technical Field

[0001] This invention relates to the fields of neuroscience and pain technology, and in particular to the application of prepyriform cortex γ-aminobutyric acid-producing neurons as analgesic targets. Background Technology

[0002] Pain and fear are two states that animals often have to balance to survive. When faced with urgent threats such as predators, the brain prioritizes the fear response and temporarily suppresses pain perception to facilitate fighting or fleeing. Although conditioned fear analgesia and its neural mechanisms have been extensively studied, the precise neural circuit basis of how innate fears, which are more ecologically significant and triggered by predator odors, suppress pain remains unclear.

[0003] The piriform cortex (PC), as the primary olfactory cortex, plays a crucial role in processing olfactory information and innate fear responses. The anterior piriform cortex (APC), receiving dense olfactory input, is particularly important for odor recognition. The dorsomedial thalamus (MD) is an important olfactory thalamic nucleus that also participates in pain information processing. However, the existence of specific functional connections between the APC and MD, and their role in innate fear analgesia and basic pain gating modulation, has not been reported. Elucidating this pathway will not only contribute to understanding the mechanisms by which the brain prioritizes among competitive survival needs but also provide potential targets for developing new analgesic strategies, especially for chronic, intractable pain. Summary of the Invention

[0004] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide the application of anterior piriform cortex γ-aminobutyric acid (GABAergic) neurons as targets in the preparation of drugs for treating and / or preventing pain. This invention discloses a neural circuit from anterior piriform cortex GABAergic neurons to the dorsomedial nucleus of the thalamus (APC). GABA →MD plays a crucial role in pain modulation. Experiments have shown that this circuit not only relieves acute pain but also has significant analgesic effects on clinically challenging chronic inflammatory pain and neuropathic pain models, demonstrating broad therapeutic application prospects.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides anterior piriform cortex γ-aminobutyric acid-producing neurons (APCs). GABA The application of this as a target in the preparation of drugs for the treatment and / or prevention of pain.

[0006] In another aspect, the present invention provides a reagent for detecting the level of γ-aminobutyric acid (GABA) in the anterior piriform cortex in the screening of drugs for the treatment and / or prevention of pain.

[0007] In another aspect, the present invention also provides the use of an activator of prepyriform cortex γ-aminobutyric acid neurons in the preparation of drugs for treating and / or preventing pain.

[0008] In one embodiment, the activator includes a direct GABA receptor agonist, a GABA receptor allosteric modulator, and / or an indirect GABAergic signaling enhancer.

[0009] In one embodiment, the GABA receptor direct agonist includes natural terpenoids, isoflavones, substituted oxalool derivatives, and / or baclofen.

[0010] In one embodiment, the GABA receptor allosteric modulator includes valerenic acid derivatives, neolignans, and / or 1,2-diphenylimidazole derivatives.

[0011] In one embodiment, the GABAergic signaling indirect enhancer includes a GABA synthesis promoter, a GABA degradation inhibitor, and / or a GABA reuptake inhibitor.

[0012] In another aspect, the present invention also provides the use of prepyriform cortex γ-aminobutyric acid in the preparation of medicaments for the treatment and / or prevention of pain.

[0013] In one embodiment, the prepyriform cortex γ-aminobutyric acid further includes pharmaceutically acceptable salts, esters, hydrates, solvates, metabolites, prodrugs, stereoisomers, tautomers, polymorphs, and / or isotope derivatives thereof.

[0014] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) Therapeutic effective amount of anterior piriform cortex γ-aminobutyric acid; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0015] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0016] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0017] In one embodiment, the pharmaceutical product is a vial or box.

[0018] In another aspect, the present invention also provides the use of the above-described pharmaceutical compositions, pharmaceutical preparations and / or pharmaceutical products in the preparation of medicaments for the prevention and / or treatment of pain.

[0019] In one embodiment, the aforementioned pain includes acute thermal pain, acute mechanical pain, formalin-induced inflammatory pain, CFA-induced chronic inflammatory pain, and / or SNI-induced neuropathic pain.

[0020] (III) Beneficial Effects This invention provides the application of anterior piriform cortex γ-aminobutyric acid (GABAergic) neurons as analgesic targets. Compared with existing technologies, it has the following beneficial effects: 1. Broad-spectrum analgesic potential: Experiments have shown that this circuit can not only relieve acute pain, but also has a significant analgesic effect on clinically challenging chronic inflammatory pain and neuropathic pain models, demonstrating broad therapeutic application prospects.

[0021] 2. Clear mechanism and precise intervention: The function of inhibitory projection in this circuit and the intervention methods (such as chemogenetics and optogenetics) have been clarified. The intervention can act on this circuit with high specificity, avoid affecting other brain regions, and have few potential side effects.

[0022] 3. Providing new strategies for non-pharmacological analgesia: Based on the discovery of this circuit, olfactory-based sensory stimulation modulation can be developed as a new non-invasive or minimally invasive method of analgesia, such as developing odor molecules with specific patterns as analgesics. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A schematic diagram comparing the pain behavior of mice exposed to water, anisole, or 2MT; Figure 2 For APC GABA The results of the neuronal response to the specific 2MT response to innate fear of odor are shown in the following figures: A is a representative image of the co-localization of tdTomato-positive neurons with inhibitory GABAergic neurons or excitatory glutamatergic neurons in the APC of TRAP2::Ai14 mice, detected by fluorescence in situ hybridization; B is the statistical results of the co-localization of the above two types of neurons with tdTomato-positive neurons; C is a schematic diagram of viral injection and fiber optic implantation in Vgat-Cre mice; D is a comparison of the average response curves, heatmaps and areas under the curves of calcium signals induced by the application of water, anisole, or 2MT. Figure 3 Chemical genetic activation of APC GABASchematic diagrams of analgesic effects of neurons in various pain models: A is a schematic diagram of the C57 mouse virus injection site; B is a representative electrophysiological recording trajectory and statistical results of membrane potential changes of depolarization of neurons expressing hM3Dq after CNO administration; CE is the effect of chemically activated APC GABA neurons on basic nociceptive sensation, acute thermal pain, and chronic neuropathic pain under conditions without 2MT exposure. Figure 4 Schematic diagrams for structural and functional verification of the APCGABA→MD neural circuit: A is a schematic diagram of the C57 mouse virus injection site; B is a representative image of mCherry positive signals in MD after virus expression; C is a schematic diagram of electrophysiological recording of the MD brain region; D is an exemplary trajectory and statistical comparison of light-induced IPSCs recorded at MD under different pharmacological conditions; E is a schematic diagram of C57 mouse virus injection and fiber optic implantation; F is a quantitative result of the firing rate of representative trajectory of ChR2-expressing neurons after blue light stimulation; GI shows the effect of optogenetic activation of APCGABA nerve terminals in MD on basic nociceptive sensation, acute thermal pain, and chronic neuropathic pain in mice under conditions without 2MT exposure. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Terms and Definitions As used in this article, the term "anterior piriform cortex" (APC) is the primary olfactory cortex located at the anterior end of the medial temporal lobe of the cerebral hemisphere. It can directly receive neural projections from the olfactory bulb, complete the initial recognition and encoding of odor information, and provide a key initial signal basis for more complex processing such as odor discrimination and memory association in higher-level areas of the brain.

[0027] As used in this article, the term "mediolar thalamic nucleus" (MD) is the most prominent nuclear structure in the medial thalamic nucleus group. It is located between the internal medullary lamina and the periventricular gray matter, and has bidirectional fiber projections with the prefrontal cortex. It is mainly involved in emotion regulation, memory integration, decision-making, and regulation of prefrontal cortex function, and has a specific association with pain information processing.

[0028] As used in this article, the term "2-methyl-2-thiazoline" (2MT) is a volatile organic compound that is a structural analog of 2,4,5-trimethylthiazoline (TMT), a secretion from the anal glands of foxes. It can mimic the scent of predators and stably induce freezing behavior in rodents, and is often used in research on innate fears.

[0029] As used in this article, the term "AAV" (Adeno-Associated Virus) refers to adeno-associated virus, a single-stranded DNA virus belonging to the genus Dependent Virus in the family Parvoviridae. It has a non-enveloped icosahedral symmetry structure, a genome of approximately 4.7 kb, and is widely used as a gene delivery vector.

[0030] As used in this article, the term "Cre" (Cyclization Recombination Enzyme) is a site-specific recombinase derived from bacteriophage P1 that recognizes a specific DNA sequence, namely the loxP sequence (locus of crossover (x) in P1). When two loxP sequences are aligned in the same direction, the Cre recombinase can catalyze recombination of the DNA sequence between them.

[0031] As used in this article, the term "DIO" (Double-floxed Inverted Open Reading Frame) is an important element used in constructing viral vectors. The presence of a DIO enables gene expression in viral vectors within specific cell types. It achieves this function by interacting with a specific Cre recombinase system within the cell. Only when the cell expresses Cre recombinase will the gene sequence between the loxP sites flanking the DIO be inverted, thereby allowing the target gene (such as mCherry, ChR2-mCherry, etc.) to be expressed.

[0032] As used in this article, the term "ChR2" refers to a light-sensitive cation channel protein that was originally cloned from the single-celled green alga Chlamydomonas reinhardtii. After genetic engineering, its coding sequence is often fused with a reporter gene (such as mCherry) and constructed in a viral vector. It is a core excitatory tool protein in optogenetic research.

[0033] As used herein, the term "NMDG solution" refers to artificial cerebrospinal fluid supplemented with N-methyl-D-glucosamine. NMDG solution can be used to perfuse the heart of mice before sectioning their brain tissue, thereby protecting the brain tissue sections and eliminating interference from blood in the experiment.

[0034] As used herein, the term "HEPES solution" refers to artificial cerebrospinal fluid supplemented with 4-hydroxyethylpiperazine ethanesulfonic acid. HEPES solution can be used to fix brain tissue after sections have been prepared from mouse brain tissue.

[0035] As used in this article, the term "ACSF solution" refers to artificial cerebrospinal fluid, also known as cerebrospinal fluid simulation solution. ACSF solution can be used to place brain slices for ex vivo electrophysiological recording of brain slices.

[0036] As used in this article, the term "tetrodotoxin" (TTX) is a typical sodium channel blocker that inhibits nerve conduction by blocking the induced postsynaptic inhibitory current (IPSC).

[0037] As used in this article, the term "4-aminopyridine" (4-AP) is a classic potassium channel blocker that can reverse the blocking effect of TTX when the induced postsynaptic current has monosynaptic characteristics.

[0038] As used herein, the term "pharmaceutical composition" refers to a composition comprising prepyriform cortex γ-aminobutyric acid formulated with one or more pharmaceutically acceptable carriers.

[0039] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonade, aromatic water, powders, syrups, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0040] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0041] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0042] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0043] The active substance in the product disclosed in this invention accounts for 0.001-99.9 wt% of the total weight of the composition, with the remainder being pharmaceutically acceptable carriers and other additives.

[0044] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0045] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0046] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0047] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0048] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0049] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0051] Example 1: 2MT-induced broad-spectrum analgesia: Adult C57 mice were exposed to 2MT (an innate fear odor), anisole (a neutral odor), or a control solvent (water), and then subjected to a series of behavioral tests for pain. Figure 1 ).

[0052] von Frey mechanical pain test: For three days prior to measuring mechanical pain in mice, they were placed in a breathable plexiglass box within the pain testing frame for acclimatization, with each acclimatization period lasting 1 hour, allowing them to adapt to the surrounding environment and eliminate interference from irrelevant factors. On the day of the behavioral test, the mice were placed in the pain testing frame for 30 minutes of acclimatization beforehand. Then, a series of von Frey nylon monofilaments of varying intensities (the mechanical filament weight ranged from 0.02 to 2 g, with higher weights indicating higher stimulation intensity) were used to stimulate the mice's hind paws. During the experiment, the von Frey filaments were poked into the base of the mouse's hind paw, keeping the filament bent for approximately 3 seconds, and the mouse's reaction was observed. If the mouse rapidly retracted its paw, licked its paw, or shook its paw, it was considered to be experiencing pain. Each test started with 0.16 g of von Frey, and each intensity of von Frey filament was used to stimulate the mouse 5 times. If the mouse exhibited two paw-lifting or other observable pain responses within the 5 stimulations, it was considered to be experiencing pain. The intensity of the von Frey wire for the next test should be adjusted according to the mouse's response: if the mouse feels pain, the intensity should be reduced by one level; otherwise, the intensity should be increased by one level. The measurement interval between different intensities of von Frey wire should be no less than 5 minutes.

[0053] Hargreaves radiant heat experiment: For three days prior to measuring radiant heat pain in mice, the mice were placed in a breathable plexiglass box on the radiant heat instrument for approximately 30 minutes each day to acclimatize and eliminate interference from irrelevant factors. On the day of behavioral testing, the mice were allowed to acclimatize again for 1 hour in the same transparent plastic box on the radiant heat instrument. During the experiment, the radiant heat emitted by the instrument was focused on the mouse's hind paw. The radiant heat beam was focused on the mouse's hind paw five times during the experiment, and the latency of each paw lift was recorded. To ensure the accuracy of the experiment and the safety of the mice, the interval between two measurements was set to be no less than 5 minutes, and the maximum irradiation time of the radiant heat beam was set to 20 seconds to prevent potential damage to the mouse's paw tissue from prolonged irradiation. After the experiment, the maximum and minimum latency of each mouse's paw lift were removed, and the average of the three remaining recorded values ​​was calculated as an indicator for assessing the mouse's heat-sensitive pain.

[0054] Acute thermal pain assessment (hot plate test): Mice are placed on a 47°C hot plate, and the latency (time required to produce a response) and number of noxious responses (jumping and licking) are recorded. To avoid potential tissue damage, the duration of the 47°C hot plate test is limited to 3 minutes.

[0055] Acute mechanical pain assessment (Pinprick test): Gently stimulate the hind paw of a mouse 10 times with a blunt needle that will not pierce or damage the skin. There should be at least a 1-minute interval between each stimulation. Record the number of paw withdrawal responses during these 10 tests and calculate the percentage of paw withdrawal responses as: (number of paw lifts / total number of tests) × 100%.

[0056] Inflammatory pain assay (formalin test): Mice were placed in a square glass box for acclimatization for 10 minutes. Formalin solution was prepared and diluted to a 1% concentration with physiological saline. Then, 50 μl of the diluted solution was injected subcutaneously into the hind paw of each mouse. Immediately after injection, the mice's licking behavior was recorded using a camera. The number of licks within 1 hour was recorded. The experiment consisted of two phases: the first phase, the acute phase, comprised the initial 10 minutes; the second phase, the inflammatory phase, included the remaining 50 minutes.

[0057] Complete Frey adjuvant (CFA) model: To induce chronic inflammation, 10 μl of CFA was injected into the hind paw of mice. Seven days after injection, the von Frey test was used to determine the paw withdrawal response threshold, and the Hargreaves test was used to determine the paw withdrawal latency.

[0058] The results showed that, compared with the control groups of anisole and water, 2MT exposure significantly increased the mechanical withdrawal threshold in the von Frey test and the thermal withdrawal latency in the Hargreaves test in mice. Figure 1 A) The latency period of noxious response in the hot plate test (in the example A) Figure 1 (B in the text), and reduced the percentage of harmful responses in the Pinprick experiment ( Figure 1 (C in the text). Furthermore, 2MT can effectively relieve formalin-induced inflammatory pain (C in the text). Figure 1 D) CFA-induced chronic inflammatory pain ( Figure 1 In the E) and nociceptive behaviors in SNI-induced neuropathic pain models ( Figure 1 (F in the middle).

[0059] These results demonstrate that innate fear states can induce a powerful analgesic effect under both physiological and pathological conditions.

[0060] Example 2 APC GABA Specific responses to fearful odors: Using TRAP2::Ai14 mice, neurons activated during 2MT exposure were selectively labeled by intraperitoneal injection of 4-OHT, and the type of this neuronal population was determined by fluorescence in situ hybridization (FISH) technology.

[0061] Labeling of activity-dependent neurons induced by innate odor phobia: 4-hydroxytamoxifen (4-OHT) was dissolved in ethanol to prepare a stock solution of 20 mg / mL. The solution was shaken at 37°C for 15 minutes, aliquoted, and stored at -20°C. Before use, the aliquoted stock solution was reconstituted at 37°C with shaking for 15 minutes, followed by the addition of corn oil and mixing to a final concentration of 10 mg / mL for 4-OHT. Ethanol was removed by vacuum centrifugation before injection. To label neurons specifically activated by innate odor phobia 2MT, TRAP2::Ai14 mice were housed separately before the odor exposure experiment. On the day of the experiment, mice were exposed to filter paper soaked in 20 μL of 2MT, while the control group received 20 μL of H2O or anisole. 1.5 hours after odor exposure, mice were intraperitoneally injected with 4-OHT (50 mg / kg) and continued to be exposed to 2MT for another 1.5 hours. Brain tissue was harvested one week later.

[0062] Fluorescence in situ hybridization: On day 1, brain sections were washed for 5 minutes in DEPC-PBS, followed by 5 minutes in DEPC-PTW (PBS containing 0.1% Tween-20), and then incubated for 30 minutes in 2x SSC containing 0.5% Triton X-100. They were then washed twice in DEPC-PTW for 5 minutes each time, followed by incubation in 3% hydrogen peroxide-methanol for 10 minutes to block endogenous peroxidase activity, and then washed twice in DEPC-PBS for 5 minutes each time. Next, the tissues were incubated for 10 minutes in triethanolamine-HCl buffer (pH 8.0) containing 0.25% acetic anhydride (ribonuclease-free) for free amine acetylation, followed by three 5-minute washes in DEPC-PBS. Tissues were pre-hybridized for 2 hours at room temperature in pre-hybridization buffer (50% formamide, 25% 2x SSC, 0.3 mg / ml yeast RNA, 0.1 mg / ml heparin, 1x Denhardt's solution, 0.1% Tween-20, 5 mM EDTA), and then hybridized for 20 hours at 65°C in the same buffer with digoxigenin-labeled CaMKII antisense cRNA probes and GAD2 antisense cRNA probes.

[0063] On the second day, brain slices treated on the first day were washed for 30 minutes in pre-hybridization buffer (65°C), followed by washing for 30 minutes in a 1:1 mixture of pre-hybridization buffer and TBST (65°C). This was followed by two washes in TBST for 5 minutes each, a brief wash in a 1:1 mixture of TBST and TAE, and then three washes in TAE for 5 minutes each. Unbound probes were removed by electrophoresis at 60V on a 2% agarose gel for 2 hours. The tissue was then incubated overnight (36 hours) at 4°C with Anti-Digoxigenin-POD antibody diluted in PBT (containing 10% sheep serum).

[0064] On the third day, the brain slices processed on the second day were washed three times in TNT buffer for 10 minutes each time, and then incubated at room temperature for 20 minutes in anti-IF 488 antibody diluted in TBST containing 3% H2O2. Immunofluorescence staining was then performed using anti-Chk pAbto mCherry antibody and Goat pAb to Chk IgY antibody.

[0065] The results showed that 2MT specifically activated a large number of neurons in the APC brain region, and compared with the control group, the 2MT-treated group showed a higher concentration of GAD2 in neurons labeled with tdTomato (TRAP2::Ai14 mice are a genetically modified strain with a gene expressing the red fluorescent protein tdTomato inserted after the c-fos promoter, thereby labeling specifically activated neurons). + The proportion of CaMKII increased significantly, while the proportion of CaMKII increased significantly. + The proportion was not significantly different from that of the control group. Figure 2 (A~B in the diagram) indicates that 2MT recruited more APCs than neutral odors. GABA .

[0066] In vivo fiber optic photometric recordings further confirmed that APC GABA It exhibits a strong calcium signaling response to 2MT ( Figure 2 (C~D in the middle).

[0067] Example 3: Activating APC GABA Sufficient to simulate the analgesic effect of 2MT: Following the aforementioned stereotactic injection method, adeno-associated virus (AAV-VGAT-hM3D(Gq)-mCherry) or control virus (AAV-VGAT-mCherry) expressing excitatory hM3Dq receptors, regulated by GABAergic neuron promoters, was injected into the bilateral APCs of C57 mice. Three weeks after viral expression, a series of behavioral tests on pain were performed on both groups of mice in the absence of 2MT. Each behavioral test was conducted over two days: on the first day, mice were injected intraperitoneally with saline, and on the second day, CNO (2 mg / kg) was injected to activate the APCs. GABA .

[0068] The results showed that APC was activated. GABA It can induce fear-freezing behavior, increase the baseline nociceptive threshold, reduce the number of nociceptive responses in acute thermal pain, and significantly alleviate mechanical and thermal hyperalgesia in SNI mice, demonstrating activation of APC. GABA Sufficient to exert a powerful analgesic effect ( Figure 3 ).

[0069] Example 4 APC GABA -MD loop connection and functional verification: Following stereotactic brain injection, AAV-VGAT-mCherry was injected into the unilateral acupoints (APCs) of C57 mice to induce acupoint stimulation (APC) responses. GABA Downstream targets are traced anterogradely.

[0070] The results showed that APC GABA It can project to the MD brain region ( Figure 4 (A~B in the original text).

[0071] The nature of this loop connectivity was further verified by whole-cell patch-clamp recording on MD brain slices two weeks after APC injection of AAV-VGAT-ChR2 and viral expression.

[0072] The results showed that light stimulation of APC GABA The fiber terminals can induce inhibitory postsynaptic currents (IPSCs) in MD neurons. This light-induced response can be completely blocked by TTX. The current can be partially restored by the potassium channel blocker 4-AP. The restored response is subsequently blocked by the GABA receptor antagonist scutellarin (Bic), confirming its monosynaptic GABAergic connection. Figure 4 (CD in the middle).

[0073] Next, the adequacy of this circuit function was verified. AAV-VGAT-ChR2-mCherry or AAV-VGAT-mCherry virus was injected bilaterally into the APCs of C57 mice using stereotactic injection. Two weeks after viral expression, optical fibers were implanted above the bilateral MDs. One week later, a brain stimulation test was performed, applying blue light (473nm, 2.5mW, 10ms, 20Hz) to the MDs via the optical fibers, directly activating the APCs. GABA At the end of MD.

[0074] The results showed that light stimulation produced good analgesic effects in various pain tests, and a significant preference for the control side was observed in the CPP test, demonstrating activation of the APC. GABA →The MD loop is powerful enough to simulate the analgesic effect of innate fear of smells. Figure 4 (E~I in the middle).

[0075] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of prepyriform cortex γ-aminobutyric acid-producing neurons as targets in the preparation of drugs for the treatment and / or prevention of pain.

2. The use of reagents for detecting γ-aminobutyric acid levels in the pre-pyriform cortex in screening for medications used to treat and / or prevent pain.

3. Application of prepyriform cortex γ-aminobutyric acid neuron activators in the preparation of drugs for the treatment and / or prevention of pain.

4. The application according to claim 3, characterized in that, The activators include direct GABA receptor agonists, GABA receptor allosteric modulators, and / or indirect GABAergic signaling enhancers.

5. Application of prepyriform cortex γ-aminobutyric acid in the preparation of drugs for the treatment and / or prevention of pain.

6. The application according to claim 5, characterized in that, The prepyriform cortex γ-aminobutyric acid also includes its pharmaceutically acceptable salts, esters, hydrates, solvates, metabolites, prodrugs, stereoisomers, tautomers, polymorphs, and / or isotope derivatives.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (1) Therapeutic effective amount of anterior piriform cortex γ-aminobutyric acid; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

8. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition of claim 7.

9. A pharmaceutical product, characterized in that, Includes the pharmaceutical preparation described in claim 8.

10. The use of the pharmaceutical composition of claim 7, the pharmaceutical formulation of claim 8, and / or the pharmaceutical product of claim 9 in the preparation of a medicament for the prevention and / or treatment of pain.

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