CN112807435A

CN112807435A - Use of acid-sensitive ion channel regulator

Info

Publication number: CN112807435A
Application number: CN202110093180.2A
Authority: CN
Inventors: 徐天乐; 李伟广; 刘明刚; 王勤
Original assignee: Shanghai Jiaotong University School of Medicine
Current assignee: Shanghai Jiaotong University School of Medicine
Priority date: 2016-11-22
Filing date: 2016-11-22
Publication date: 2021-05-18
Also published as: CN108079300B; CN112618721A; CN112618722A; CN108079300A; CN112717135A

Abstract

The invention discloses an application of an acid-sensitive ion channel regulator in preparing a medicament for treating bulimia nervosa and obesity, wherein the acid-sensitive ion channel is an acid-sensitive ion channel containing ASIC1a subunits. The acid-sensitive ion channel regulator of the invention promotes the extinction of conditional memory by promoting the activity of the acid-sensitive ion channel containing ASIC1a subunit, provides a new medicine or a treatment target for treating or relieving nervous bulimia and obesity, and has very wide application prospect.

Description

Use of acid-sensitive ion channel regulator

The application is a divisional application of the invention with the application date of 2016, 11, and 22, the application number of 201611031313.9, and the name of the invention is 'application of acid-sensitive ion channel regulator'.

Technical Field

The invention relates to the technical field of medicines, in particular to a new application of an acid-sensitive ion channel regulator.

Background

The acid-sensing ion channel (ASIC) family contains six subtypes encoded by four genes, namely

ASICs

1a,1b,2a,2b,3 and 4, etc. These proton-gated channels are composed of three identical or different subunits, and ion channels formed by different combinations exhibit differential pH sensitivity, ion selectivity, and pharmacological properties. In the central nervous system, the channel containing ASIC1a was the most prominent ASIC member, and neurons with ASIC1a gene deletion failed to exhibit acid-induced currents. The importance of ASIC1a has been well studied in rodent models, such as ischemic neuronal death, chronic pain, seizure termination, and neurodegenerative diseases. Although neuronal expression of ASIC1a and its study of pathological significance have made significant progress, the physiological function of ASIC1a in brain neurons remains unclear.

Increasing evidence supports that ASIC1a plays a critical role in synaptic transmission and plasticity. Based on the post-synaptic positioning of ASIC1a, it is believed that ASIC1a may be activated by protons released by synaptic activity during normal synaptic transmission. ASIC1a channel, which is the receptor of protons functioning in synapses, plays a role in regulating hippocampal and amygdala high frequency stimulation or Theta Burst Stimulation (TBS) induced synaptic long-term potentiation (LTP) plasticity processes and thus contributes to the process of fear learning. ASIC1a promotes density of dendritic spines in hippocampal regions, but inhibits dendritic spine density in nucleus accumbens, alters the function of excitatory synaptic receptors, and limits cocaine-induced synaptic plasticity, which advances suggest that ASIC1a may play a more complex role in regulating synaptic plasticity and behavior adaptability.

The complexity of the ASIC1a function may be reflected in its participation in different forms of plasticity and behavioral modification in different brain regions. ASIC1a is highly expressed in islet cortex neurons, but its channel function is unclear. The islet cortex is involved in a number of cognitive and affective regulatory functions, including sensory integration, chronic pain, affective processing, and taste recognition memory. The best known function of this brain region is its role in taste learning. In particular, the islet cortex plays an important role in negative mood-driven taste learning known as Conditioned Taste Aversion (CTA). During the learning of conditional taste aversion, the learning subject associates a completely new taste (conditioned stimulus, CS) with the subsequent visceral discomfort (unconditioned stimulus, US), thereby creating an aversive emotional memory of this particular taste. At the synaptic level, long-term synaptic potentiation of the islet cortex contributes to the acquisition of conditioned taste aversion memory. However, the molecular and synaptic mechanisms that mediate the resolution of conditioned taste aversion memory are unclear.

Anorexia, also known as anorexia nervosa, is an abnormal eating behavior often seen in adolescent women, and is characterized in that the diet is intentionally limited to reduce the body weight to be obviously lower than the normal standard, so that people are often worried about getting fat, even if the people have obviously thinned, the people still think of being too fat, and even if the doctor explains the situation, the people are ineffective. There is still a lack of effective anorexia remedies.

Many people with drug addiction, tobacco addiction or alcohol addiction often suffer from the physical pain after drug withdrawal, smoking cessation or alcohol withdrawal and also re-take the drug, smoke or drink. Therefore, there is a great need for nervous system therapeutics for the successful abstinence of drugs or smoking or alcohol.

Anxiety is also a disease of the nervous system with a global incidence of 7.3% and an individual's prevalence of up to 11.6% throughout life. Anxiety patients often exhibit excessive fear, anxiety or excessive avoidance of perceived danger in certain settings, such as social or strange settings. These responses far exceed the normal fear or anxiety response caused by actual danger. The occurrence of anxiety can significantly reduce the quality of life of the patient and place a significant economic burden on the patient's family and the entire society. However, there is a lack of drugs currently available for treating or effectively alleviating anxiety disorders.

Disclosure of Invention

The invention aims to solve the technical problem of lack of medicaments for treating mental disorders such as anorexia, anxiety, drug addiction, tobacco addiction and the like at present, and provides application of an acid-sensitive ion channel regulator for treating or relieving mental disorders such as anorexia, anxiety, drug addiction, tobacco addiction and the like.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the invention, there is provided a use of a modulator of an acid sensitive ion channel comprising an ASIC1a subunit in the manufacture of a medicament for the treatment of a psychotic disorder.

The modulator comprises an acid-sensitive ion channel promoter or inhibitor.

The acid-sensitive ion channel enhancer includes any compound or recombinant vector that increases expression and/or activity of ASIC1a subunit protein. Preferably, the acid-sensitive ion channel enhancer compound comprises the polypeptide MitTx- α/β, and/or spermine.

The acid-sensitive ion channel inhibitor comprises any compound or recombinant vector which specifically inhibits the expression and/or activity of ASIC1a channel-related protein. Preferably, the acid sensitive ion channel inhibitor compound comprises the polypeptide PcTX1, amiloride, amidine A-317567, nafamostat mesilate, non-steroidal anti-inflammatory drugs (NSAIDs), compound 5b (2-oxo-2H-chromene-3-carboxamine derivative), a basic neutralizing agent, or a combination thereof. The basic neutralizing agent comprises sodium bicarbonate, ammonium chloride, an alkali metal salt, an alkaline earth metal salt, or a combination thereof.

The acid sensitive ion channel promoter is used for treating mental disorder diseases and comprises: anorexia, anxiety, addictive disorders, bulimia nervosa and obesity, and any other neurological disorder with abnormally intractable pathological memory. Wherein, the addiction diseases comprise drug addiction, tobacco addiction, wine addiction and the like.

By adopting the acid-sensitive ion channel promoter and assisting repeated regression training, the symptoms of patients with anorexia or addiction diseases can be obviously improved; the acid-sensitive ion channel promoter can be used for assisting exposure therapy, and can effectively treat anxiety.

The acid sensitive ion channel inhibitor is used for treating mental disorder diseases including Alzheimer disease and any other nervous system diseases accompanied with cognitive disorder and memory impairment.

By adopting the acid-sensitive ion channel inhibitor and assisting cognitive training, the symptoms of the Alzheimer disease patient can be improved.

In the present invention, the term "acid-sensing ion channels" (ASICs) refers to a class of cation-permeable protein complexes widely present on cell membranes, belonging to the epithelial sodium channel/degenerated protein superfamily, and having important roles in sensing the pH of body fluid and regulating multiple physiological functions such as pain sensation and mechanical sensation.

Molecular cloning has shown that ASIC has at least six subunits encoded by four genes (ASIC1a,1b,2a,2b,3 and 4) that form homotrimeric or heterotrimeric functional complexes. The diversity of ASIC subunit expression and distribution, as well as the variation of subunit composition and channel gating, allows for diversity in channel function.

ASIC1a has a distribution in both the central and peripheral nervous systems, primarily involved in synaptic transmission and plasticity. ASIC1a can be used for treating various neurological diseases such as ischemic cell death, epilepsy, and neurodegenerative diseases. The ASIC1a channel referred to herein refers to a homopolymer or a heteromer composed primarily of ASIC1a subunits.

In the present invention, the term "acid-sensitive ion channel regulating agent" refers to a substance capable of regulating an acid-sensitive ion channel, and the regulating agent may be a small molecule compound or a large molecule compound.

In the present invention, the modulator includes an enhancer (or agonist), or an inhibitor (or antagonist).

Representative acid-sensitive ion channel modulators include the polypeptides PcTX1, MitTx-alpha/beta, spermine, amiloride, amidine A-317567, nafamostat (nafamostat mesilate), non-steroidal anti-inflammatory drugs (NSAIDs), compound 5b (2-oxo-2H-chromene-3-carboxamidine derivative), and the like.

As used in the examples of the present invention, the polypeptide MitTx- α/β, a toxin derived from Texas coral snake, is capable of directly activating the ASIC1a channel.

As used in the present embodiments, the pennisetum toxin PcTX1, is effective in inhibiting ASIC1a channel.

In the present invention, the term "anorexia" refers to an eating disorder characterized by an individual intentionally making and maintaining a weight significantly lower than the normal standard by means of diet or the like, which is mainly characterized by strong fear of weight gain and obesity, extreme attention to weight and body type, significant weight loss with frequent malnutrition, metabolic and endocrine disorders, severe patients with cachexia due to extreme malnutrition, body failure or even life threatening, 5% to 15% of patients ultimately dying from cardiac complications, multiple organ failure, secondary infection or the like.

In the present invention, the term "anxiety disorder" means that a patient tends to exhibit excessive fear, anxiety or excessive avoidance of perceived danger in a particular environment (such as a social environment or a strange environment). These responses far exceed the normal fear or anxiety response caused by actual danger. Anxiety disorders include: post-traumatic stress disorder, dissociative anxiety disorder, selective mutism, specific phobia, social phobia, panic disorder, agoraphobia, generalized anxiety disorder, anxiety disorder associated with another disease, drug induced anxiety disorder, hypochondriasis, and the like.

In another aspect of the present invention, there is also provided a pharmaceutical composition for treating psychotic disorders, comprising a safe and effective amount of a modulator of an acid sensitive ion channel, which is an acid sensitive ion channel comprising the subunit of ASIC1a, and a pharmaceutically acceptable carrier.

The acceptable carrier is non-toxic, can be administered adjunctively, and does not adversely affect the therapeutic efficacy of the acid sensitive ion channel modulator. Such carriers can be any solid excipient, liquid excipient, semi-solid excipient, or in aerosol compositions, gaseous excipient, commonly available to those skilled in the art.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional methods in the pharmaceutical field. For example, the acid-sensitive ion channel modulating agent is mixed with one or more carriers and then formulated into a desired dosage form, such as tablets, pills, capsules, semisolids, powders, sustained release dosage forms, solutions, suspensions, formulations, aerosols, and the like.

In another aspect of the present invention, there is also provided a method for screening a candidate drug for treating a psychotic disorder, comprising the steps of:

detecting the influence of a compound to be detected on an acid sensitive ion channel containing ASIC1a subunits, and selecting the compound to be detected with a regulating and controlling effect on the acid sensitive ion channel as a primary screening compound;

and detecting the treatment or alleviation effect of the prescreened compound on the mental disorder disease, and selecting a compound with a curative effect as a candidate drug.

Preferably, the step of detecting to obtain a prescreened compound comprises: culturing the cells in the presence of a test compound, detecting the expression quantity, activity and/or ASIC-like current of the acid-sensitive ion channel 1a protein in the cells, and detecting the expression quantity, activity and/or ASIC-like current of the acid-sensitive ion channel 1a protein in the same cells in a control group which does not have the test compound and has the same other conditions, and comparing; if the detection value Vt of the experimental group is obviously higher or obviously lower than the detection value Vc of the control group, the compound to be detected is the compound to be detected which has the regulation and control function on the acid-sensitive ion channel.

The acid-sensitive ion channel regulator of the invention can promote or inhibit the regression of conditioned memory by promoting or inhibiting the activity of the acid-sensitive ion channel containing ASIC1a subunit, thereby providing a new medicine or a new treatment target for treating or relieving mental disorder diseases such as anorexia, anxiety, drug addiction, tobacco addiction and the like, and having very wide application prospect.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a graph showing the results of expression of ASIC1a in the cortex of mouse islet of wild type and ASIC1a knockout mice according to example 1 of the present invention;

FIG. 2 is a graph of the experimental results necessary for ASIC1a of example 2 of the present invention to induce long-term synaptic inhibition (LTD) induced by low frequency stimulation;

FIG. 3 is a graph showing the results of experiments conducted in example 3 of the present invention to block low frequency stimulation-long term synaptic inhibition when wild-type mice interfere with the dynamic change of extracellular pH;

FIG. 4 is a graph showing the results of the experiment for rescuing low frequency stimulation-long-term synaptic inhibition in ASIC1a knockout mice by re-expressing ASIC1a in example 4 of the present invention;

FIG. 5 is a graph of the experimental results necessary for the long-term synaptic inhibition induced by DHPG in the islet cortex of ASIC1a of example 5 of the present invention;

FIG. 6 is a graph of experimental results that ASIC1a of example 6 did not contribute to long-term synaptic potentiation (LTP) induction in the cortex of the island;

FIG. 7 is a graph of the results of experiments in which the selectivity of islet cortical long-term synaptic inhibition is counteracted by a conditioned taste aversion resolution process in example 7 of the present invention;

FIG. 8 is a graph of the experimental results of conditioned taste aversion memory regression with deletion selective impairment ASIC1a of example 8 of the present invention;

FIG. 9 is a graph of the experimental results of the functional ASIC1a of the islet cortex of example 9 of the present invention having a key effect on conditioned taste aversion resolution;

FIG. 10 is a graph of the results of experiments in which long-term synaptic inhibition of the islet cortex is required for conditioned taste aversion resolution in example 10 of the present invention;

fig. 11 is a graph showing the results of experiments in which pharmacological blockade or activation of the ventral hippocampus ASIC1a inhibits and promotes fear regression, respectively, in example 11 of the present invention;

fig. 12 is a graph of experimental results of conditional knockout or overexpression of the ventral hippocampus ASIC1a to inhibit and promote fear regression, respectively, in example 12 of the invention.

Detailed Description

The invention utilizes a multi-electrode array brain slice recording means to identify that ASIC1a plays a crucial role in island cortex long-term synaptic depression (LTD) plasticity, and utilizes a behavioral determination means to analyze the contribution of the LTD in conditioned taste aversion learning and memory regression. Unlike the known contribution of ASIC1a to long-term synaptic potentiation (LTP) in other brain regions and thus to promote joint learning and memory, ASIC1a in the present invention is a key regulator of long-term synaptic depression (LTD) in the islet cortex, a mechanism that is important for the regression of established taste aversion memory.

General methods and materials

1. Animal(s) production

Animal care and experimental procedures were approved by the animal ethics committee of the medical college of shanghai university of transportation. To reduce variability in the experiment, age-matched littermates were used for the experiments. All behavioral assays were performed on conscious, free-moving, male, 2-3 month old, C57BL/6J background mice. Conventional ASIC1a full-body knockout mice are a gift from professor Michael j.welsh, University of Iowa, loving; conditional ASIC1a knockout mice (ASIC1 a)^flox/flox) Provided by professor Cheng-Chang Lien, university of taiwan yangming, china; a tool mouse (CaMKII-Cre) highly expressing Cre recombinase in forebrain excitatory neurons was given by professor Joe Z.Tsien, Georgia Regents University, USA. Adult C57BL/6J mice were purchased from Shanghai Spikel. Animals are kept in a 12-hour period of day-night alternating environment (illumination time: 7: 00-19: 00), the temperature of the rearing room is constant at 21 ℃, the humidity is maintained at 50-60%, and the animals can freely eat drinking water during non-experimental period. Animals were randomly selected for double-blind experiments. In all behavioral experiments, the experimenters transferred mice to the behavioral feeding room at least two weeks earlierAnd (3) breeding, namely placing the mouse in a behavior detection chamber for adapting to the environment 1 hour before the experiment so as to fully reduce the influence of environmental change on the behavior result. All behavioral tests were performed and evaluated under conditions that were double-blind to genotype or drug treatment.

2. Model for conditioned taste aversion regression

The construction of the conditioned taste aversion memory model is to carry out timed drinking continuous training on male mice for 6-8 days so as to adapt to drinking at the same time point every morning. On the day of establishment of conditioned taste aversion memory, mice were given a 0.5% saccharin sodium solution instead of water for 30 minutes, and 40 minutes after cessation of drinking, experimental mice were injected intraperitoneally with lithium chloride (LiCl, 0.3 mol/l). Wherein the saccharin sodium solution is used for conditional stimulation, and the LiCl injected into abdominal cavity is used for unconditional stimulation. The intensity of taste aversion memory is indicated by an aversion index (aversive index). The aversion index is water intake/(water intake + sugar intake) × 100%. After the conditioned taste aversion memory is established, the learning model for taste aversion fading is obtained through repeated tests.

3. Animal models of anxiety and behavioral paradigms associated with anxiety

The most common animal model for studying fear behavior is fear conditioning, which involves repeatedly coupling a harmless stimulus (conditioned stimuli, CS) to a noxious stimulus (unconditioned stimuli, US) to produce a fear response (CR) to CS, which is manifested as a physical and autonomic response, including a rigidity response (calming), a heart beat acceleration, a blood pressure rise, analgesia, etc. Thereafter, if CS alone is repeatedly administered to the animal without US, the animal's conditioned fear response to CS gradually decreases, a process called fear regression (fear extinction).

The behavioral assays of the invention were performed on conscious, freely moving C57BL/6J mice or ASIC1a Flox transgenic mice (both 2-3 months of age, C57BL/6J genetic background). The animal model of anxiety employed was a clue-dependent fear conditioned reflex. Animals were palpated for three days and adapted to the experimental cages before the start of the experiment. The formal experiment was completed within three consecutive days: on the first day, animals are subjected to fear study in an environment A (one side of an experimental cage is transparent, the other three sides of the experimental cage are pasted with wallpaper with black and white lattices at intervals, the bottom of the experimental cage is provided with a conductive iron fence, and 75% alcohol is sprayed), in the experiment, the animals are firstly adaptive to the experimental cage for 3 minutes, then the animals are subjected to 30 seconds, 76 decibels and 4000 hertz sounds (harmless stimulation or conditional stimulation, CS) and 0.5 milliampere foot shock (harmful stimulation or unconditional stimulation, US) is given to the animals 2 seconds before the sounds are finished, 5 times of combined sounds and foot shocks are given totally, and the time interval between each sound is 20-120 seconds; in the next day, animals are allowed to fade and learn in an environment B (one side of the experimental cage is transparent, the other three sides are pasted with gray wallpaper, a smooth plastic plate is laid at the bottom, 4% acetic acid is sprayed), and CS is continuously given to the animals for 20 times in the experiment without US; animals were given CS 8 times in B environment on the third day and animals were tested for fear response to clues after withdrawal from learning. The fear response of animals to CS was reflected in three-day experiments by measuring the percentage of time that the animals were immobilized during CS (fleezing). Evaluation of fear response was achieved by analyzing the percentage of time that the animal was immobilized (fleezing). The behavioral experiments in the examples were performed and evaluated under conditions that were double-blind for genotype or drug treatment.

4. Brain slice preparation and patch clamp recordings

The references (Kim, J.I.et al. (2011). PI3Kgamma is required for NMDA receptor-dependent Long-term expression and behavioral flexibility. Nat Neurosci 14, 1447. 1454. Liu, M.G.et al. (2013b) Long-term expression of synthetic transmission in the expression vector in the visual expression Eur J Neurosci 38, 3128. Qia, S.I.et al. (2013) An initial in synthetic receptors in the expression vector in the mouse, Sigra 34. the expression vector in the mouse.

5. Research of synaptic plasticity using multi-electrode array recording and analyzing system

In order to research the synaptic plasticity of the islet leaf cortex of the adult mouse, a planar microelectrode array recording technology (MED64 multichannel recording system) is adopted to carry out multichannel excitatory postsynaptic field potential (fEPSP) recording and analysis on the acutely separated islet leaf. Wild type or mutant mice were used with a background of male C57/BL6J at 7-10 weeks of age. After anesthesia and decapitation, the brain was rapidly removed and placed in oxygenated frozen artificial cerebrospinal fluid for 1-2 minutes. The brain tissue blocks were then transferred to a frozen microtome and 2-3 islets of 300 μm were cut. After incubating the cut islets in artificial cerebrospinal fluid at 26-30 ℃ for 2 hours, one islet leaf was selected and placed under a microscope on the MED64 electrode and moved to the designated position, allowing the 8 x 8 array of electrodes to cover the entire islet cortex as completely as possible. The system noise was adjusted and the mobilus operating software was turned on to begin baseline recording. After 20-30 minutes from stabilization of the isobaseline response, brain slices were given different stimulation patterns to induce different types of synaptic plasticity, including LTP and LTD. LTP or LTD may be recorded for 1-3 hours, depending on the experimental requirements. The offline analysis of data collected by the MED64 system mainly used mobilus software to emphasize the time course change of the recorded slope of fEPSP before and after different synaptic plasticity induction parameters were given.

6. Data analysis

All results are expressed as mean ± sem. Statistical comparisons were performed using either unpaired or paired Student's t test or two-way repeated measures of variance analysis. Among them, P <0.05, P <0.01, and P <0.001 were considered to be significantly different.

Example 1 ASIC1a was significantly expressed in mouse islet cortex

The expression of ASIC1a in the cortex of adult mouse island leaf was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting (Western blotting) experiments. mRNA for ASIC1a subunit was most abundant on wild-type island cortex compared to other ASIC subunits, but was completely absent on ASIC1a knockout mice (fig. 1a, b). At the protein level, ASIC1a was abundantly expressed in the islet cortex of wild type mice, comparable to the prefrontal cortex and amygdala, significantly higher than the hippocampus, but completely absent in ASIC1a knockout mice (fig. 1 c). These data demonstrate that ASIC1a has significant expression in the islet cortex.

In FIG. 1, (a) RT-PCR showed expression of different ASIC subunits in wild type and ASIC1a knockout mouse islet cortex tissues. (b) Quantitative statistics of mRNA levels revealed by real-time quantitative reverse transcription PCR. n is 3. N.s. no statistical difference when comparing the Wild Type (WT) and ASIC1a gene knock-out (ASIC1a KO) groups using the unpaired Student's t-test;^***P<0.001, when compared using the unpaired Student's t-test, showed significant statistical differences between the Wild Type (WT) and ASIC1a gene knock-outs (ASIC1a KO).^###P<0.001, when compared to the two groups using the unpaired Student's t-test, showed significant statistical differences between the other ASIC subunits and ASIC1a groups, respectively. (c) ASIC1a protein levels were compared in the islet cortex and other brain regions. (upper), representative immunoblot pictures of ASIC1a protein at the prefrontal lobe, hippocampus, amygdala and islet cortex. (lower), statistical analysis of the relative amount of ASIC1a protein. Data are shown as relative optical density ratios, normalized to hippocampal data. n is 4.^*P<0.05，^**P<0.01, significant statistical differences were shown when each brain region was compared to the hippocampus using the paired Student's t-test.

Example 2 ASIC1a is necessary for Low frequency stimulus induced Long-term synaptic inhibition (LTD)

A64-channel multi-electrode array recording system is adopted to analyze various synaptic plasticity models. In which a 64-channel multi-electrode array was placed on an island cortical brain sheet as shown in figures 2a, b. Synaptic responses were recorded with multiple electrodes covering the entire area by stimulating the deep regions (layers V-VI, as shown in fig. 2 b) of the island leaf on the mouth side (rostral) close to the corpus callosum level. Injection of current at the stimulation site causes about 10 channels in a 64-channel electrode array to exhibit significant field excitatory postsynaptic potentials (fEPSPs), which are defined as activated channels. Consistent with past reports (Liu, M.G.et al. (2013b). Long-term suppression of synthetic transmission in the additive mouse reactor core in vitro. Eur J Neurosci 38, 3128-. This low frequency stimulation-long-term synaptic inhibition can be inhibited by an antagonist of the NMDA receptor, D-AP5, and is therefore also referred to as NMDA receptor-dependent long-term synaptic inhibition. The ratio of the number of channels positive for long-term synaptic inhibition to all channels of activation, defined as the induction rate, was 70.5 ± 4.9% in wild-type mice (fig. 2d), but decreased to 20.9 ± 5.7% in ASIC1a knockout mice (P ═ 1.156E-05, ASIC1a KO vs. wt, fig. 2 d).

To further establish the role of ASIC1a in island leaf long-term synaptic inhibition, a conditional ASIC1a knockout mouse (ASIC1 a) was used^flox/flox) (Wu, P.Y.et al. (2013), Acid-sensing ion channel-1a is not required for normal hippopal LTP and spatial memory. J Neurosci 33(5), 1828-1832). One month before brain slice recording, adeno-associated virus (AAV) carrying Cre recombinase or control green fluorescent protein was injected bilaterally into the islet cortex. Immunoblotting showed that Cre virus injection significantly reduced the expression of ASIC1a protein compared to control virus. The induction rate of long-term synaptic inhibition decreased from 80.0 ± 4.3% of the control virus injection to 15.9 ± 5.5% of the Cre virus injection (P ═ 2.177E-06vs. aav-Ctrl, fig. 2E, f).

The effect of the pharmacological inhibition ASIC1a channel on low frequency stimulation-long term synaptic inhibition was next examined. ASIC1a homopolymer and ASIC1a/2b heteromeric channel inhibitor, pennisetum toxin (Psalmotoxin 1, pctx1,100 nmol/liter), were administered starting 15 minutes prior to low frequency stimulation until the entire low frequency stimulation process was completed. Heat inactivated pennisetum toxin (PcTX1) was used as a control. As a result, neither the spilotoxin nor heat-inactivated spilotoxin was found to have an effect on basal excitatory synaptic transmission (fig. 2 g); however, the induction rate of long-term synaptic inhibition of the islet cortex after PcTX1 administration was reduced to 17.9 ± 9.3%, whereas the induction rate of long-term synaptic inhibition of heat-inactivated PcTX 1-treated brain slices was 72.4 ± 8.4% (P ═ 0.0018, fig. 2 h). These results confirm the critical role of ASIC1a in islet cortex long-term synaptic depression, and also rule out the possibility that ASIC1a knockout mice exhibit long-term synaptic depression-inducing abnormalities resulting from developmental compensation.

In fig. 2, (a) (left) multi-electrode array recording electrode corresponds to a position schematic diagram of an island cortex coronal slice; the position arrangement of the (right) multi-electrode array recording electrodes is (8 × 8). (b) The multi-electrode array records a photo-mirror photograph of the electrodes on an island cortical brain slice, with the illustration also labeled with different hierarchical information (from layer I to layer V-VI). Wherein the red dots are the stimulation sites. (c, e, g) time-course dependent changes in the slope of the excitatory postsynaptic potential of the island cortex field caused by low frequency stimulation (1 Hz, 900 pulse stimulations) under the different conditions illustrated. Illustration is shown: a representative field excitatory post-synaptic potential trace is shown at time points. Scale bar: the ordinate is 100 microvolts and the abscissa is 10 milliseconds. (d, f, h) a summary of the number of channels activated per islet cortical brain slice and the number of channels exhibiting long-term synaptic inhibition. N.s., no statistical difference was observed when comparing the two groups using unpaired Student's t-test;^*P<0.05，^***P<0.001, comparing the two groups using the unpaired Student's t-test, showed that the two groups exhibited significant statistical differences. (d) 7-9 brain slices (7-8 mice); (f) n-6-7 brain slices (4 mice per group); (h) n-5-6 brain slices (3-5 mice).

Example 3 the pH dynamics Process is necessary for Low frequency stimulus-induced Long-term synaptic inhibition (LTD)

To examine the effect of the dynamic course of pH changes by which ASIC1a is activated on the induction of long-term synaptic inhibition, the pH buffering capacity of extracellular fluid during induction of low frequency stimulation was enhanced, which was shown in the past study (Kreple c.j.et al (2014.) Acid-sensing on channels to synthetic transmission and inhibition co-expressed pathology. Nat Neurosci 17(8), 1083. sup. 1091) to reduce activation of neuronal ASICs. This treatment significantly reduced the probability of induction of long-term synaptic inhibition by low-frequency stimulation in wild-type mice (fig. 3). These results demonstrate that ASIC1a modulates the rate of induction of long-term synaptic inhibition by sensing changes in extracellular pH.

In FIG. 3, by increasing bicarbonate ion (HCO) in the extracellular solution₃ ^–At a concentration of 70 millimoles per liter (mM); conventionally 25mM) and CO₂At a concentration of 15%, conventionally 5%, to enhance the pH buffering capacity, the inducibility of long-term synaptic inhibition of the island cortex was blocked. The time course of the slope of the field excitatory postsynaptic potential induced by the low frequency stimulus under the treatment conditions of enhanced pH buffering capacity changes dependently. n-56 activation channels (7 brain slices, 6 wild-type mice).

Example 4 Re-expression of ASIC1a in ASIC1a Gene knockout mice rescues Low frequency stimulation-Long term synaptic inhibition

To test whether restoring expression of ASIC1a on the islet cortex of ASIC1a knockout mice could rescue the induction of long-term synaptic inhibition, AAV viruses were prepared that contained both green fluorescent protein and mouse ASIC1a gene sequences, linked between these two functional protein sequences with a "self-splicing" 2A polypeptide sequence, which was used to overexpress ASIC1 a. In addition, AAV viruses containing only green fluorescent protein were also prepared as a control. To achieve neuron-specific expression, both control virus and ASIC1a overexpressing virus were expressed on neurons driven by the human synapsin i (human synapsin i) promoter. Control or ASIC1a overexpression virus was injected separately into the islet cortex 1 month before brain slice recording (FIGS. 4 a-c). As a result, it was found that the injection of ASIC1a over-expressing virus was able to successfully rescue the low frequency stimulation-long term synaptic inhibition (fig. 4d), while the control virus did not have a similar effect. Wherein, after the island cortex is injected with the control virus, the inductivity of the long-term synaptic inhibition is 14.9 +/-6.3%; whereas, the induction of long-term synaptic inhibition was 69.9 ± 5.4% (P ═ 2.513E-05vs. aav-Ctrl, fig. 4E) after ASIC1a overexpression. Furthermore, overexpression of a mutant virus of ASIC1a, AAV-ASIC1a-HIF (i.e., all histidine-isoleucine-phenylalanine residues at positions 32 to 34 were mutated to alanine, fig. 4a), which did not have ion permeability, failed to rescue the abnormality of long-term synaptic inhibition in ASIC1a knockout mice (fig. 4d), with an induction rate of 24.0 ± 8.2%, which was not significantly different from the group injected with the control virus (P ═ 0.3976, fig. 4 e). These results support the key contribution of ASIC1a and its ion permeability to island cortex long-term synaptic inhibition, while also further excluding the contribution of developmental stage compensation to explain the impaired long-term synaptic inhibition in ASIC1a gene-deficient mice.

In FIG. 4, (a) schematic diagram of the AAV vector. hsynapsin I, which represents the human synapsin I promoter, is mainly used to drive gene expression selectively in neurons. (b) AAV-mediated expression of fluorescent proteins in the islet cortex (left). The brain slices were negatively stained with DAPI. For a clearer presentation, a schematic diagram of the brain atlas at the corresponding position is placed on the right side. (c) High magnification pictures of fluorescent protein expressed at the level of individual neurons of the islet cortex. (d) Under the different conditions illustrated, the time course of the island cortex field excitatory postsynaptic potential slope changes with low frequency stimulation (1 hz, 900 pulse stimulations). Illustration is shown: a representative field excitatory post-synaptic potential trace is shown at time points. Scale bar: the ordinate is 100 microvolts and the abscissa is 10 milliseconds. (e) A summary of the number of channels activated per islet cortical brain sheet and the number of channels exhibiting long-term synaptic inhibition. N.s., no statistical difference was observed when comparing the two groups using unpaired Student's t-test;^*P<0.05，^***P<0.001, comparing the two groups using the unpaired Student's t-test, showed that the two groups exhibited significant statistical differences. n-6-8 brain slices (4-8 mice).

Example 5 ASIC1a is essential for Long-term synaptic inhibition induced by island leaf cortex DHPG

To investigate the role of ASIC1a in another form of synaptic depression plasticity, an agonist of the metabotropic glutamate receptor type I, 3,5-dihydroxyphenylglycine (DHPG, 100 μm/l), was administered, and DHPG induced synaptic depression insensitive to NMDA receptor inhibitors and therefore also referred to as NMDA receptor independent long-term synaptic depression. Significantly, DHPG-induced long-term synaptic inhibition was also deficient in ASIC1a gene-deficient animals (fig. 5 a). In wild animals, the long-term synaptic inhibition inductivity induced by DHPG is 82.2 +/-5.6%; in ASIC1a knockout animals, the induction rate of DHPG-induced long-term synaptic inhibition was 20.4 ± 9.2%, which is significantly different from that of wild-type animals (P ═ 0.0004, fig. 5 b). Importantly, re-expression of ASIC1a by AAV virus on ASIC1a knockout mice was able to restore DHPG-induced long-term synaptic inhibition. Among them, the induction rate of DHPG-long-term synaptic inhibition was 15.3 ± 9.4% and the induction rate of overexpression ASIC1a virus was 82.2 ± 5.8% after the control virus injection, which were significantly different from each other (P ═ 0.0001, fig. 5c, d). Therefore, ASIC1a is necessary for the induction of both NMDA receptor-dependent and NMDA receptor-independent long-term synaptic depression in mouse islet cortex.

In fig. 5, (a, c) under the conditions shown, DHPG (100 μ M,20min, a, c) caused a time-course dependent change in the island cortex field excitatory postsynaptic potential slope. Illustration is shown: a representative field excitatory post-synaptic potential trace is shown at time points. Scale bar: the ordinate is 100 microvolts and the abscissa is 10 milliseconds. (b, d) number of channels activated per islet cortical brain slice and summarised with the number of channels exhibiting long-term synaptic inhibition (b, d). N.s., no statistical difference was observed when comparing the two groups using unpaired Student's t-test;^*P<0.05，^**P<0.01, comparison of the two groups using the unpaired Student's t-test showed significant statistical differences. (b) n-5 brain slices (4 mice per group); (d) 5-6 brain slices (5-6 mice).

Example 6 ASIC1a did not contribute to the induction of long-term synaptic potentiation in the cortex of an island

The effect of ASIC1a gene knock-out or inhibition on synaptic plasticity of another form of the islet cortex was examined. Theta Burst Stimulation (TBS) on wild-type island cortical brain slices was able to induce a rapid and persistent enhancement of synaptic responses at multiple sites (fig. 6a), long-term synaptic potentiation (LTP). However, theta-string stimulation induced similar levels of long-term synaptic enhancement on brain slices of wild-type and ASIC1a knockout mice (fig. 6a), with induction rates of long-term synaptic enhancement of 75.5 ± 7.4% on wild-type mice and 71.2 ± 6.3% on ASIC1a knockout mice, with no significant difference between the two (P0.6711, fig. 6 b). In addition, ASIC1a inhibitor PcTX1 did not affect long-term synaptic potentiation by wild-type mouse theta string stimulation (fig. 6c), with induction of 60.9 ± 6.6% (fig. 6d) and no difference compared to untreated wild-type brain slices (P0.1822). These results indicate that ASIC1a is unlikely to participate in theta-string stimulus-induced long-term synaptic potentiation, in stark contrast to the aforementioned significant contribution of ASIC1a in long-term synaptic depression.

In FIG. 6, (a, c) under the conditions shown, the time-course dependent change in the slope of the excitatory postsynaptic potential of the island cortex field caused by the theta-series stimulation (10 series stimulation at a frequency of 5 Hz, each series comprising 4 pulse stimulation at a frequency of 100 Hz, the theta-burst stimulation, TBS, a, c). Illustration is shown: a representative field excitatory post-synaptic potential trace is shown at time points. Scale bar: the ordinate is 100 microvolts and the abscissa is 10 milliseconds. (b, d) number of channels activated per islet cortical brain slice and summarised of the number of channels exhibiting long-term synaptic potentiation (b, d). N.s., no statistical difference was observed when comparing the two groups using unpaired Student's t-test;^*P<0.05，^**P<0.01, comparison of the two groups using the unpaired Student's t-test showed significant statistical differences. (b) n-6 brain slices (6 mice per group); (d) 5-6 brain slices (5-6 mice). Where the control data in panel (d) is derived from panel (b), the repeated display is for better comparison.

Example 7 island cortex Long time-course synaptic inhibition selectivity is counteracted by the conditioned taste aversion resolution Process

To induce conditioned taste aversion, mice were given sugar water as Conditioned Stimulus (CS) followed by intraperitoneal injection of LiCl to induce abdominal pain in mice as Unconditioned Stimulus (US). The establishment of conditioned taste aversion was evaluated by calculating an aversion index [ water intake/(water intake + sugar intake) × 100% ] based on a comparison of the amounts of water and sugar consumed, respectively, in the following days after the mice underwent the conditioned taste aversion training. With this procedure, most animals received between 80-90% aversion index after training. To completely resolve the conditioned taste aversion, these animals were allowed only sugar water for the next 7 days after training, while control animals were allowed only water to maintain aversive memory to a particular taste (sugar water) (fig. 7 a). As shown in fig. 7b, the conditioned taste aversion control mice maintained a higher aversion index, however the aversion index of the fully resolved animals had decreased significantly, indicating that repeated consumption of sugar water without re-coupling with LiCl injection would result in resolution of conditioned taste aversion.

By analyzing the islet cortical brain slices of the mice, it was found that the conditioned taste aversion control animals exhibited normal long-term synaptic inhibition, wherein the induction of low frequency stimulation-long-term synaptic inhibition (fig. 7c) was 68.5 ± 7.5% (fig. 7 d); the induction rate of DHPG-long-term synaptic inhibition (FIG. 7e) was 80.3. + -. 2.6% (FIG. 7 f). These two values were not different from the data obtained on naive mice (fig. 2d and 5b), indicating that island cortex long-time synaptic inhibition was not involved in the acquisition and retention of conditioned taste aversion. On the other hand, brain slices obtained from mice with complete resolution of conditional taste aversion exhibited a decrease in the induction rate of long-term synaptic inhibition by low-frequency stimulation (fig. 7c) or DHPG (fig. 7E), wherein the induction rate of long-term synaptic inhibition by low-frequency stimulation was 19.3 ± 11.4% (fig. 7d, P ═ 0.0049) and the induction rate of DHPG-long-term synaptic inhibition was 11.4 ± 5.5% (fig. 7f, P ═ 1.982E-07), which were significantly different from those of the conditional taste aversion animals. These results indicate that the resolution process of conditioned taste aversion offsets long-term synaptic inhibition of the stator vane cortex.

Comparing the input-output curves for the excitatory postsynaptic currents of both conditioned taste aversion and its control tissues, it was found that the withdrawal process reduced excitatory synaptic transmission in the stator vane cortex (fig. 7g, h). Therefore, islet cortical long-time synaptic inhibition is involved in the regression process of conditioned taste aversion memory. The process of complete regression of taste aversion memory results in a saturation effect that counteracts the long-term synaptic inhibition induction process that follows.

In fig. 7, (a) a behavioral operation step. On day 0, mice established a combined taste aversion memory by combining sugar water with abdominal discomfort caused by intraperitoneal injection of LiCl. On subsequent days 1-7, one group of mice will be fed only sugar water to completely resolve the established conditioned taste aversion memory; another group of mice will be fed water only to retain the established conditioned taste aversive memory. On day 8, mice were sacrificed for brain slice preparation and electrophysiological recording was performed. (b) The obtained aversion index was calculated by comparing the water intake and the sugar intake of the mice. The aversion index is water intake/(water intake + sugar intake) × 100%. n is 5-7.^***P<0.001, unpaired Student's t-test. (c-f) the induction of long-term synaptic inhibition of islet leaf cortex disappeared in mice with complete withdrawal of taste aversion memory, but remained in mice that retained taste aversion memory. (c, e) complete regression of taste aversion memory or time-course dependent changes in the field excitatory postsynaptic potential elicited after the mouse islet cortical brain sheet remains in response to low frequency stimuli (c) or DHPG (g). Illustration is shown: a representative field excitatory post-synaptic potential trace is shown at time points. Scale bar: the ordinate is 100 microvolts and the abscissa is 10 milliseconds. (d, f) a summary of the number of channels activated per islet cortical brain slice and the number of channels exhibiting long-term synaptic inhibition. N.s., no statistical difference was observed when comparing the two groups using unpaired Student's t-test;^*P<0.05，^***P<0.001, comparing the two groups using the unpaired Student's t-test, showed that the two groups exhibited significant statistical differences. (d) n-6 brain slices (4 mice per group); (f) n-5-7 brain slices (4 mice per group). (g, h) input-output curves of excitatory postsynaptic currents of mouse islet cortical brain neurons with complete regression or retention of taste aversion memory. (g) Representative trajectory. (h) The data is summarized. n-20-23 cells (7 mice per group).^*P<0.05, ^**P<0.01,^***P<0.001 comparison of the two groups using the unpaired Student's t-test showed that the two groups exhibited significant statistical differences.

Example 8 deletion of ASIC1a Selective impairment resolution of conditioned taste aversion memory

ASIC1a plays an important role in the induction of islet cortical long-term synaptic depression, with the expectation that ASIC1a gene-deficient mice may exhibit normal acquisition and retention of conditioned taste aversion memory, but may be impaired in conditioned taste aversion regression. The results show that wild-type and ASIC1a knockout mice do perform equivalently in the acquisition of conditioned taste aversion memory. On the day of conditioning training, both mice ingested similar amounts of sugar water (wild type: 1.93 ± 0.11 g, n ═ 15; ASIC1a knockout mouse: 1.74 ± 0.15 g, n ═ 14; P ═ 0.3020). On the first day after conditioning training, both groups developed similar aversive responses (fig. 8a), indicating that ASIC1a did not play a significant role in the acquisition and extraction of conditioned taste aversive memory. The memory retention strength of wild-type and ASIC1a knockout mice was found to be similar when subjected to the conditional taste aversion memory strength test on subsequent 3, 7, 14 and 28 days (fig. 8b), indicating that ASIC1a knockout mice, like wild-type mice, were able to retain normal conditional taste aversion memory for at least 1 month.

In contrast, ASIC1a knockout mice failed to exhibit significantly reduced aversion in subsequent conditional taste aversion regression tests, whereas wild-type mice exhibited significantly reduced memory strength the second to three days after taste selectivity testing (fig. 8 c). Similarly, ASIC1a knockout mice also showed significant impairment in regression compared to wild-type mice if regression training was performed on long-term conditioned taste aversion memory (fig. 8 d). These results indicate that ASIC1a selectively contributes to the resolution of conditioned taste aversion memory, but has no significant effect on memory acquisition or retention.

As a control, to confirm that ASIC1a gene-deficient animals have complete taste discrimination, mice were examined for their preference for sweet, salty, bitter and sour solutions, respectively, using an unconditioned two-vial taste preference test (Yu, h., et al. (2009) Variant BDNF Val66Met polymorphism extracts of conditioned aversive medium j Neurosci 29, 4056) 4064, and no significant difference was found between wild-type and ASIC1a knockout mice.

In FIG. 8, wild type and ASIC1a knockout mice were subjected to conditional taste aversion training on day 0 and to a taste aversion memory acquisition test on day 1; using different batches of mice to perform taste aversion memory tests, defined as tests of memory intensity retention, on

days

3, 7, 14 and 28 after performing conditional taste aversion training, respectively; with respect to the regression test, mice that have established conditional taste aversion memory will be given a bottle of sugar water and a bottle of water for a selection test, a continuous taste selection test 1-7 days after memory establishment, referred to as the regression of recent taste aversion memory; continuous taste selection tests 15-21 days after memory establishment, called regression of distant taste aversion memory. The data for day 1 in graph (c) are the same as the data in graph (a), and are presented repeatedly for better comparison. n is 8-16. N.s., no statistical difference was observed when comparing the two groups using unpaired Student's t-test;^*P<0.05，^**P<0.01，^***P<0.001, comparing the two groups using the unpaired Student's t-test, showed that the two groups exhibited significant statistical differences.

Example 9 Key Effect of island cortex ASIC1a on conditioned taste aversion resolution

To clarify the effect of the supracortical ASIC1a on conditioned taste aversion, the previously described ASIC1a conditioned knockout mouse, ASIC1a, was again used^flox/floxA mouse. ASIC1a for AAV control virus injection versus AAV control virus injection^flox ^/floxMouse islet leaf cortex injection of AAV-Cre virus (fig. 9a) resulted in animals exhibiting significantly reduced conditioned taste aversion (fig. 9b), which supports the specific role of islet leaf expressed ASIC1a in conditioned taste aversion.

To further test whether acute inhibition of ASIC1a in the islet cortex affected the resolution of conditioned taste aversion, ASIC1a inhibitor, PcTx1(10 picomoles) or its solvent control, was administered immediately after the two-vial selection test in animals with established conditioned taste aversion memory (fig. 9 c). Administration of PcTX1 (fig. 9d) significantly blocked the resolution of conditioned taste aversion (fig. 9e) compared to solvent control.

To test whether re-expression of ASIC1a in the cortex of island of ASIC1a knockout mouse can rescue regression abnormalities in gene-deficient animals. The intervention strategy shown in example 4 was used, except that in the experiments herein, conditioned taste aversion training was performed on animals 1 month after AAV virus injection, followed by a taste selectivity test 3-6 days after training (fig. 9 f). As expected, injection of ASIC1a overexpressing virus in the islet cortex of ASIC1a gene deficient mice resulted in animals with a level of conditioned taste aversion resolution close to that of wild-type mice, whereas injection of control or ASIC1a-HIF mutant overexpressing virus did not have a similar effect (fig. 9 g). Therefore, functional ASIC1a on the islet cortex is a key deletion component of ASIC1a knockout mice, which supports just the conditioned taste aversion resolution learning process in animals.

Using another strategy to eliminate ASIC1a expression, an AAV viral vector expressing short hairpin RNA (shRNA) targeting ASIC1a was constructed and expression driven by the U6 promoter (fig. 9 i). After validation of ASIC1a-shRNA on Chinese hamster ovary cells, AAV-ASIC1a-shRNA or negative control virus was injected in the islet cortex of wild type mice (FIG. 9 h). AAV-ASIC1a-shRNA virus significantly reduced the expression of island cortex AISC1a protein (P5.651E-05, n 4-6, fig. 9j) compared to AAV negative control virus, also resulting in significant blockade of the conditioned taste aversion process (fig. 9 k). On the basis of ASIC1a-shRNA, a rescue strategy for excitatory neuron type-specific re-expression of ASIC1a was increased (FIG. 9 l). The AAV viral vector herein comprises, in addition to ASIC1a-shRNA element, mCherry-2A-ASIC1a (denoted as ASIC1 a) insensitive to shRNA^*) The expression sequence in the reverse Direction (DIO) was controlled by double-flox, which was normally expressed only in the presence of Cre recombinant endonuclease (FIG. 9 m). This AAV virus was injected into the islet cortex of CaMKII-Cre tool mice (Cre recombinase was expressed only in pyramidal neurons of the forebrain), and significant expression of mCherry (fig. 8n) and ASIC1a (fig. 9o) was found in a population of neurons in the islet cortex.However, when the same virus was injected into wild-type mice, neither mCherry (fig. 9n) nor ASIC1a (fig. 9o) was significantly expressed. The ASIC1a-shRNA sequence contained in this AAV viral vector (FIG. 9m) was also effective, as evidenced by a marked abnormal resolution of conditioned taste aversion, i.e., a higher aversion index, in wild-type mice injected with the virus (FIG. 9 p). In contrast, in CaMKII-Cre tool mice, injection of AAV virus resulted in a resolution of conditioned taste aversion in the animals that was close to that of normal control animals (fig. 9 p). In summary, the above results establish a key role for island cortex ASIC1a in conditioned taste aversion resolution.

In FIG. 9, (a, c, f, h, l) is a behavioral step. (b, e, g, k, p) is a time course dependent change in the aversion index caused by a resolution of conditional taste aversion.^*P<0.05，^**P<0.01，^***P<0.001, comparing the two groups using the unpaired Student's t-test, showed that the two groups exhibited significant statistical differences. (b) n is 7. (e) n is 10-11. (g) n is 8-13.^*P<0.05，^***P<0.001，WT+AAV-Ctrl vs.KO+AAV-Ctrl；^#P<0.05，^##P<0.01，KO+AAV-ASIC1a vs. KO+AAV-Ctrl.(k)n＝9–12。(p)n＝11–12。^*P<0.05，^**P<0.01，WT+AAV-NC-Ctrl vs.WT +AAV-shRNA-DIO-ASIC1a^*；^#P<0.05，^##P<0.01，WT vs.CaMKII-Cre for AAV-shRNA-DIO-ASIC1a^*Rejected, unpaired Student's t-test. (d) Schematic representation of the diffusion region after site-specific injection of drug into the cortex of island. (i, m) schematic representation of the construction of AAV vectors. (j, o) representative immunoblot pictures. (n) pictures of the island cortex mCherry expression, here negatively stained with DAPI.

Example 10 Long-term synaptic inhibition of islet cortex is essential for conditioned taste aversion resolution

To further establish the significance of island cortex ASIC1 a-dependent long-term synaptic inhibition for conditioned taste aversion resolution, the behavioral consequences of in vivo blockade of island cortex long-term synaptic inhibition were studied. Endocytosis of AMPA receptors is a common mechanism for long-term synaptic inhibition, which can be blocked by either intracellular administration of a GluA2 mimetic short peptide, GluA2-3Y, or extracellular administration of a GluA2 mimetic short peptide with the ability to cross the model, FITC-Tat-GluA2-3Y (Tat-3Y, FIG. 10 a). Wherein, the penetrating ability of the polypeptide is realized by containing a Tat short peptide derived from Human Immunodeficiency Virus (HIV). As a negative control, FITC-Tat-GluA2-3A (Tat-3A, FIG. 10a) also had a transtemplating ability, but failed to exert an effect of blocking the endocytosis of AMPA receptors. Administration of Tat-3Y (1. mu.M) before and during low frequency stimulation significantly blocked the induction of long-term synaptic inhibition in the island cortex, whereas the control polypeptide Tat-3A (1. mu.M) did not have this effect (FIG. 10 b). The induction rate of long-term synaptic inhibition of brain slice of treated Tat-3Y polypeptide was 16.0 ± 7.1%, while that of control polypeptide Tat-3A polypeptide was 78.0 ± 6.1%, which were significantly different from each other (P-5.900E-05, fig. 10 c). These results indicate that GluA2 subunit-dependent AMPA receptor endocytosis is a critical step in island cortex long-term synaptic inhibition.

The effect of in vivo administration of island cortex Tat-3Y polypeptide (100 picomoles) on the resolution of conditional taste aversion is shown in FIG. 10 d. The effectiveness and specificity of administration of the Tat-3Y polypeptide was confirmed based on FITC green fluorescence exhibited by the layers of the island cortex (FIG. 10 e). Animals receiving the Tat-3Y polypeptide showed a marked abnormality in the resolution of conditional taste aversion compared to mice receiving either the control polypeptide Tat-3A or solvent control (FIG. 10 f). This result supports that long-term synaptic inhibition of the islet cortex is essential for conditioned taste aversion resolution. In summary, the island cortex ASIC1a plays a key role in conditioned taste aversion resolution.

In fig. 10, (a) interfering peptide sequences that block endocytosis of AMPA receptors, the key tyrosine residues (Y) are indicated in red; the polypeptide formed when the critical tyrosine residue is mutated to alanine (a) loses its effect of blocking interference with AMPA receptor endocytosis and thus serves as a control peptide. (b) Acute administration of Tat-3Y interfering peptide (1. mu. mol/l, starting 20 minutes prior to the low frequency stimulation step and lasting 35 minutes) blocked the long-term synaptic inhibition caused by low frequency stimulation, but no significant effect was observed with the administration of the control polypeptide (Tat-3A). (c) Every island cortex brainSummary of the number of sheet-activated channels and the number of channels exhibiting long-term synaptic inhibition. N.s., no statistical difference was observed when comparing the two groups using unpaired Student's t-test;^***P<0.001, comparing the two groups using the unpaired Student's t-test, showed that the two groups exhibited significant statistical differences. (d) And (5) performing a behavioral step. (e) Schematic representation of the location of the injection of interfering polypeptides and their invasion into the interior of the cell. (f) Conditioned taste aversion resolution causes a time course dependent change in the aversion index. n is 10-14.^***P<0.001, when compared to the two groups using the unpaired Student's t-test, showed significant statistical differences between Tat-3A and Tat-3Y.

Example 11 pharmacological blockade or activation of the ventral hippocampus ASIC1a inhibits and promotes fear resolution, respectively

To examine the physiological significance of ASIC1a in fear regression, the fear response of animals to CS during the second day of regression learning and the third day of fear detection was observed and quantified by microinjecting the bilateral ventral hippocampus with saline, ASIC1a agonist MitTx- α/β, or ASIC1a blocker PcTX1 (both injected volumes were 1 microliter/side, fig. 11a, b) half an hour prior to the second day of regression learning. Data are presented as mean ± sem. The number of animals was 8-14 mice per group.

The experimental formulation was selected from the group consisting of:

(i) MitTx- α/β,1 μmol/l, dissolved in physiological saline.

(ii) PcTX1, 10. mu. mol/l, dissolved in physiological saline.

The results were as follows (the saline treatment groups are shown in blank figures and the MitTx- α/β or PcTX1 treatment groups are shown in filled figures):

in the fear learning process, the fear response of the animal to CS is gradually increased, the fear response of the animal to CS in the learning process is gradually decreased the next day, and the animal can maintain the low fear response to CS on the third day. The animals regressed the ventral hippocampus before learning and injected with ASIC1a agonist MitTx- α/β significantly promoted fear regressions compared to saline group, i.e. animals maintained a lower fear response to CS in the MitTx- α/β treated group on the third day (fig. 11c, d); in contrast, animal regression learning the ventral hippocampus before injection of ASIC1a blocker PcTX1 significantly inhibited fear regression.

Example 12 conditional knockout or overexpression of ventral hippocampus ASIC1a inhibits and promotes fear resolution, respectively

AAV-GFP/AAV-ASIC1a (injection volume is 1 microliter/side) is injected into ventral hippocampus of wild type C57BL/6J mouse at fixed point, or ASIC1a (conditional ASIC1a gene knockout) -ASIC1a^flox/floxMice were injected with AAV-GFP/AAV-Cre (injection volume was 1 μ l/side) at a site located in the ventral hippocampus, and animals were observed and quantified for fear response to CS during fear learning, regressive learning, and third day fear detection one month after virus expression (figure 12 a). Data are presented as mean ± sem. The number of animals was 13-19 mice per group.

The experimental formulation was selected from the group consisting of:

(i) AAV-GFP titer over 10¹²Viral Genomes (VG)/ml (ml) were dissolved in sterile PBS.

(ii) AAV-Cre, titer over 10¹²VG/ml, dissolved in sterile PBS.

(iii) AAV-ASIC1a, titer over 10¹²VG/ml, dissolved in sterile PBS.

The results are as follows (indicated by blank plots for the AAV-GFP treated groups and filled plots for the AAV-Cre or AAV-ASIC1a treated groups):

ventral hippocampus overexpressing ASIC1a, animal fear learning was unaffected on day one, but subsequently regressed learning was enhanced (fig. 12b, c); conditioned knockout of the ventral hippocampus ASIC1a, animal fear learning was also unaffected on the first day, but subsequently regressive learning was impaired (fig. 12d, e).

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Use of an acid sensitive ion channel promoter in the manufacture of a medicament for the treatment of bulimia nervosa and obesity, said acid sensitive ion channel being an acid sensitive ion channel comprising ASIC1a subunits.

2. The use of claim 1, wherein said acid-sensitive ion channel enhancer comprises any compound or recombinant vector that increases expression and/or activity of ASIC1a subunit protein.

3. Use according to claim 2, wherein the acid-sensitive ion channel enhancer compound comprises the polypeptide MitTx-a/β, and/or spermine.