CN108079300A - Uses of Acid Sensitive Ion Channel Modulators - Google Patents

Abstract

The invention discloses an application of an acid-sensitive ion channel regulator in preparing a medicament for treating mental disorder diseases, wherein the acid-sensitive ion channel is an acid-sensitive ion channel containing ASIC1a subunits. The invention also discloses a pharmaceutical composition containing the acid-sensitive ion channel regulator. 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.

Description

Uses of Acid Sensitive Ion Channel Modulators

technical field

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

Background technique

The acid-sensing ion channel (ASIC) family contains six subtypes encoded by four genes, namely ASIC1a, 1b, 2a, 2b, 3 and 4. These proton-gated channels are composed of three identical or different subunits, and ion channels formed by different combinations exhibit different pH sensitivity, ion selectivity, and pharmacological properties. In the central nervous system, ASIC1a-containing channels are the most dominant ASIC members, and neurons lacking the ASIC1a gene fail to exhibit acid-induced currents. The importance of ASIC1a has been well studied in rodent models such as ischemic neuronal death, chronic pain, epileptic termination and neurodegenerative diseases. Although the research on the neuronal expression of ASIC1a and its pathological significance has made important progress, however, the physiological function of ASIC1a in brain neurons is still unclear.

Accumulating evidence supports the critical role of ASIC1a in synaptic transmission and plasticity. Based on the postsynaptic localization of ASIC1a, it is thought that ASIC1a is activated by protons released by synaptic activity during normal synaptic transmission. As the ASIC1a channel that serves as a proton receptor in the synapse, it plays a role in regulating the synaptic long-term potentiation (LTP) induced by high-frequency stimulation of the hippocampus and amygdala or theta burst stimulation (theta burst stimulation, TBS) play a role in the process of plasticity and thus contribute to the process of fear learning. ASIC1a promotes dendritic spine density in the hippocampus, but inhibits dendritic spine density in the nucleus accumbens, alters the function of excitatory synaptic receptors, and limits cocaine-induced synaptic plasticity, these developments suggest that ASIC1a plays a role in regulating synaptic plasticity Adaptive aspects of behavior and behavior may play a more complex role.

The complexity of ASIC1a function may be reflected in its participation in different forms of plasticity and behavior regulation in different brain regions. ASIC1a is highly expressed in insular cortex neurons, but its channel function is unclear. The insular cortex is involved in many cognitive and emotional regulation functions, including sensory integration, chronic pain, emotional processing, and taste recognition memory. The best known function of this brain region is its role in taste learning. In particular, the insular cortex plays an important role in a type of negative emotion-driven taste learning called conditioned taste aversion (CTA). In the learning process of conditioned taste aversion, the learning subject establishes a connection between a brand-new taste (conditioned stimulus, CS) and the visceral discomfort (unconditioned stimulus, unconditioned stimulus, US) that is subsequently felt, so as to have a better understanding of this taste. Certain tastes produce aversive emotional memories. At the synaptic level, long-term synaptic potentiation in the insular cortex contributes to the acquisition of conditioned taste aversive memories. However, the molecular and synaptic mechanisms mediating conditioned taste aversion memory extinction are poorly understood.

Anorexia, also known as anorexia nervosa, is an abnormal eating behavior that is more common in adolescent girls. Fat, even if the doctor explains it, it will not work. There is still a lack of effective drugs for the treatment of anorexia.

Many people who are addicted to drugs, smoking or alcohol often take drugs or smoking or drinking again because they cannot bear the physical pain after quitting drugs or smoking or drinking. Therefore, drugs that treat the nervous system are very necessary for successful detoxification from drugs or smoking or alcohol.

Anxiety disorders are also neurological disorders with a global prevalence of 7.3% and an individual lifetime prevalence of up to 11.6%. People with anxiety disorders often exhibit excessive fear, anxiety, or excessive avoidance of perceived danger in specific environments (such as social or unfamiliar environments). And these responses go well beyond the normal fear or anxiety responses elicited by actual danger. The occurrence of anxiety disorders can significantly reduce the quality of life of patients and bring a great economic burden to patients' families and the whole society. However, there are still very few drugs currently used to treat or effectively relieve anxiety disorders.

Contents of the invention

The present invention aims to solve the technical problem of the current lack of drugs for the treatment of mental disorders such as anorexia, anxiety, drug addiction, and smoking addiction, and provides an acid-sensitive ion channel regulator for treating or alleviating anorexia, anxiety, drug addiction, etc. Addiction, smoking and other mental disorders.

In order to solve the problems of the technologies described above, the present invention is realized through the following technical solutions:

In one aspect of the present invention, a use of an acid-sensitive ion channel regulator in the preparation of a drug for treating mental disorders is provided, and the acid-sensitive ion channel is an acid-sensitive ion channel comprising an ASICIa subunit.

The regulators include promoters or inhibitors of acid-sensitive ion channels.

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

The acid-sensitive ion channel inhibitor includes any compound or recombinant carrier that specifically inhibits the expression and/or activity of ASICIa channel-related proteins. Preferably, the acid-sensitive ion channel inhibitor compound includes polypeptide PcTX1, amiloride (amiloride), amidine A-317567, nafamostat (nafamostat mesilate), non-steroidal anti-inflammatory drug (non-steroidal anti-inflammatory drugs, NSAIDs), compound 5b (2-oxo-2H-chromene-3-carboxamidine derivative), alkaline neutralizer, or a combination thereof. The alkaline neutralizing agent includes sodium bicarbonate, ammonium chloride, alkali metal salts, alkaline earth metal salts, or combinations thereof.

The mental disorders that the acid-sensitive ion channel enhancer is used for treatment include: anorexia, anxiety, addictive diseases, bulimia nervosa and obesity, and any other neurological diseases accompanied by abnormal intractable pathological memory. Among them, addictive diseases include drug addiction, smoking addiction, alcohol addiction and so on.

Using the acid-sensitive ion channel enhancer of the present invention and assisting repeated extinction training can significantly improve the symptoms of patients with anorexia or addictive diseases; using the acid-sensitive ion channel enhancer of the present invention and assisting exposure therapy can effectively treat anxiety disease.

The mental disorders that the acid-sensitive ion channel inhibitors are used to treat include Alzheimer's disease and any other neurological diseases accompanied by cognitive impairment and memory impairment.

The symptoms of Alzheimer's patients can be improved by using the acid-sensitive ion channel inhibitor of the invention and assisting cognitive training.

In the present invention, the term "acid-sensing ion channels" (acid-sensing ion channels, ASICs) refers to a class of protein complexes widely present on the cell membrane to permeate cations, belonging to the epithelial sodium channel/degenin protein superfamily, It plays an important role in sensing the pH value of body fluids and regulating many physiological functions such as pain and mechanosensation.

Molecular cloning revealed that ASIC has at least six subunits (ASIC1a, 1b, 2a, 2b, 3, and 4) encoded by four genes that form homo- or heterotrimeric functional complexes. Differences in the expression and distribution of ASIC subunits, as well as changes in subunit composition and channel gating, result in a diversity of channel functions.

ASIC1a is distributed in both the central and peripheral nervous systems and is mainly involved in synaptic transmission and plasticity. Abnormal function of ASIC1a is associated with various neurological diseases such as ischemic cell death, epilepsy, neurodegenerative diseases and so on. The ASIC1a channel referred to in the present invention refers to homomers or heteromers mainly composed of ASIC1a subunits.

In the present invention, the term "acid-sensitive ion channel regulator" refers to a substance capable of regulating acid-sensitive ion channels, and the regulator may be a small molecular compound or a macromolecular compound.

In the present invention, the regulating agent includes a promoter (or agonist), or an inhibitor (or antagonist).

Representative acid-sensitive ion channel modulators include polypeptide PcTX1, MitTx-α/β, spermine, amiloride, amidine A-317567, nafamostat mesilate, non-steroidal anti-inflammatory Drugs (non-steroidal anti-inflammatory drugs, NSAIDs), compound 5b (2-oxo-2H-chromene-3-carboxamidine derivatives), etc.

As used in the embodiments of the present invention, the polypeptide MitTx-α/β is a toxin derived from Texas coral snake, which can directly activate the ASIC1a channel.

As used in the examples of the present invention, the tarantula toxin PcTX1 can effectively inhibit the ASIC1a channel.

In the present invention, the term "anorexia" refers to an eating mental disorder characterized by intentionally causing and maintaining a body weight significantly lower than normal standards by means such as dieting. Its main feature is a strong fear of weight gain and obesity. Extremely concerned about body shape. While significantly losing weight, there are often malnutrition, metabolism and endocrine disorders. Severe patients may experience cachexia, body failure and even life-threatening due to extreme malnutrition. 5% to 15% of patients finally die of heart disease. Complications, multiple organ failure, secondary infection, etc.

In the present invention, the term "anxiety disorder" refers to patients who tend to show excessive fear, anxiety or excessive avoidance of perceived danger in a specific environment (such as a social environment or an unfamiliar environment). And these responses go well beyond the normal fear or anxiety responses elicited by actual danger. Anxiety disorders include: post-traumatic stress disorder, separation anxiety disorder, selective mutism, specific phobias, social phobia, panic disorder, agoraphobia, generalized anxiety disorder, anxiety disorder associated with another disorder, Drug-induced anxiety disorder, hypochondria, etc.

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

The above-mentioned acceptable carrier is non-toxic, can assist administration and has no adverse effect on the therapeutic effect of the acid-sensitive ion channel modulator. Such carriers can be any solid, liquid, semisolid or, in aerosol compositions, gaseous excipients 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 field of pharmacy. For example, the acid-sensitive ion channel modulator is mixed with one or more carriers, and then it is made into a desired dosage form, such as tablet, pill, capsule, semi-solid, powder, sustained-release dosage form, solution, suspension , formulations, aerosols, etc.

In another aspect of the present invention, there is also provided a method for screening candidate drugs for the treatment of mental disorders, comprising the following steps:

Detect the effect of the test compound on the acid-sensitive ion channel containing the ASIC1a subunit, and select the test compound that has a regulatory effect on the acid-sensitive ion channel as the primary screening compound;

Detect the therapeutic or alleviating effects of the primary screened compounds on mental disorders, and select compounds with curative effects as candidate drugs.

Preferably, the detection step of obtaining the primary screening compound comprises: cultivating the cells in the presence of the compound to be tested, and detecting the expression level, activity and/or ASIC-like current of the acid-sensitive ion channel 1a protein in the cells, and in the absence of the In the control group with the compound to be tested and other conditions the same, detect the expression level, activity and/or ASIC-like current of the acid-sensitive ion channel 1a protein in the same cells, and compare them; if the detection value Vt of the experimental group is significantly higher than or Significantly lower than the detection value Vc of the control group, it indicates that the test compound is a test compound that has a regulatory effect on the acid-sensitive ion channel.

The acid-sensitive ion channel regulator of the present invention promotes or inhibits the activity of the acid-sensitive ion channel containing ASIC1a subunits, thereby promoting or inhibiting the extinction of conditioned memory, and is used for treating or alleviating anorexia, anxiety, drug addiction, smoking addiction, etc. Mental disorders provide new drugs or therapeutic targets with broad application prospects.

Description of drawings

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

1 is a graph showing the expression results of ASIC1a in the insular cortex of wild-type and ASIC1a knockout mice according to Example 1 of the present invention;

Fig. 2 is the experimental result figure that ASIC1a of embodiment 2 of the present invention is necessary for long-term synaptic depression (LTD) induced by low-frequency stimulation;

Fig. 3 is a diagram of the experimental results of blocking low-frequency stimulation-long-term synaptic inhibition in the process of interfering with the dynamic change of extracellular pH in wild-type mice in Example 3 of the present invention;

Figure 4 is a diagram of the experimental results of re-expressing ASIC1a to rescue low-frequency stimulation-long-term synaptic inhibition in ASIC1a knockout mice according to Example 4 of the present invention;

Fig. 5 is a graph showing that ASIC1a of Example 5 of the present invention is necessary for the long-term synaptic inhibition induced by DHPG in the insular cortex;

Figure 6 is a diagram of the experimental results that ASIC1a of Example 6 of the present invention does not contribute to the induction of long-term synaptic potentiation (LTP) in the insular cortex;

Fig. 7 is a diagram of the experimental results of the long-term synaptic inhibition selectivity of the insular cortex in Example 7 of the present invention being offset by the extinction process of conditioned taste aversion;

Fig. 8 is a graph showing the experimental results of the loss of ASIC1a selectively impairing conditioned taste aversion memory loss in Example 8 of the present invention;

Fig. 9 is a graph showing the experimental results that the functional ASIC1a of the insular cortex in Example 9 of the present invention plays a key role in the extinction of conditioned taste aversion;

Figure 10 is a graph showing the experimental results that long-term synaptic inhibition of the insular cortex in Example 10 of the present invention is necessary for the extinction of conditioned taste aversion;

Fig. 11 is a graph showing the experimental results of pharmacologically blocking or activating ventral hippocampal ASIC1a respectively inhibiting and promoting fear extinction in Example 11 of the present invention;

Fig. 12 is a graph showing experimental results of conditional knockout or overexpression of ASIC1a in the ventral hippocampus of Example 12 of the present invention, respectively inhibiting and promoting fear extinction.

Detailed ways

The present invention identified ASIC1a to play a crucial role in the plasticity of long-term depression (LTD) in the insular cortex by means of multi-electrode array brain slice recording, and at the same time analyzed the Its contribution in conditioned gustatory aversion learning and its memory extinction. Unlike the known ASIC1a involved in long-term synaptic enhancement (long-term potentiation, LTP) in other brain regions and further promoting the contribution of joint learning and memory, ASIC1a in the present invention is a long-term synaptic synapse in the insular cortex Key regulator of Taste Depression (LTD), a mechanism important for the extinction of established taste aversive memories.

General Methods and Materials

1. Animals

Animal care and experimental procedures were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. To reduce experimental variability, experiments were performed with age-matched littermate control mice. All behavioral assays were performed on conscious, freely moving, male, 2–3 month old mice on a C57BL/6J background. Conventional ASIC1a whole-body knockout mice were donated by Professor Michael J. Welsh from the University of Iowa; conditional ASIC1a knockout mice (ASIC1a ^flox/flox ) were donated by Cheng-Chang Lien of Yangming University, Taiwan Provided by the professor; the tool mouse that highly expresses Cre recombinase in the excitatory neurons of the forebrain—(CaMKII-Cre) was donated by Professor Joe Z.Tsien of Georgia Regents University (Georgia Regents University). Adult C57BL/6J mice were purchased from Shanghai Slack Company. Animals were kept in a 12-hour cycle of alternating day and night (lighting time: 7:00-19:00), the temperature of the feeding room was kept at 21°C, and the humidity was maintained at 50-60%. Animals were free to eat during non-experimental periods drinking water. Animals were randomly selected for double-blind experiments. In all behavioral experiments, the experimenter transferred the mice to the behavioral feeding room at least two weeks in advance, and placed the mice in the behavioral testing room to adapt to the environment one hour before the experiment, so as to fully reduce the impact of environmental changes on the behavioral results. influences. All behavioral tests were performed and evaluated double-blind to genotype or drug treatment.

2. Conditioned taste aversion extinction model

The construction of the conditioned taste aversion memory model is to train the male mice to drink water regularly for 6-8 days in order to adapt to drinking water at the same time every morning. On the day when the conditioned taste aversion memory was established, the mice were given 0.5% saccharin sodium solution instead of water, and the drinking time was 30 minutes. ). Among them, saccharin sodium solution was the conditioned stimulus, and intraperitoneal injection of LiCl was the unconditioned stimulus. The strength of taste aversion memory is indexed by aversive index. Aversion index = water consumption/(water consumption+sugar consumption)×100%. After the conditioned taste aversion memory is established, through repeated testing, it is a learning model of taste aversion extinction.

3. Animal models of anxiety disorders and behavior paradigms related to anxiety disorders

The most commonly used animal model for studying fear behavior is fear conditioning, which involves repeatedly coupling harmless stimuli (conditioned stimulus, CS) with noxious stimuli (unconditioned stimulus, US) so that Make animals produce fear response (conditional response, CR) to CS, which manifests as body and autonomic response, including freezing, rapid heartbeat, elevated blood pressure, loss of pain sensation, etc. Afterwards, if the animal is repeatedly given CS without US, the conditioned fear response of the animal to CS will gradually decrease, and this process is called fear extinction.

The behavioral assays of the present invention were carried out on conscious, freely moving C57BL/6J mice or ASIC1a Flox transgenic mice (both 2-3 months old, C57BL/6J genetic background). The adopted animal model of anxiety disorder is cue-dependent fear conditioning. The animals were touched and acclimatized to the experimental cage for three days before the start of the experiment. The formal experiment was completed in three consecutive days: on the first day, the animals were subjected to fear learning in environment A (one side of the experimental cage was transparent, the other three sides were pasted with black and white checked wallpaper, the bottom was a conductive iron fence, and 75% alcohol was sprayed), In the experiment, let the animals adapt to the experimental cage for 3 minutes, then give the animals 30 seconds, 76 decibels, 4000 Hz sound (harmless stimulus or conditioned stimulus, CS), and give the animal 0.5 mA 2 seconds before the end of the sound Plantar electric shock (noxious stimulus or unconditioned stimulus, US), a total of 5 combined sounds and plantar electric shocks were given, and the interval between each sound was 20-120s; One side is transparent, the other three sides are pasted with gray wallpaper, the bottom is covered with a smooth plastic plate, and 4% acetic acid is sprayed) for extinction study. During the experiment, animals are given CS 20 times in a row without US; on the third day, animals are given CS in B environment. 8 CS, to detect the animal's fear response to the cue after extinction learning. The animal's fear response to CS was measured by measuring the percentage of time the animal was freezing during CS during the three-day experiment. Evaluation of the fear response was achieved by analyzing the percentage of time the animals spent freezing. Behavioral experiments in the Examples were carried out and evaluated under conditions of double-blind genotype or drug treatment.

4. Brain Slice Preparation and Patch Clamp Recording

References (Kim, J.I.et al.(2011).PI3Kgamma is required for NMDAreceptor-dependent long-term depression and behavioral flexibility.NatNeurosci 14,1447-1454; Liu,M.G.et al.(2013b).Long-term depression of synaptic transmission in the adult mouse insular cortex in vitro. Eur J Neurosci 38, 3128-3145; Qiu, S. et al. (2013). An increase in synaptic NMDA receptors in the insular cortex contributes to neuropathic pain. Sci Signal 6, ra34) Brain slice preparation and patch-clamp recording were performed from differently treated wild-type and mutant mice.

5. Using a multi-electrode array recording and analysis system to study synaptic plasticity

In order to study the synaptic plasticity in the insular cortex of adult mice, the planar microelectrode array recording technique (MED64 multi-channel recording system) was used to conduct multi-channel excitatory postsynaptic potential (fieldexcitatory postsynaptic potential) on acutely isolated insular brain slices. , fEPSP) recording and analysis. Male C57/BL6J background wild-type or mutant mice aged 7-10 weeks were used. After anesthetized decapitation, the brain was quickly removed and placed in oxygenated frozen artificial cerebrospinal fluid for 1-2 minutes. Then the blocks were trimmed on ice, and the brain tissue blocks were transferred to a frozen microtome to cut 2-3 300 μm insular brain slices. After the cut insular lobe slices were incubated in artificial cerebrospinal fluid at 26-30°C for 2 hours, select a slice of insular lobe brain and place it on the MED64 electrode under a microscope and move it to the designated position so that the 8×8 array of electrodes Cover the entire insular cortex as completely as possible. Adjust the system noise, open the Mobius operating software to start the baseline recording. After the baseline response stabilized for 20-30 minutes, brain slices were given different stimulation patterns to induce different types of synaptic plasticity, including LTP and LTD. According to different experimental needs, LTP or LTD can record for 1-3 hours. The off-line analysis of the data collected by the MED64 system mainly uses Mobius software, focusing on calculating the time-course changes of the slope of the recorded fEPSP before and after giving different synaptic plasticity induction parameters.

6. Data analysis

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

Example 1 ASIC1a is significantly expressed in the mouse insular cortex

The expression of ASIC1a in the insular cortex of adult mice was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Compared with other ASIC subunits, the mRNA of ASIC1a subunit was most abundant in wild-type insular cortex, but was completely absent in ASIC1a knockout mice (Fig. 1a,b). At the protein level, ASIC1a was abundantly expressed in the insular cortex of wild-type mice, comparable to the prefrontal cortex and amygdala, and significantly higher than that in the hippocampus, but completely absent in ASIC1a knockout mice (Fig. 1c). These data suggest that ASIC1a is significantly expressed in the insular cortex.

In Figure 1, (a) RT-PCR shows the expression of different ASIC subunits in the insular cortex of wild-type and ASIC1a knockout mice. (b) Quantitative statistics of mRNA levels shown by real-time quantitative reverse transcription PCR. n=3. NS, there was no statistical difference between wild type (WT) and ASIC1a knockout (ASIC1a KO) groups using unpaired Student's t-test; ^*** P<0.001, compared using unpaired Student's t-test Finally, it was shown that there was a significant statistical difference between the wild type (WT) and ASIC1a knockout (ASIC1a KO) groups. ^### P<0.001, using the unpaired Student's st-test to compare the two groups, it shows that there are significant statistical differences between the other ASIC subunits and the ASIC1a group. (c) Comparison of ASIC1a protein levels in the insular cortex and other brain regions. (Upper), Representative western blot images of ASIC1a protein in the prefrontal cortex, hippocampus, amygdala, and insular cortex. (Bottom), Statistical analysis of relative amounts of ASIC1a protein. Data shown are relative optical density ratios, normalized to hippocampal data. n=4. ^* P<0.05, ^** P<0.01, using the paired Student's t-test to compare each brain region with the hippocampal region, showing significant statistical differences.

Example 2 ASIC1a is necessary for long-term synaptic depression (LTD) induced by low-frequency stimulation

Various models of synaptic plasticity were analyzed using a 64-channel multielectrode array recording system. Among them, the 64-channel multi-electrode array was placed on the insular cortex brain slice as shown in Figure 2a,b. By stimulating the rostral (rostral) deep region of the insular slice near the level of the corpus callosum (layer V-VI, as shown in Figure 2b), synaptic responses covering the entire region were recorded with multiple electrodes. Current injection at the stimulation site caused about 10 channels in the 64-channel electrode array to exhibit significant field excitatory postsynaptic potentials (fEPSPs), which were defined as activated channels. Consistent with past reports (Liu, M.G. et al. (2013b). Long-term depression of synaptic transmission in the adult mouse insular cortex in vitro. Eur JNeurosci 38, 3128-3145), low-frequency stimulation (long-frequency stimulation, LFS) A decrease in excitatory postsynaptic potential was induced in most of the activated channels in the insular cortex slices of wild-type mice (Fig. 2c), which was defined as low-frequency stimulation-long-term synaptic depression. This low-frequency stimulation-long-term synaptic depression can be inhibited by the NMDA receptor antagonist D-AP5, so it is also called NMDA receptor-dependent long-term synaptic depression. The ratio of the number of channels positive for long-term synaptic inhibition to the number of all activated channels, defined as the induction ratio, was 70.5±4.9% in wild-type mice (Figure 2d), but in ASIC1a knockout The reduction in mice was 20.9±5.7% (P=1.156E-05, ASIC1aKO vs. WT, Figure 2d).

To further establish the role of ASIC1a in long-term synaptic inhibition in the insula, a conditional ASIC1a knockout mouse (ASIC1a ^flox/flox ) (Wu,PYet al.(2013).Acid-sensing ion channel- 1a is not required for normal hippocampal LTP and spatial memory. Model of J Neurosci 33(5), 1828-1832). One month before brain slice recording, adeno-associated virus (adeno-associated virus, AAV) carrying Cre recombinase or control green fluorescent protein was injected bilaterally in the insular cortex. Western blot showed that Cre virus injection significantly decreased the expression of ASIC1a protein compared to control virus. The induction rate of long-term synaptic depression decreased from 80.0±4.3% injected with control virus to 15.9±5.5% injected with Cre virus (P=2.177E-06 vs. AAV-Ctrl, Fig. 2e,f).

The effect of pharmacological inhibition of ASIC1a channels on low-frequency stimulation-long-term synaptic depression was next examined. The ASIC1a homomer and ASIC1a/2b heteromer channel inhibitor - tarantula toxin (Psalmotoxin 1, PcTX1, 100 nmol/L) was administered from 15 minutes before low-frequency stimulation until the end of the whole low-frequency stimulation process. Heat-inactivated tarantula toxin (PcTX1) was used as a control. It was found that neither tarantula toxin nor heat-inactivated tarantula toxin had any effect on basal excitatory synaptic transmission (Fig. 2g); but the induction rate of long-term synaptic depression in the insular cortex after administration of PcTX1 was reduced to 17.9±9.3%, while the induction rate of long-term synaptic inhibition in heat-inactivated PcTX1-treated brain slices was 72.4±8.4% (P=0.0018, Figure 2h). These results confirm the critical role of ASIC1a in long-term synaptic depression in the insular cortex and rule out the possibility that ASIC1a-knockout mice exhibit abnormalities in the induction of long-term synaptic depression due to developmental compensation.

In Fig. 2, (a) (left) Schematic diagram of the location of the multi-electrode array recording electrodes corresponding to the coronal slice of the insular cortex; (right) the positional arrangement of the multi-electrode array recording electrodes (8×8). (b) The light microscope photograph of the multi-electrode array recording electrodes on the insular cortex brain slice, and the different layer information (from layer I to layer V–VI) is also marked in the diagram. The red dots are the stimulation sites. (c,e,g) Time-course-dependent changes in the slope of excitatory postsynaptic potentials in the insular cortex evoked by low-frequency stimulation (1 Hz, 900 pulses) under different conditions shown. Inset: Representative field excitatory postsynaptic potential trajectories for time points illustrated. Scale bar: 100 microvolts on the ordinate, 10 ms on the abscissa. (d,f,h) Summary of the number of activated channels and the number of channels exhibiting long-term synaptic depression per slice of the insular cortex. NS, there is no statistical difference after comparing the two groups using the unpaired Student's t-test; ^* P<0.05, ^*** P<0.001, after comparing the two groups using the unpaired Student's t-test, it shows that the two groups have significant statistical difference. (d) n=7–9 brain slices (7–8 mice); (f) n=6–7 brain slices (4 mice per group); (h) n=5–6 brain slices slices (3–5 mice).

Example 3 The dynamic change process of pH is necessary for long-term synaptic depression (LTD) induced by low-frequency stimulation

In order to detect the effect of the pH dynamic change process on which ASIC1a is activated on the induction of long-term synaptic inhibition, the pH buffering capacity of the extracellular fluid during the induction of low-frequency stimulation was enhanced. This operation was carried out in the past research (Kreple C.J.et al. ). Acid-sensing ion channels contribute to synaptic transmission and inhibit cocaine-evoked plasticity. Nat Neurosci 17(8), 1083-1091) showed that it can reduce the activation of neuron ASIC. This treatment significantly reduced the probability of induction of long-term synaptic depression by low-frequency stimulation in wild-type mice (Fig. 3). These results suggest that ASIC1a regulates the rate of induction of long-term synaptic depression by sensing changes in extracellular pH.

In Fig. 3, by increasing the concentration of bicarbonate ion (HCO ₃ ^– , the concentration is 70 millimoles/liter (mM); conventionally 25 mM) and CO ₂ (concentration is 15%; conventionally 5%) in the extracellular solution to thereby Enhanced pH buffering capacity blocks the induction of long-term synaptic depression in the insular cortex. Time-course-dependent changes in the slope of field excitatory postsynaptic potentials induced by low-frequency stimulation under treatments that enhance pH buffering capacity. n = 56 activated channels (7 brain slices, 6 wild-type mice).

Example 4 Re-expression of ASIC1a in ASIC1a knockout mice rescues low-frequency stimulation-long-term synaptic depression

To test whether restoring ASIC1a expression in the insular cortex of ASIC1a-knockout mice could rescue the induction of long-term synaptic depression, AAV viruses containing both the green fluorescent protein and mouse ASIC1a gene sequences, two functional proteins, were prepared. The sequences are connected with a "self-cleaving" 2A polypeptide sequence, and this virus is used to overexpress ASIC1a. In addition, an AAV virus containing only green fluorescent protein was also prepared as a control. In order to achieve neuron-specific expression, both the control virus and the ASIC1a overexpression virus were expressed on neurons driven by the human synapsin I (human synapsin I) promoter. One month before brain slice recordings, control or ASIC1a-overexpressing viruses were injected in the insular cortex on both sides (Fig. 4a–c). It was found that injection of ASIC1a overexpression virus could successfully rescue low-frequency stimulation-long-term synaptic inhibition (Figure 4d), while the control virus did not have a similar effect. Among them, after injecting the control virus in the insular cortex, the induction rate of long-term synaptic inhibition was 14.9±6.3%; after injecting ASIC1a overexpression virus, the induction rate of long-term synaptic inhibition was 69.9±5.4% (P = 2.513E-05 vs. AAV-Ctrl, Figure 4e). In addition, overexpression of an ion-impermeable ASIC1a mutant virus, namely AAV-ASIC1a-HIF (that is, the histidine-isoleucine-phenylalanine residues at positions 32 to 34 are all mutated Alanine, Figure 4a), cannot rescue the abnormality of long-term synaptic inhibition in ASIC1a knockout mice (Figure 4d), and its induction rate is 24.0±8.2%, which is not significantly different from the group injected with the control virus (P=0.3976, Figure 4e). These results support the critical contribution of ASIC1a and its ion permeability to long-term synaptic inhibition in the insular cortex, while further excluding developmental stage compensation for the impairment of long-term synaptic inhibition in ASIC1a-deficient mice contribute.

In Fig. 4, (a) A schematic diagram of the composition of the AAV vector. hsynapsin I, representing the human synapsin I promoter, is mainly used to drive gene expression selectively in neurons. (b) AAV-mediated expression of fluorescent proteins in the insular cortex (left). The brain slices were negatively stained with DAPI. For a clearer display, a schematic diagram of the brain map at a corresponding position is placed on the right. (c) High magnification images of fluorescent proteins expressed at the level of individual neurons in the insular cortex. (d) Time-course-dependent changes in the slope of excitatory postsynaptic potentials in the insular cortex evoked by low-frequency stimulation (1 Hz, 900 pulses) under the different conditions shown. Inset: Representative field excitatory postsynaptic potential trajectories for time points illustrated. Scale bar: 100 microvolts on the ordinate, 10 ms on the abscissa. (e) Summary of the number of activated channels and the number of channels exhibiting long-term synaptic depression per slice of the insular cortex. NS, there is no statistical difference after comparing the two groups using the unpaired Student's t-test; ^* P<0.05, ^*** P<0.001, after comparing the two groups using the unpaired Student's t-test, it shows that the two groups have significant statistical difference. n = 6–8 brain slices (4–8 mice).

Example 5 ASIC1a is required for DHPG-induced long-term synaptic depression in the insular cortex

To study the role of ASIC1a in another form of synaptic inhibitory plasticity, an agonist of type I metabotropic glutamate receptors, 3,5-dihydroxyphenylglycine (DHPG, 100 micromolar /L), DHPG-induced synaptic depression is insensitive to NMDA receptor inhibitors, so it is also called NMDA receptor-independent long-term synaptic depression. Remarkably, DHPG-induced long-term synaptic depression was also defective in ASIC1a-deficient animals (Fig. 5a). In wild-type animals, the induction rate of DHPG-induced long-term synaptic inhibition was 82.2±5.6%; in ASIC1a knockout animals, the induction rate of DHPG-induced long-term synaptic inhibition was 20.4±9.2%, which was higher than that of wild-type animals There was a significant difference compared to (P=0.0004, Figure 5b). Importantly, reexpression of ASIC1a by AAV virus in ASIC1a knockout mice restored DHPG-induced long-term synaptic depression. Among them, after injection of the control virus, the induction rate of DHPG-long-term synaptic inhibition was 15.3±9.4%, and the induction rate of the overexpressed ASIC1a virus was 82.2±5.8%, which was significantly different (P=0.0001, Fig. 5c,d). Thus, ASIC1a is required for both NMDA receptor-dependent and NMDA receptor-independent induction of long-term synaptic depression in the mouse insular cortex.

In Fig. 5, (a, c) DHPG (100 μM, 20 min, a, c) induced time-course-dependent changes in the slope of the excitatory postsynaptic potential in the insular cortex under the conditions shown. Inset: Representative field excitatory postsynaptic potential trajectories for time points illustrated. Scale bar: 100 microvolts on the ordinate, 10 ms on the abscissa. (b,d) Summary of the number of activated channels per slice of insular cortex and the number of channels exhibiting long-term synaptic depression (b,d). NS, there is no statistical difference after comparing the two groups using the unpaired Student's t-test; ^* P<0.05, ^** P<0.01, after comparing the two groups using the unpaired Student's t-test, it shows that there is a significant difference statistical difference. (b) n = 5 brain slices (4 mice per group); (d) n = 5–6 brain slices (5–6 mice).

Example 6 ASIC1a does not contribute to the induction of long-term synaptic potentiation in the insular cortex

Examine the effect of ASIC1a knockout or inhibition on another form of synaptic plasticity in the insular cortex. After theta burst stimulation (TBS) on the wild-type insular cortex slices, it can induce rapid and long-lasting enhancement of synaptic responses at multiple sites (Figure 6a), that is, long-term synaptic enhancement ( long-termpotentiation, LTP). However, theta-string stimulation induced similar levels of long-term synaptic potentiation in wild-type and ASIC1a knockout mouse brain slices (Fig. 6a), where the induction rate of long-term synaptic potentiation in wild-type mice was 75.5±7.4%, 71.2±6.3% in ASIC1a knockout mice, there was no significant difference between the two (P=0.6711, Figure 6b). In addition, the ASIC1a inhibitor PcTX1 did not affect the long-term synaptic potentiation induced by theta string stimulation in wild-type mice (Fig. 6c), and the induction rate of long-term synaptic potentiation was 60.9±6.6% (Fig. There was no difference between treated wild-type brain slices (P=0.1822). These results suggest that ASIC1a is unlikely to be involved in long-term synaptic strengthening induced by theta train stimulation, which is in sharp contrast to the aforementioned important contribution of ASIC1a in long-term synaptic depression.

In Figure 6, (a, c) under the conditions shown, theta burst stimulation (a total of 10 trains of stimulation at a frequency of 5 Hz, each train of stimulation contains 4 times of pulse stimulation at a frequency of 100 Hz, theta-burst stimulation, TBS, a,c) Evoked time-course-dependent changes in the slope of excitatory postsynaptic potentials in the insular cortex. Inset: Representative field excitatory postsynaptic potential trajectories for time points illustrated. Scale bar: 100 microvolts on the ordinate, 10 ms on the abscissa. (b,d) Summary of the number of channels activated per insular cortex slice and the number of channels exhibiting long-term synaptic potentiation (b,d). NS, there is no statistical difference after comparing the two groups using the unpaired Student's t-test; ^* P<0.05, ^** P<0.01, after comparing the two groups using the unpaired Student's t-test, it shows that there is a significant difference statistical difference. (b) n = 6 brain slices (6 mice per group); (d) n = 5–6 brain slices (5–6 mice). Among them, the control data in Figure (d) are derived from Figure (b), and are repeated for better comparison.

Example 7 Long-term synaptic inhibition in the insular cortex is selectively counteracted by the extinction process of conditioned taste aversion

To induce conditioned taste aversion, mice were fed sugar water as a conditioned stimulus (CS), followed by abdominal pain induced by intraperitoneal injection of LiCl, which served as an unconditioned stimulus (US). Based on comparing the amount of water and sugar water consumed by mice in the next few days after conditioned taste aversion training, the aversion index [drinking water amount/(drinking water amount + sugar drinking amount)×100%] was calculated to evaluate conditioned taste Establishment of taste aversion. Using this procedure, most animals achieved an aversive index between 80-90% after training. To completely extinguish the conditioned taste aversion, these animals were allowed to drink only sugar water for the next 7 days after training, while the control animals were allowed to drink only water to maintain the aversive memory for the specific taste (sugar water) (Fig. 7a). As shown in Figure 7b, the conditioned gustatory aversive control mice maintained a high aversion index, whereas the aversive index of the completely regressed animals had been significantly reduced, indicating that repeated consumption of sugar water without re-coupling with LiCl injections would lead to Regression of conditioned taste aversion.

By analyzing the brain slices of the insular cortex of the above mice, it was found that the conditioned taste aversion control animals showed normal long-term synaptic depression, and the induction rate of low-frequency stimulation-long-term synaptic depression (Figure 7c) was 68.5 ±7.5% (Fig. 7d); the induction rate of DHPG-long-term synaptic depression (Fig. 7e) was 80.3±2.6% (Fig. 7f). These two values were not different from the data obtained in natural mice (Fig. 2d and 5b), indicating that long-term synaptic depression in the insular cortex is not involved in the acquisition and retention of conditioned taste aversion. On the other hand, brain slices obtained from mice with complete extinction of taste aversion conditioned showed a decrease in the induction rate of long-term synaptic inhibition to low-frequency stimulation (Fig. 7c) or DHPG (Fig. The induction rate of synaptic depression was 19.3±11.4% (Fig. 7d, P=0.0049), and the induction rate of DHPG-long-term synaptic depression was 11.4±5.5% (Fig. 7f, P=1.982E-07). Significant differences compared with sexual gustatory aversive animals. These results suggest that the extinction process of conditioned taste aversion counteracts long-term synaptic depression in the inductive cortex.

Comparing the input-output curves of excitatory postsynaptic currents between complete extinction of conditioned taste aversion and control tissues, it was found that the extinction process reduced excitatory synaptic transmission in the inductive cortex (Fig. 7g,h). Thus, long-term synaptic depression in the insular cortex is involved in the extinction of conditioned taste aversion memories. The complete extinction of the taste aversion memory resulted in a saturation effect that counteracted the subsequent induction of long-term synaptic depression.

In Fig. 7, (a) Behavioral operation steps. On day 0, mice established associative gustatory aversive memory through abdominal discomfort induced by sucrose water and intraperitoneal injection of LiCl. On the following days 1–7, one group of mice will be fed only sugar water to completely extinguish the established conditioned taste aversion memory; the other group of mice will be fed only water to retain the established memory. Conditioned taste aversion memory. On day 8, the mice were sacrificed to prepare brain slices for electrophysiological recording. (b) The aversion index calculated by comparing the amount of water and sugar consumed by mice. Aversion index = water consumption/(water consumption+sugar consumption)×100%. n=5–7. ^*** P<0.001, unpaired Student's t-test. (cf) Induction of long-term synaptic depression in the insular cortex is abolished in mice with complete loss of taste aversion memory, but persists in mice with retained taste aversion memory. (c,e) Time-course-dependent changes of field excitatory postsynaptic potentials in insular cortex slices of mice whose taste aversion memory completely disappeared or remained in response to low-frequency stimulation (c) or DHPG (g). Inset: Representative field excitatory postsynaptic potential trajectories for time points illustrated. Scale bar: 100 microvolts on the ordinate, 10 ms on the abscissa. (d,f) Summary of the number of activated channels and the number of channels exhibiting long-term synaptic depression per slice of the insular cortex. NS, there is no statistical difference after comparing the two groups using the unpaired Student's t-test; ^* P<0.05, ^*** P<0.001, after comparing the two groups using the unpaired Student's t-test, it shows that the two groups have significant statistical difference. (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 in neurons in the insular cortex of mice whose taste aversive memory completely disappeared or remained. (g) Representative trajectories. (h) Aggregated Data. n = 20–23 cells (7 mice per group). ^* P<0.05, ^** P<0.01, ^*** P<0.001 After using unpaired Student's t-test to compare the two groups, it shows that the two groups present a significant statistical difference.

Example 8 Deletion of ASIC1a selectively impairs extinction of conditioned taste aversion memory

ASIC1a plays an important role in the induction of long-term synaptic inhibition in the insular cortex. It is expected that ASIC1a gene-deficient mice may show normal acquisition and retention of conditioned taste aversion memory, but may have obstacles in conditioned taste aversion extinction. The results showed that wild-type and ASIC1a knockout mice were indeed comparable in the acquisition of conditioned taste aversion memories. On the day of conditioning training, the two strains of mice ingested similar amounts of sugar water (wild type: 1.93±0.11 g, n=15; ASIC1a knockout mice: 1.74±0.15 g, n=14; P= 0.3020). On the first day after conditioning, both groups of animals developed similar aversive responses (Fig. 8a), suggesting that ASIC1a does not play a significant role in the acquisition and retrieval of conditioned taste aversive memories . The conditioned taste aversion memory strength test was carried out on the following 3 days, 7 days, 14 days and 28 days, and it was found that the memory retention strength of wild-type and ASIC1a knockout mice was similar (Fig. Mice, like wild-type mice, were able to retain normal conditioned taste aversion memory for at least 1 month.

In contrast, ASIC1a-knockout mice failed to show significantly reduced aversiveness in the subsequent conditioned taste aversion extinction test, while wild-type mice showed it two to three days after the taste-selection test. Significantly reduced memory strength (Fig. 8c). Similarly, ASIC1a knockout mice also showed significantly impaired extinction compared with wild-type mice when extinction training was performed on long-term conditioned taste aversion memory (Fig. 8d). These results suggest that ASIC1a selectively contributes to the extinction of conditioned taste aversion memories, but has no significant effect on memory acquisition or retention.

As a control, in order to confirm that ASIC1a gene-deficient animals have complete taste discrimination ability, an unconditioned two-bottle taste preference test (Yu, H., et al.. (2009). Variant BDNF Val66Met polymorphism effects extinction of conditioned aversive memory. J Neurosci 29, 4056-4064), examined the preferences of mice for sweet, salty, bitter and sour solutions separately and found no significant difference between wild-type and ASIC1a knockout mice.

In Figure 8, wild-type and ASIC1a knockout mice were trained on the 0th day of conditioned taste aversion, and on the first day were tested for the acquisition of taste aversive memory; using different batches of mice, they were conditioned On the 3rd, 7th, 14th, and 28th days after the taste aversion training, the taste aversion memory test was performed, which was defined as the test of memory strength retention; for the extinction test, the mice that had established the conditioned taste aversion memory were treated with Given a bottle of sugar water and a bottle of water for a choice test, the continuous taste choice test 1–7 days after memory establishment is called the extinction of recent taste aversion memory; the continuous taste choice test 15–21 days after memory establishment is called far Phase extinction of gustatory aversive memory. Among them, the data on the first day in Figure (c) is the same as the data in Figure (a), and they are presented repeatedly for better comparison. n=8–16. NS, there is no statistical difference after comparing the two groups using the unpaired Student's t-test; ^* P<0.05, ^** P<0.01, ^*** P<0.001, using the unpaired Student's t-test to compare the two groups Afterwards, there was a statistically significant difference between the two groups.

Example 9 The key role of ASIC1a in the insular cortex for the extinction of conditioned taste aversion

In order to clarify the role of ASIC1a on the insular cortex for the extinction of conditioned taste aversion, the aforementioned ASIC1a conditional gene knockout mice, ie ASIC1a ^flox/flox mice, were used again. Injection of AAV-Cre virus into the insular cortex of ASIC1a ^{flox /flox} mice (Fig. 9a) resulted in animals showing significantly reduced extinction of conditioned taste aversion (Fig. 9b) compared to injection of AAV control virus, supporting the insular expression of ASIC1a plays a specific role in extinction of conditioned taste aversion.

To further test whether acute inhibition of ASIC1a in the insular cortex affects extinction of conditioned taste aversion, animals with established conditioned taste aversion memory were given the ASIC1a inhibitor, PcTx1 (10 pmol) immediately after the two-bottle choice test Or its solvent control (Fig. 9c). Administration of PcTX1 (Fig. 9d) significantly blocked extinction of conditioned taste aversion compared to vehicle control (Fig. 9e).

To test whether reexpression of ASIC1a in the insular cortex of ASIC1a-knockout mice could rescue the extinction abnormalities in gene-deficient animals. Using the intervention strategy shown in Example 4, the difference is that in the experiments here, the animals were trained 1 month after AAV virus injection for conditioned taste aversion, followed by taste selectivity 3-6 days after training Test (Fig. 9f). As expected, injection of ASIC1a-overexpressing virus into the insular cortex of ASIC1a-deficient mice resulted in animals with extinction levels of conditioned taste aversion close to wild-type mice, whereas injection of control or ASIC1a-HIF mutant overexpressing virus did not. A similar effect was observed (Fig. 9g). Thus, functional ASIC1a in the insular cortex is the key missing component in ASIC1a knockout mice that supports the animals' conditioned taste aversion extinction learning.

Using another strategy to eliminate ASIC1a expression, an AAV viral vector expressing short hairpin RNA (shRNA) targeting ASIC1a was constructed and driven by the U6 promoter (Fig. 9i). After confirming the effectiveness of ASIC1a-shRNA on Chinese hamster ovary cells, AAV-ASIC1a-shRNA or negative control virus was injected into the insular cortex of wild-type mice (Fig. 9h). Compared with the AAV negative control virus, the AAV-ASIC1a-shRNA virus significantly reduced the expression of AISC1a protein in the insular cortex (P=5.651E-05, n=4–6, Figure 9j), and also caused the extinction process of conditioned taste aversion to be suppressed. Significantly blocked (Fig. 9k). On the basis of ASIC1a-shRNA, a rescue strategy was added to excitatory neuron type-specific reexpression of ASIC1a (Fig. 9l). The AAV viral vector here includes, in addition to the ASIC1a-shRNA element, the expression sequence of the shRNA-insensitive mCherry-2A-ASIC1a (denoted as ASIC1a ^* ) double-flox control reverse (double-floxedinverted orientation, DIO), the latter It can be expressed normally only in the presence of Cre recombinant endonuclease (Fig. 9m). This AAV virus was injected into the insular cortex of CaMKII-Cre tool mice (Cre recombinase is only expressed in the pyramidal neurons of the forebrain), and it was found that there were mCherry (Figure 8n) and Significant expression of ASIC1a (Fig. 9o). 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 (Figure 9m) contained in the AAV virus vector is also effective, showing that the wild-type mice injected with the virus have significantly abnormal conditioned taste aversion extinction, that is, they show a higher disgust index (Figure 9p) . In contrast, in CaMKII-Cre tool mice, injection of AAV virus resulted in the extinction of conditioned taste aversion close to that of normal control animals (Fig. 9p). Taken together, the above results establish a critical role for ASIC1a in the insular cortex in extinction of conditioned taste aversion.

In Figure 9, (a,c,f,h,l) are behavioral steps. (b,e,g,k,p) are the time course-dependent changes in the disgust index caused by conditioned taste aversion extinction. ^* P<0.05, ^** P<0.01, ^*** P<0.001, after using the unpaired Student's t-test to compare the two groups, it shows that the two groups present a significant statistical difference. (b) n=7. (e) n=10–11. (g) n=8–13. ^* P<0.05, ^*** P<0.001, WT+AAV-Ctrl vs. KO+AAV-Ctrl; ^#P <0.05, ^## P<0.01, KO+AAV-ASIC1avs .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 ^* injected, unpaired Student's t-test. (d) Schematic diagram of the diffusion area after targeted injection of drugs in the insular cortex. (i,m) Schematic diagram of the composition of AAV vectors. (j,o) Representative western blot pictures. (n) Image of mCherry expression in the insular cortex, here negatively stained with DAPI.

Example 10 Long-term synaptic depression in the insular cortex is necessary for extinction of conditioned taste aversion

To further establish the significance of ASIC1a-dependent long-term synaptic inhibition in the insular cortex for the extinction of conditioned taste aversion, the behavioral consequences of blocking long-term synaptic inhibition in the insular cortex in vivo were studied. The endocytosis of AMPA receptors is a common mechanism of long-term synaptic inhibition, which can be administered from intracellularly by a short GluA2-mimetic peptide - GluA2-3Y, or by extracellularly administered GluA2 with the ability to penetrate the model Blocked by a short mimic peptide - FITC-Tat-GluA2-3Y (Tat-3Y, Figure 10a). Wherein, the ability of the polypeptide to pass through the mold is realized by including a short Tat peptide derived from Human Immunodeficiency Virus (Humanimmunodeficiency virus, HIV). Its negative control, FITC-Tat-GluA2-3A (Tat-3A, FIG. 10a ), although it also has the ability to penetrate the mold, cannot exert the effect of blocking AMPA receptor endocytosis. Administration of Tat-3Y (1 micromol/L) before and during low-frequency stimulation significantly blocked the induction of long-term synaptic depression in the insular cortex, while the control polypeptide Tat-3A (1 micromol/L) did There was no such effect (Fig. 10b). Among them, the induction rate of long-term synaptic inhibition in the brain slices treated with Tat-3Y polypeptide was 16.0±7.1%, while the induction rate of brain slices treated with the control polypeptide Tat-3A polypeptide was 78.0±6.1%. Sexual difference (P=5.900E-05, Figure 10c). These results suggest that GluA2-subunit-dependent endocytosis of AMPA receptors is a critical step in long-term synaptic inhibition in the insular cortex.

The effect of administering insular cortex Tat-3Y polypeptide (100 pmol) in vivo on the extinction of conditioned taste aversion is shown in Fig. 10d. Based on the FITC green fluorescence exhibited by the insular cortex, the effectiveness and specificity of Tat-3Y polypeptide administration was confirmed ( FIG. 10 e ). Animals receiving the Tat-3Y polypeptide showed significantly abnormal extinction of conditioned taste aversion compared to mice receiving the control polypeptide Tat-3A or vehicle control (Fig. 1Of). This result supports that long-term synaptic depression in the insular cortex is required for extinction of conditioned taste aversion. In conclusion, insular cortex ASIC1a plays a key role in conditioned taste aversion extinction.

In Figure 10, (a) Interfering peptide sequence that blocks AMPA receptor endocytosis, the key tyrosine residue (Y) is indicated in red; when the key tyrosine residue is mutated to alanine (A) The resulting polypeptide lost the effect of blocking and interfering with AMPA receptor endocytosis, so it was used as a control peptide. (b) Acute administration of Tat-3Y interfering peptide (1 μmol/L, starting 20 minutes before the low-frequency stimulation step and continuing for 35 minutes) blocked the long-term synaptic depression induced by low-frequency stimulation, but administration of the control peptide ( Tat-3A) had no significant effect. (c) Summary of the number of activated channels and the number of channels exhibiting long-term synaptic depression per insular cortex slice. NS, there is no statistical difference after comparing the two groups using the unpaired Student's t-test; ^*** P<0.001, after comparing the two groups using the unpaired Student's t-test, it shows that the two groups present a significant statistical difference difference. (d) Behavioral steps. (e) Schematic diagram of where the interfering peptide is injected and its invasion into the interior of the cell. (f) Conditioned gustatory aversion extinction caused time-course-dependent changes in the aversion index. n=10–14. ^*** P<0.001, using the unpaired Student's t-test to compare the two groups, it shows that there is a significant statistical difference between the Tat-3A and Tat-3Y groups.

Example 11 Pharmacological blockade or activation of ventral hippocampus ASIC1a inhibits and promotes fear extinction, respectively

To examine the physiological significance of ASIC1a in fear extinction, microinjection of saline, ASIC1a agonist MitTx-α/β or ASIC1a blocker PcTX1 (both injection volume 1 μl/side, Fig. 11a,b), observation and quantitative statistics of animals' fear response to CS during extinction learning on the second day and fear test on the third day. Data are presented in the form of mean ± standard error. The number of animals was 8-14 mice per group.

The experimental formulations were selected from the group:

(i) MitTx-α/β, 1 micromol/liter, dissolved in physiological saline.

(ii) PcTX1, 10 μmol/L, dissolved in physiological saline.

The results are as follows (saline-treated groups are represented by blank graphs, MitTx-α/β or PcTX1-treated groups are represented by filled graphs):

During fear learning, animals' fear responses to CS gradually increased, and animals' fear responses to CS gradually decreased on the second day during extinction learning, while animals could maintain a low fear response to CS on the third day. Compared with the normal saline group, the injection of the ASIC1a agonist MitTx-α/β in the ventral hippocampus before animals extinction learning can significantly promote fear extinction, that is, the MitTx-α/β-treated animals maintained lower fear responses to CS on the third day (Fig. 11c,d); in contrast, injection of the ASIC1a blocker PcTX1 into the ventral hippocampus of animals before extinction learning significantly inhibited fear extinction.

Example 12 Conditional knockout or overexpression of ASIC1a in the ventral hippocampus inhibits and promotes fear extinction, respectively

Inject AAV-GFP/AAV-ASIC1a in the ventral hippocampus of wild-type C57BL/6J mice (injection volume is 1 μl/side), or in the ventral side of conditional ASIC1a gene knockout-ASIC1a ^flox/flox mice The hippocampus was injected with AAV-GFP/AAV-Cre (injection volume was 1 microliter/side), and the expression of the virus was observed and quantitatively counted one month after the animal was in fear learning, extinction learning, and fear detection on the third day. Fear response of CS (Fig. 12a). Data are presented in the form of mean ± standard error. The number of animals was 13-19 mice per group.

The experimental formulations were selected from the group:

(i) AAV-GFP with a titer exceeding 10 ¹² viral genomes (viral genomes, VG)/milliliter (ml), dissolved in sterile PBS.

(ii) AAV-Cre with a titer exceeding 10 ¹² VG/ml, dissolved in sterile PBS.

(iii) AAV-ASIC1a with a titer exceeding 10 ¹² VG/ml, dissolved in sterile PBS.

The results are as follows (the AAV-GFP treatment group is represented by a blank graph, and the AAV-Cre or AAV-ASIC1a treatment group is represented by a filled graph):

Overexpression of ASIC1a in the ventral hippocampus did not affect fear learning on the first day, but the subsequent extinction learning was enhanced (Fig. effect, but subsequent extinction learning was impaired (Fig. 12d,e).

The above-mentioned embodiments only express the implementation manner of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. The use of an acid-sensitive ion channel modulator in the preparation of a medicament for treating mental disorders, the acid-sensitive ion channel being an acid-sensitive ion channel comprising an ASIC1a subunit. 2. The use according to claim 1, characterized in that the regulating agent comprises an acid-sensitive ion channel promoter or inhibitor. 3. purposes according to claim 2, is characterized in that, the mental disorder disease that described acid-sensitive ion channel enhancer is used for treatment is any nervous system disease that is accompanied by abnormal obstinate pathological memory, comprises: anorexia, anxiety syndrome, addictive disorders, or bulimia nervosa and obesity. 4. purposes according to claim 2, is characterized in that, the mental disorder disease that described acid-sensitive ion channel inhibitor is used for treatment is any neurological disease accompanied with cognitive impairment and memory impairment, including Alzheimer's disease. sick. 5. The use according to claim 2, characterized in that, the acid-sensitive ion channel enhancer includes any compound or recombinant vector that increases the expression and/or activity of the ASICIa subunit protein. 6. The use according to claim 5, characterized in that, the acid-sensitive ion channel enhancer compound comprises polypeptide MitTx-α/β, and/or spermine. 7. The use according to claim 2, wherein the acid-sensitive ion channel inhibitor includes any compound or recombinant vector that specifically inhibits the expression and/or activity of ASIC1a channel-related proteins. 8. The use according to claim 7, wherein the acid-sensitive ion channel inhibitor compound comprises polypeptide PcTX1, amiloride, amidine A-317567, nafamostat, non-steroidal anti-inflammatory drugs , an alkaline neutralizing agent or a combination thereof, the alkaline neutralizing agent comprising sodium bicarbonate, ammonium chloride, an alkali metal salt, an alkaline earth metal salt, or a combination thereof. 9. A pharmaceutical composition for treating mental disorders, comprising a safe and effective amount of an acid-sensitive ion channel modulator and a pharmaceutically acceptable carrier, the acid-sensitive ion channel being an acid-sensitive ion comprising an ASIC1a subunit aisle. 10. A method for screening candidate drugs for the treatment of mental disorders, comprising the following steps: Detect the effect of the test compound on the acid-sensitive ion channel containing the ASIC1a subunit, and select the test compound that has a regulatory effect on the acid-sensitive ion channel as the primary screening compound; Detect the therapeutic or alleviating effects of the primary screened compounds on mental disorders, and select compounds with curative effects as candidate drugs.