CN111789951A - Methods and pharmaceutical compositions for preventing fear memory and related diseases - Google Patents

Abstract

The present invention provides methods of preventing and treating fear memory and related disorders such as anxiety disorders by increasing astrocyte excitability. The present invention also provides a pharmaceutical composition containing a preparation for increasing astrocyte excitability, which can be used for the prevention and treatment of fear memory and related diseases.

Description

Methods and pharmaceutical compositions for preventing fear memory and related diseases

technical field

The present invention relates to the field of disease treatment and medicine. In particular, the present invention relates to methods and pharmaceutical compositions for the prevention and/or treatment of fear memory and related disorders such as anxiety disorders.

Background technique

Astrocytes are widely present in the central nervous system and are the most numerous glial cells. Providing energy metabolism and structural support for neurons, maintaining water, ion, neurotransmitter homeostasis, regulating blood flow and blood-brain barrier, etc. are considered to be the traditional functions of astrocytes (Allaman, I., et al., Trends Neurosci., 2011.). In recent years, more and more studies have shown that the processes of astrocytes and the presynaptic and postsynaptic processes of neurons form a tripartite synapse (Zhang, J.M. et al. Neuron., 2003). Astrocytes respond to neuronal synaptic activity with an increase in cytoplasmic calcium concentration, leading to the release of glial transmitters that regulate synaptic activity and plasticity, such as long-term potentiation. potentiation, LTP) and long-term depression (long-term depression, LTD). LTP and LTD are considered molecular mechanisms of memory processing. Studies in animals and humans have shown that astrocytes undergo morphological and molecular changes in hippocampus-dependent scene or spatial memory processing, suggesting that astrocytes may be involved in memory processing (Choi, M., Mol. Brain., 2016; Sagi, Y., Neuron., 2012). The involvement of pyramidal neurons and interneurons in the hippocampus in the formation of fear memory has received extensive attention, but there is still a lack of sufficient evidence whether astrocytes participate in and regulate fear memory.

Learning is the process of acquiring information and knowledge, while memory is the process of preserving the learned information. The content of short-term memory can be transformed into long-term and even permanent storage through the process of memory consolidation, but the process of memory consolidation does not necessarily require short-term memory as an intermediary. Long-term memory and short-term memory may coexist in parallel (Mark F. Bear et al., Neuroscience: Explroing the Brain, ^2nd Edition, 2001).

Memory consolidation process Memory consolidation is a process in which new memories are gradually stabilized after they are acquired. During this process, processing at the molecular and cellular levels occurs, enabling intracellular signaling cascades, activating transcription factors leading to gene expression and protein synthesis, enhancing synaptic plasticity in the hippocampus and other brain regions (long-term potentiation, LTP) and Synaptic connections, a process also known as synaptic consolidation (Mednick, S.C., et al., Trends Neurosci. 2011). LTP is widely regarded as the molecular mechanism of learning and memory. Studies in isolated brain slices, anesthetized and freely moving animals have shown that LTP can be divided into two phases, early LTP independent of protein synthesis and late LTP dependent on protein synthesis.

Traumatic memories can lead to inappropriate behavioral responses and severe physical or psychological harm (Izquierdo, I., et al., Physiol. Rev., 2016). In humans, traumatic fear memory causes a variety of psychiatric disorders, including schizophrenia, posttraumatic stress disorder (PTSD), anxiety disorders, phobias (Luthi, A., et al., Nat. Neurosci., 2014; Garfinkel, S., N. et al., J. Neurosci., 2014). The estimated lifetime prevalence of fear- and stress-related disorders is approximately 29% (Kessler, R.C., et al., Arch. Gen. Psychiatry., 2005). Fear extinction, also known as exposure therapy, is a behavioral approach to reducing fear. But exposure therapy is a type of learning that relies on contextual cues and cannot interrupt and eliminate the fear memory that is initially generated. Studies have shown that fear memories spontaneously recover or renew when patients undergoing exposure therapy are re-exposed to similar or identical situations as when the trauma occurred (Britton, J.C., et al., Depress. Anxiety. , 2011; Tovote, P., et al., Nat. Rev. Neurosci., 2015). Therefore, it is necessary to find a new strategy that can specifically interrupt or prevent the formation of fear memory, or reduce or eliminate the formed fear memory.

There is also a need in the art for safer methods and medicaments that are effective in the prevention and/or treatment of fear memory or its related disorders, such as anxiety disorders, without producing or reducing side effects.

SUMMARY OF THE INVENTION

The present invention finds for the first time that the activation of hippocampal CA1 astrocytes plays an important role in regulating the memory consolidation of fear memory, and even in reducing fear memory and preventing and/or treating fear memory related diseases such as anxiety disorders. Specifically, the inventors found that astrocyte activation can consistently and significantly eliminate or reduce the formation of fearful memories, without affecting the formation of new memories. The inventors further found that activation of astrocytes reduces fear memory through ATP degradation products adenosine and adenosine A1 receptors, and found that local injection or intraperitoneal injection of astrocyte excitability can reduce fear memory and Related disorders such as anxiety disorders. The present invention thus provides methods and medicaments for preventing and treating fear memory or its related diseases such as anxiety disorders by increasing the excitability of astrocytes.

In one of its aspects, the present invention provides a method of modulating memory consolidation of fear memory in a mammal, comprising increasing astrocyte excitability in said subject, thereby inhibiting the occurrence of lactation following an event (eg, fear stimulus) Steps in the formation of fear memory in animals.

In one of its aspects, the present invention provides a method of preventing and treating fear memory or a related disease thereof in a subject comprising increasing astrocyte excitability, such as by light stimulation or administration Stimulant preparations.

Correspondingly, the present invention provides the use of a preparation for increasing the excitability of astrocytes in the preparation of a medicament for regulating the memory consolidation of fear memory in mammals. Correspondingly, the present invention also provides the use of the preparation for increasing the excitability of astrocytes in the preparation of a medicament for preventing and treating fear memory or its related diseases in a subject. wherein the agent that increases astrocyte excitability inhibits the formation of fear memory in mammals following an event, such as a fearful stimulus.

The present invention also provides a pharmaceutical composition for preventing and treating fear memory or its related diseases, which comprises a preparation for increasing the excitability of astrocytes.

A subject in need of the methods and medicaments (pharmaceutical compositions) described herein can be mammals, including humans or non-human primates such as monkeys. Mammals can be other animals such as rats, mice, rabbits, pigs, dogs, and the like. The mammal may be a domestic animal such as a cat or a dog.

Manifestations of astrocyte excitability include astrocyte calcium signaling responses. In the absence of neuronal activity, astrocytes can produce spontaneous calcium elevations; during synaptic activity, neurotransmitters released also trigger astrocyte calcium elevations. Synaptic regulation of astrocyte calcium signaling is based on astrocytes expressing a large number of functional neurotransmitter receptors. Most of these receptors are metabotropic and G protein-coupled, and once activated, stimulate phospholipase C to form IP ₃ , which in turn increases intracellular calcium ion concentration by releasing calcium ions through IP ₃ -sensitive calcium stores in cells.

Agents that increase astrocyte excitability include optogenetics, chemogenetics, or chemical drugs.

In the present invention, the optogenetic agent is an agent that can express light-sensing genes (eg, ChR2, eBR, NaHR3.0, Arch or OptoXR) in astrocytes.

In the present invention, the chemogenetic agent is an agent that can express astrocyte excitatory protein in astrocytes, such as an agent that expresses neurotransmitter receptors, including acetylcholine ( acetylcholine, ACh), glutamate (glutamate), ATP, adenosine (adenosine), γ-aminobutyric acid (GABA), norepinephrine (norepinephrine), dopamine (dopamine), endogenous Cannabinoids (endocannabinoids), nitric oxide (nitric oxide), histamine (histamine).

In the present invention, the chemical drugs that can increase the excitability of astrocytes include but are not limited to:

a. Neurotransmitters, such as acetylcholine (ACh), glutamate (glutamate), ATP, adenosine (adenosine), γ-aminobutyric acid (GABA), norepinephrine (norepinephrine) , dopamine, endocannabinoids, nitric oxide or histamine;

b. Neurotransmitter receptor agonists, such as agonists of Gq GPCRs, including endocannabinoid receptors (CB1Rs) agonists; metabotropic glutamate type I and II receptor agonists and AMPA receptor agonists ; purinergic receptor agonists; GABAergic receptor agonists; alpha-adrenergic receptor agonists, dopaminergic receptor agonists; histaminergic receptor agonists; protease-activated receptors ) agonist;

or

c. Agonists of ion channels, such as transient receptor potential cation channel, subfamily V, agonist of member 1 (transientreceptor potential cation channel, subfamily V, TRPV1); Na ⁺ /Ca ^{2 +} exchangers (NCXs) agonist.

In the present invention, fear memory particularly includes traumatic fear memory. Fear memory-related diseases refer to various mental diseases caused by fear memory, including schizophrenia, posttraumatic stress disorder (PTSD), anxiety disorder (anxiety disorder), and phobia (phobias). and depression, etc.

In the present invention, fear memory can be fear memory mediated in different brain regions, and can particularly refer to "hippocampal CA1-mediated fear memory". The inventors of the present application have found and proved that the activity of astrocytes in hippocampal CA1 plays an important role in the formation of fear memory and anxiety disorders: astrocyte activation can significantly reduce fear memory continuously and significantly without affecting the development of new memory. form. In addition, the inventors found that astrocyte activation reduces fear memory and anxiety by increasing the activity of adenosine A1 receptor-involved channels.

The method of the present invention was shown to be more effective in the consolidation phase of fear memory. When the brain obtains external information, it forms short-term memory (STM), and when short-term memory enters long-term memory (LTM), the information is strengthened and integrated, and it becomes stable after being stored in specific brain areas. This is the consolidation phase of memory. Reconsolidation of memory refers to the process in which the consolidated memory is reactivated and modified under similar induction, and the original stable memory becomes fragile and volatile; the reactivated memory is re-stably preserved.

Memory consolidation is the process by which new memories are gradually stabilized after acquisition. During this process, processing at the molecular and cellular levels occurs, enabling intracellular signaling cascades, activating transcription factors leading to gene expression and protein synthesis, enhancing synaptic plasticity in the hippocampus and other brain regions (long-term potentiation, LTP) and Synaptic contact, a process also known as synaptic consolidation, typically occurs within 6 hours of memory acquisition (Mednick, S.C., et al, Trends Neurosci., 2011). LTP is widely regarded as the molecular mechanism of learning and memory. Studies in isolated brain slices, anesthetized and freely moving animals have shown that LTP can be divided into two phases, early LTP independent of protein synthesis, lasting approximately 2 hours, and late LTP dependent on protein synthesis. With prolonged post-LTP induction, such as drug treatment or other interventions 1-2 hours later, it is difficult to affect LTP maintenance (Fujii et al., Brain Research. 1991; Abraham, W.C. et al, Neurobiol. Learn. Mem. 2008 ;). Behavioral studies have shown that when the intervention occurs within a relatively short period of time after learning (such as 1 hour), it is easier to block memory formation, while if it occurs in a longer time after learning (such as more than 6 hours), it is more likely to block memory formation. Does not easily interfere with memory formation (Mednick, S.C., et al, Trends Neurosci., 2011; Dudai, Y. Rev. Psychol. 2004). Therefore, once LTP or memory enters the protein synthesis-dependent stage, memory is difficult to be erased due to consolidation, so interventions should be taken to block the formation of fear memory in the early stage of synaptic consolidation.

The methods of the present invention can be used to prevent and treat fear memory or its related diseases. Intervention within a certain time window after the occurrence of a specific event leading to fear memory can significantly block and reduce the fear memory and related anxiety caused by this event without recurrence. The methods of the present invention are thus suitably administered within about 6 hours of exposure to the fearful stimulus, preferably within about 2 hours of exposure to the fearful stimulus.

In another aspect of the present invention, the method or pharmaceutical composition of the present invention is particularly suitable for emergency prevention and treatment of fear memory or its related diseases. The methods or pharmaceutical compositions of the present invention can be used for emergency treatment, such as prophylactic treatment of a subject within a relatively short period of time after experiencing an occasional accident or a sudden mass injury event. In another aspect of the present invention, the present invention is suitable for preventing people, especially fear-susceptible people (such as those commonly known as "timid" people) from acquiring fear memories and even developing related diseases such as anxiety after experiencing a generally fear-inducing event disease, etc.

In one aspect of the present invention, the methods and pharmaceutical compositions of the present invention for treating fear memory or its related diseases are suitable for use in patients for whom other methods and drugs for treating fear memory are ineffective. The inventors of the present application discovered and proved for the first time that the astrocytes of the hippocampus, especially the hippocampal CA1, have an important role in fear memory, thus providing the treatment (inhibition) of fear memory by stimulating the astrocytes of the hippocampus CA1 Methods and medicines for or related diseases. This is the known mechanism in the art for the treatment of fear memory or its related diseases and the pathological mechanism that the drugs fail to target, and the target tissue of the brain for treatment or the target at the molecular level.

In one embodiment of the invention, an agent that increases astrocyte excitability can be administered systemically. Agents that increase astrocyte excitability can be administered intraperitoneally, intravenously, orally, intramuscularly, or subcutaneously to achieve this systemic effect. The most suitable systemic delivery route depends, at least in part, on the pharmacological properties of the agent chosen to increase astrocyte excitability. In a more preferred embodiment, the dosage of the agent that increases astrocyte excitability is 0.05-50 mg/kg.

If desired, formulations that increase astrocyte excitability can be formulated for topical administration to the brain, particularly the hippocampus. For example, formulations are prepared for local injection through a cannula. The advantage of topical formulations is that any possible systemic side effects of the drug can be avoided.

In one aspect of the present invention, in the method for treating and preventing fear memory and related diseases of the present invention and the medicament (pharmaceutical composition) for treating and preventing fear memory and related diseases of the present invention, the method, medicament or The pharmaceutical compositions are locally acting in the hippocampus, particularly hippocampal CA1, that is, methods and medicaments for administration in the hippocampus, particularly hippocampal CA1. For drugs intended for use on neural tissue, particularly in the brain, eg, the hippocampus, it is beneficial to limit the effect of the drug to the target tissue. For topical administration in the hippocampus, both the method of treatment and the preparation of the drug are limiting technical features. The method or drug used in the hippocampus, especially hippocampal CA1, needs to consider whether the method or drug can exert the effectiveness of the drug in the hippocampus, especially hippocampal CA1, including whether the drug can reach the hippocampus, especially hippocampal CA1, and whether the drug can reach the hippocampus, especially hippocampal CA1, and whether the drug can reach the hippocampus, especially hippocampal CA1. Whether it can reach the effective concentration in hippocampal CA1 and so on. In the present invention, the medicament or pharmaceutical composition is in the form of topical administration in the hippocampus, especially the hippocampal CA1. The effect of the drug can be limited to the target tissue by local administration, for example, by preparing the preparation for increasing astrocyte excitability into a dosage form that can be implanted into the hippocampus, especially hippocampal CA1, for local administration through a cannula. For another example, the drug is made into a dosage form for sustained release after implantation in tissue, and the like. In addition, the above-mentioned drugs can be formulated in the form of tissue-specific targeted drug delivery systems. For example, an agent that increases astrocyte excitability can be encapsulated in hippocampus-targeted exosomes.

The active ingredient in the pharmaceutical composition provided by the present invention is a preparation for increasing the excitability of astrocytes. Although the active ingredient in the pharmaceutical compositions of the present invention suitable for use in therapy may be administered in the form of the starting compound, it is preferred that the active ingredient, optionally in the form of a physiologically acceptable salt, be combined with one or more adjuvants Agents, excipients, carriers, buffers, diluents and/or other conventional pharmaceutical adjuvants are incorporated into the pharmaceutical composition.

The pharmaceutical compositions of the present invention may be administered by any convenient route suitable for the desired therapy. Preferred routes of administration include oral administration, especially in the form of tablets, capsules, lozenges, powders and liquids; and parenteral administration, especially dermal, subcutaneous, intramuscular and intravenous injection. The pharmaceutical compositions of the present invention can be prepared by those skilled in the art using standard methods and conventional techniques appropriate to the desired formulation. If desired, compositions suitable for sustained release of the active ingredient can be used.

The pharmaceutical compositions of the present invention may be those suitable for oral, rectal, bronchial, nasal, pulmonary, topical (including buccal and sublingual), transdermal, vaginal or parenteral (including dermal, subcutaneous, intramuscular, intraperitoneal, intravenous Pharmaceutical compositions for intra-arterial, intracerebral, intraocular injection or infusion) administration, or those suitable for administration by inhalation or insufflation (including powder and liquid aerosol administration) or suitable for administration by sustained release Pharmaceutical compositions in the form of systemic administration. Examples of suitable sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compounds of the present invention, which matrices may be in the form of shaped articles such as films or microcapsules.

Thus, the active ingredients in the pharmaceutical compositions of the present invention can be formulated with conventional adjuvants, carriers or diluents in the form of pharmaceutical compositions and unit dosages thereof. Such forms include solids, especially tablets, filled capsules, powders and pellets, as well as liquids, especially aqueous or non-aqueous solutions, suspensions, emulsions, elixirs and filled capsules, all of which are For oral administration, suppositories for rectal administration, and sterile injectable solutions for parenteral administration. Such pharmaceutical compositions and unit dosage forms thereof may include conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount commensurate with the desired daily application dosage range the active ingredient.

For preparing pharmaceutical compositions from the active ingredients in the pharmaceutical compositions of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

When desired, compositions suitable to provide sustained release of the active ingredient may be employed.

The pharmaceutical formulation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

A therapeutically effective dose means the amount of active ingredient that relieves a symptom or condition. Therapeutic efficacy and toxicity, such as ED50 and LD50, can be determined by standard pharmacological procedures in cell cultures or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed by the ratio LD50/ED50.

The dose administered must, of course, be carefully adjusted with respect to the age, weight and condition of the individual being treated, as well as the route, dosage form and regimen of administration, and the desired results, and the exact dose should of course be determined by the physician.

The actual dosage will depend on the nature and severity of the disease being treated, the exact mode of administration and dosage form, and may be varied, within the judgment of the physician, by escalating dosages to produce the desired treatment in accordance with the circumstances of the present invention. Effect.

In one aspect of the invention, in the methods and pharmaceutical compositions of the invention, the agent that increases astrocyte excitability is administered systemically. The dosage is about 0.01-20 mg/kg, preferably about 0.05-5 mg/kg, more preferably about 0.01-1 mg/kg.

In another aspect of the present invention, in the methods and pharmaceutical compositions of the present invention, the agent that increases astrocyte excitability is administered topically to the hippocampus, particularly to hippocampal CA1. The dosage is about 0.001-0.1 mg/kg, preferably about 0.005-0.05 mg/kg, more preferably about 0.01 mg/kg.

In another aspect of the present invention, the preparation for increasing astrocyte excitability provided by the present invention is used to prepare memory consolidation for modulating fear memory in mammals, or prevent and treat fear memory or its relatedness in a subject. Use in a medicament for a disease, wherein the medicament is prepared in a dosage form for systemic administration of a preparation that increases astrocyte excitability. Preferably, the dose of the drug is about 0.01-20 mg/kg, more preferably about 0.05-5 mg/kg, for example about 0.01-1 mg/kg.

In another aspect of the present invention, the preparation for increasing astrocyte excitability provided by the present invention is used to prepare memory consolidation for modulating fear memory in mammals, or prevent and treat fear memory or its relatedness in a subject. In the use in medicine for disease, the medicine is prepared so that the preparation of astrocyte excitability is a dosage form for topical administration. For example topical application to the hippocampus, in particular to hippocampal CA1. Preferably, the dose of the drug is about 0.001-0.1 mg/kg, more preferably about 0.005-0.05 mg/kg, for example about 0.01 mg/kg.

In another aspect of the present invention, the preparation for increasing astrocyte excitability provided by the present invention is used to prepare memory consolidation for modulating fear memory in mammals, or prevent and treat fear memory or its relatedness in a subject. In the use in medicine for a disease, the medicine is an emergency pharmaceutical composition.

Description of drawings

Figure 1. Optogenetic activation of hippocampal CA1 astrocytes reduces fear memory and fear-related anxiety. a,b Schematic diagram of the CA1 optical fiber implantation in the hippocampus of rats, the white column refers to the optical fiber implantation position, and the lower part of the dotted line refers to the CA1 position. c Experimental design and procedures for fear conditioning, photoactivation paradigms, and memory and open field testing. Because the experiment wanted to explore the effect of photoactivation of astrocytes on fear memory during the memory consolidation phase, photoactivation of astrocytes was performed immediately after fear conditioning. d During fear conditioning training, rats in the control and light-activated groups had similar levels of freezing, indicating similar learning abilities. e, In the contextual fear memory test, the freezing level of rats in the light-activated astrocytes group immediately after fear conditioning training was significantly reduced, indicating that light-activated astrocytes significantly reduced contextual fear memory. f In the cued fear memory test, the level of freezing in the light-activated astrocytes group was similar to that of the control group, indicating that light-activated astrocytes did not affect cued memory. g Representative heatmap showing the trajectories of rats in the open field in the control group (without fear conditioning training), fear conditioning training group, and light-activated astrocytes group after fear conditioning training. h, Histograms show that conditioned fear training induces an anxiety phenotype compared to the control group; light-activated astrocytes after conditioned fear training effectively reverse the anxiety phenotype: significantly increase the distance traveled in the open field (h) and the exploration time (i) in the center of the open field. All data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2. Light-activated astrocytes have a long-term reducing effect on contextual fear memory and do not affect new memory formation. a Experimental design and procedures for fear conditioning training, photoactivation paradigms, and memory testing. b In the contextual fear test, the freezing level of the rats in the light-activated astrocytes group was significantly reduced immediately after fear conditioning training, which could last until 26 days after the photo-activation, indicating that the light-activated astrocytes continued and significantly decreased Scenario fear memory. c During fear conditioning training, the level of freezing of rats in the light-activated group that underwent fear conditioning again was similar to that of control rats that underwent fear conditioning for the first time, indicating similar learning abilities and also suggesting that astrocyte activation was not affect new learning. d In the contextual fear memory test, the level of freezing of the rats in the light-activated group and the rats in the control group were similar to those of the control group, which indicated that they had similar levels of fear memory, and also suggested that astrocyte activation did not affect the formation of new memories.

Figure 3. Photoactivation of astrocytes within a certain time window significantly reduces contextual fear memory. a, d, g Experimental design and procedures for fear conditioning training, photoactivation paradigms, and memory testing. b, e, h During fear conditioning training, rats in the control and light-activated groups had similar freezing levels, indicating similar learning abilities. c After light-activated astrocytes 1 hour after fear conditioning training, the level of freezing in rats was significantly reduced, indicating a significant reduction in fear memory. f,i After light-activated astrocytes 2 or 3 hours after fear conditioning training, the level of freezing in rats was similar to that in the control group, indicating no effect on fear memory.

Figure 4 Activation of adenosine and adenosine A1 receptors mediates the reduction of contextual fear memory. a Schematic diagram of bilateral cannula implantation in the hippocampal CA1 of rats. b Experimental design and process of fear conditioning training, drug injection and memory testing. c-e During fear conditioning training, rats in the control group (solvent injection) and the drug injection group had similar learning abilities. f The freezing levels of rats in the local bilateral injection of ATPrS (2 mM, 9 mM, 1 μl/side) and MesADP (5 mM, 1 μl/side) and control rats (injected with the corresponding solvent) were similar, indicating that ATPrS and MesADP Does not affect contextual fear memory.g The freezing level of rats in the group of local bilateral injections of NECA (2mM, 1μl/side) and CCPA (5mM, 1μl/side) was significantly reduced, indicating a significant decrease in contextual fear memory.h Local bilateral The freezing level of rats in the group injected with CGS 21680 (5mM, 1μl/side) was similar to that of the control group, indicating that CGS 21680 did not affect contextual fear memory.

Figure 5. Local bilateral injection of NECA in the hippocampal CA1 brain region, CCPA did not affect the spontaneous movement of animals. a Flow chart of the experiment for drug injection and open field testing. b, d Local bilateral injection of NECA (2mM, 1μl/side) and CCPA (5mM, 1μl/side) did not affect the animal's movement distance in the open field. c, e local bilateral injection of NECA in brain regions, CCPA did not affect the exploration time of animals in the center of the open field.

Figure 6 Intraperitoneal injection of adenosine A1 receptor agonists significantly reduces fear memory and anxiety. a Experimental design and flow chart for fear conditioning training, drug injection, and memory testing. Drugs were injected immediately after fear conditioning, and contextual fear memory tests were performed on days 2 and 3, respectively. b-e In the contextual fear test, rats in the control (solvent-injected) and drug-injected groups had similar levels of freezing, indicating that the animals had similar learning abilities. f Intraperitoneal injection of low-dose CCPA (0.03 mg/kg) group rats had similar freezing levels to control rats, indicating no effect on fear memory. g-i intraperitoneal injection of high doses of CCPA (0.1/0.3/1 mg/kg) in rats showed a significant reduction in freezing levels, indicating a significant reduction in fear memory. j,m Representative heatmaps showing control (no fear conditioning training), fear conditioning Movement trajectories of rats in the training group in the open field at 3 and 24 hours after the injection of solvent or drug. Compared with the control group, the conditioned fear-trained rats significantly reduced the distance of movement in the open field and the exploration time of the center of the open field, which induced an anxiety phenotype. Adenosine A1 receptor agonists were injected intraperitoneally after fear conditioning training. CCPA (0.1 mg/kg) effectively reversed the anxiety phenotype: significantly increased movement distance in the open field and exploration time in the center of the open field.

Figure 7 Intraperitoneal injection of low-dose CCPA did not affect the spontaneous movement of animals. a Representative heat map showing the trajectories of rats in the open field after injection of different doses of CCPA (0.1/0.3/1 mg/kg). b-e Low doses of CCPA (0.1 mg/kg) did not affect the spontaneous movement of animals, and high doses (0.3/1 mg/kg) significantly reduced the animals' movement distance, but returned to normal levels 5 hours or 24 hours after injection, respectively.

Detailed ways

The substance and beneficial effects of the present invention will be further described below with reference to the embodiments, which are only used to illustrate the present invention and not to limit the present invention.

Example 1 Materials and Methods

Experimental methods and materials

animal material

Male GFAP-ChR2-EYFP SD rats (8-12 weeks old), wild-type SD rats (8-12 weeks old). GFAP-ChR2-EYFP rats were produced by the Institute of Neuroscience, Chinese Academy of Sciences. There were 4-5 rats/cage, with free access to water and food, and were reared in a 12-hour light-dark cycle (with light from 7:00 to 19:00). Rats after surgery were housed in single cages. All animal experiments were approved by the Animal Care and Use Committee of Zhejiang University.

Stereotaxic surgery embedded fiber optic cannula

Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (1%, wt/vol) and fixed on a stereotaxic apparatus. A heating pad is used to control body temperature during surgery to keep body temperature stable. Cut the scalp with ophthalmic scissors. Drill holes at the target site with a skull drill. In the hippocampal CA1 brain area bilaterally (anterior-posterior distance bregma: 3.75mm (AP), left and right lateral opening ± 2.46mm (ML), the skull surface vertically downward -2.63mm (DV)) embedded fiber optic sleeves (core diameter 200μm, NA 0.37, Newdoon, China). The fiber optic sleeve is connected to the laser through fiber jumpers (Newdoon, China). The output power of the fiber tip was measured with an optical power meter (LP1, sanwa, Japan) before each experiment.

Stereotaxic surgery implanted drug delivery cannula

For the pharmacological experiments, two drug delivery cannulas (RWD Life Science, China) were embedded in the bilateral hippocampal CA1 (anteroposterior distance bregma: 3.75mm (AP), left and right lateral opening ± 2.46mm (ML), the skull surface down -2.63mm(DV)). The optical fiber and drug delivery cannula were fixed to the skull surface with dental cement. After the dental cement has completely set, insert a core of the same length as the casing into the casing, and tighten the nut to prevent the core from falling off. Animals recovered 7 days after surgery to begin behavioral testing. After the experiment, the location of the fiber and the cannula was histologically identified. Animals with incorrect fiber and cannula positions were excluded.

conditioned fear experiment

Fear conditioning experiments were performed in a 25 × 25 × 25 cm fear box (Panlab Harvard Apparatus, Spain). The fear conditioning experiment consists of three stages: fear conditioning, hippocampus-dependent contextual fear test (Kim, J.J et al., Science 256, 675-677, 1992) and non-hippocampus-dependent contextual fear test The hippocampus-independentcued fear test (Phillips, R.G. et al., Behav. Neurosci. 106, 274-285, 1992). Rodents display characteristic immobility responses when they are frightened. Animals were defined as freezing if they were immobile for more than 2 seconds. Three days before the start of the experiment, the animals were placed in the experimental environment and handled once a day. On the first day of the experiment, the conditioned fear box was wiped with 70% alcohol, and the box contained a stainless steel floor grill that could be used to deliver the shock. The rats were gently put into the box to acclimate for 2 minutes and the basic freezing level during this period was recorded. Then, a 30-second sound stimulation (2kHz, 85dB) was given. When the sound continued to the 29th second, the rats were given a foot shock (0.6mA) for 2 seconds. The sound and the shock ended at the same time. A total of 3 times of sound-shock pairing training was given, with an interval of 60 seconds between each time. Rats remained in the training box for 90 seconds after the last shock and then returned to the cage. Fear conditioning training lasted 7 minutes in total, and the whole process was completed in a relatively dark box. Twenty-four hours after the conditioned fear training, a contextual fear memory test was performed, and the rats were placed in the same training box for 5 minutes to detect their freezing time. After 2 hours, a cued fear memory test was performed. The cued fear test was performed in a brighter box with a different environment and smell than the training box, wiped with 1% acetic acid. After 1 minute of acclimatization, rats were given three 30-second sound stimuli (2kHz, 85dB), with 30-second intervals between each sound stimulation, and the freezing level induced by the sound stimulation was recorded. The entire recording process was automatically recorded with commercial software (FREEZING, Panlab Harvard Apparatus, Spain). The level of fear is expressed as a percentage of freezing time. In the contextual fear memory test, the level of freezing was calculated as the percentage of freezing time during the 5-minute contextual test. In the cued fear memory test, the freezing level was calculated as the percentage of freezing time under 3 sound stimuli.

To explore the effect of astrocyte activation on fear memory, rats were given optogenetic blue light stimulation for 15 minutes (473 nm, 10 Hz, the light intensity of the fiber tip was 1-3mW, 30s light on, 30s light off).

To explore the effect of drug injection on fear memory, rats were injected with drugs and the corresponding control solvent in the hippocampus or intraperitoneal region immediately after fear conditioning training.

Anxiety-like behavior test

Open field test: The open field test is a classic method to detect anxiety-like behavior and locomotor activity in rodents (Cai, Y.Q et al., J. Neurosci. 2018). Animals are less active in the central area because they are afraid of the new and empty environment, and mainly move in the surrounding areas. However, due to curiosity, animals will spontaneously develop the characteristics of exploring the central area. Animals with anxiety-like behavior spent less time exploring the center of the open field than normal animals. The length×width×height of the rat’s open field is 100×100×40(cm), and the inner wall is black. The area of 50 cm × 50 cm in the center of the open field was defined as the central area of the open field, and the rest of the area was defined as the peripheral area. At the beginning of the experiment, the rats were placed in a corner of the open field, and the rats were allowed to explore freely for 5 minutes and recorded with the upper camera. The behavioral activities of rats in the open field were analyzed with an automated behavioral tracking software (ANY-maze, Stoelting Co., USA). After each animal was tested, the open field apparatus was wiped down with a paper towel and 75% alcohol before testing the next animal, this was done to avoid residual odor affecting the behavior of the next animal. The total distance of the rat in the open field within 5 minutes was defined as the total distance, and the exploration time in the central area was defined as the central area time.

drug injection

The drugs ATP-γ-S, NECA, CCPA, CPT, SCH58261 and ARL67156 trisodium salt used in the experiment were purchased from Sigma-Aldrich Company. MesADP and CGS 21680 were purchased from Tocris. NECA, CCPA, CPT, SCH58261, and CGS 21680 were dissolved in DMSO to form a stock solution, and diluted with sterile normal saline to the final concentration during the experiment. ATP-γ-S, MesADP and ARL67156 were dissolved in sterile normal saline to form stock solutions, which were diluted to final concentrations with sterile normal saline during experiments. The drug injection in the brain area is injected through the cannula that has been embedded previously. During the experiment, the inner tube was inserted into the guide cannula, the inner tube was 2 mm longer than the cannula, and 1 microliter of drug was injected into the nucleus on each side. The control group was injected with the same amount of solvent. For intraperitoneal injection, animals were injected with different doses of CCPA and the corresponding solvent.

Statistical Analysis

All statistics were done with GraphPad Prism (Version 6.01). Data were analyzed using one-way ANOVA, two-way ANOVA, post hoc Bonferroni or Newman-Keuls tests, and two-tailed unpaired t-tests. Experimental animals were randomized into groups. Data are presented as mean ± standard error. P<0.05 was considered statistically significant.

Example 2 Activation of hippocampal CA1 astrocytes reduces fear memory and fear-related anxiety

The GFAP-ChR2-EYFP and WT rats were subjected to stereotaxic surgery to implant optical fiber cannulae according to the method described in Example 1, and after fear stimulation training, the effects of different treatments on the fear memory and fear-related anxiety of the rats were observed.

The experimental method and results are shown in Figure 1.

Among them, Fig. 1a and Fig. 1b are schematic diagrams of CA1 optical fiber implantation in rat hippocampus, in which the white column refers to the optical fiber implantation position, and the lower part of the dotted line refers to the CA1 position. Figure 1c shows the experimental design and process of fear conditioning, photoactivation paradigm, memory, and open field tests in rats. According to the method described in Example 1, the rats were subjected to conditioned fear training, hippocampal-dependent contextual fear testing and non-hippocampal-dependent cued fear testing. Because the experiments explored the effect of light-activated astrocytes on fear memory during the memory consolidation phase, astrocytes were light-activated immediately after fear conditioning or at intervals of 1, 2, and 3 hours.

Figure 1d-Figure 1i are the experimental results. Figure 1d shows that rats in the control and light-activated groups had similar learning abilities during fear conditioning training. Figure 1e shows that in the contextual fear memory test, the freezing level of the rats in the light-activated astrocytes group immediately after fear conditioning training was significantly reduced, indicating that photo-activated astrocytes significantly reduced contextual fear memory. Figure 1f shows that in the cued fear memory test, the level of freezing in the light-activated astrocytes group was similar to that of the control group, indicating that light-activated astrocytes did not affect cued memory. Figure 1g is a representative heatmap showing the trajectories of rats in the open field in the control group (no fear conditioning training), the fear conditioning training group, and the light-activated astrocytes group after fear conditioning training. The histogram in Figure 1h shows that fear conditioning training induces an anxiety phenotype compared to the control group; light activation of astrocytes after fear conditioning training effectively reverses the anxiety phenotype: significantly increases the movement distance in the open field (h ) and the exploration time (i) in the center of the open field.

All data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Example 3 Photoactivation of astrocytes has a long-term reducing effect on contextual fear memory and does not affect new memory formation

According to the method described in Example 1, GFAP-ChR2-EYFP and WT rats were subjected to stereotaxic surgery to embed optical fiber cannulae, and after fear stimulation training, the effects of different treatments on the rats' contextual fear memory were observed.

Figure 2a shows the experimental design and process of conditioned fear, photoactivation paradigm, memory, and open field tests in rats. According to the method described in Example 1, the rats were respectively subjected to fear conditioning training and retraining, and the hippocampus-dependent contextual fear test.

Figure 2b shows that in the contextual fear test, the freezing level of the rats in the light-activated astrocytes group was significantly reduced immediately after the fear conditioning training, which could last until 26 days after the photo-activation, indicating that the light-activated astrocytes persisted and significantly reduced episodic fear memory. Figure 2c shows that during fear conditioning training, the level of freezing of rats in the light-activated group that underwent fear conditioning again was similar to that of control rats that underwent fear conditioning for the first time, indicating similar learning abilities, also suggesting astrocytes Cell activation does not affect new learning. Figure 2d shows that in the contextual fear memory test, the level of freezing of the rats in the light-activated group that was reconditioned with fear training was similar to that of the control group, indicating similar levels of fear memory, and also suggesting that astrocyte activation did not affect new memory formation.

Example 4 Photoactivation of astrocytes within a certain time window can significantly reduce contextual fear memory

The GFAP-ChR2-EYFP and WT rats were subjected to stereotaxic surgery according to the method described in Example 1, and the fiber-optic cannula was implanted. After the training of fear stimulation, the effect of the treatment on the rats at different times on the situational fear memory of the rats was observed. influences.

Figure 3a,d,g show the experimental design and process of the photoactivation paradigm and memory test 1 hour, 2 hours and 3 hours after fear conditioning training, respectively. The results in Figure 3b,e,h show that rats in the control and light-activated groups had similar levels of freezing during fear conditioning training, indicating similar learning abilities. Figure 3c shows that astrocytes were light-activated 1 h after fear conditioning training, and the freezing level of rats was significantly reduced, indicating a significant reduction in fear memory. Figures 3f and 3i show that light-activated astrocytes 2 or 3 hours after fear conditioning training resulted in similar levels of freezing in rats to controls, indicating no effect on fear memory. These results suggest that activation of astrocytes during the early stages of memory consolidation after a traumatic event can significantly reduce fear memory.

Example 5 Activation of adenosine and adenosine A1 receptors mediates the reduction of contextual fear memory

According to the method described in Example 1, GFAP-ChR2-EYFP and WT rats were surgically implanted with administration cannulae, and after fear stimulation training, it was observed that the effect of different adenosine A1 by cannula administration in hippocampal CA1 was observed. Effects of somatic agonist processing on episodic fear memory in rats.

The ATP receptor agonists tested included the non-hydrolyzable analog of ATP, ATPrS, the agonist of the P2Y receptor, MesADP, the non-hydrolyzable analog of adenosine, NECA, the adenosine A1 receptor agonist CCPA, and the adenosine A2A receptor agonist CGS 21680.

Figure 4a is a schematic diagram of bilateral cannula implantation in the rat hippocampus CA1. Figure 4b is the experimental design and flow chart of conditioned fear training, drug injection and memory testing in rats.

The results in Figure 4c-e show that rats in the control (solvent-injected) and drug-injected groups had similar learning abilities during fear conditioning training.

Figure 4f shows that the freezing levels of rats in the local bilateral injection of ATPrS (2 mM, 9 mM, 1 μl/side) and MesADP (5 mM, 1 μl/side) and control rats (injected with the corresponding solvent) were similar, indicating that ATPrS and MesADP did not affect episodic fear memory.

Figure 4g shows that freezing levels were significantly reduced in rats in the group of local bilateral injections of the non-hydrolyzed analog of adenosine (2mM, 1 μl/side) and the adenosine A1 receptor agonist CCPA (5mM, 1 μl/side), indicating that Scenario fear memory was significantly reduced.

Figure 4h shows that the freezing level of rats in the group with local bilateral injection of adenosine A2a receptor agonist CGS 21680 (5mM, 1μl/side) was similar to that of the control group, indicating that CGS 21680 did not affect contextual fear memory.

Example 6 Local bilateral injection of NECA in the hippocampal CA1 brain region, CCPA does not affect the spontaneous movement of animals

GFAP-ChR2-EYFP and WT rats were surgically implanted with administration cannulae according to the method described in Example 1, and the rats were observed to be treated by administering different adenosine A1 receptor agonists through the cannula in hippocampal CA1 Effects on spontaneous activity in rats.

Figure 5a is the experimental flow chart of drug injection and open field test in rats.

Figure 5b,d shows that local bilateral injection of NECA (2mM, 1μl/side) in the rat brain region, CCPA (5mM, 1μl/side) did not affect the animal's movement distance in the open field. Figure 5c,e Local and bilateral injection of NECA in the brain region, CCPA did not affect the animals' exploration time in the center of the open field.

Example 7 Intraperitoneal injection of adenosine A1 receptor agonists significantly reduces fear memory and anxiety

After training GFAP-ChR2-EYFP and WT rats with fear stimulation, the effects of intraperitoneal administration of adenosine A1 receptor agonists on the memory of rats were observed.

Figure 6a shows the experimental design and flow chart of fear conditioning training, drug injection, and memory testing in rats. The experiment explored the effect of injection of adenosine A1 receptor agonist CCPA on fear memory during the memory consolidation stage, injected the drug immediately after fear conditioning, and conducted contextual fear memory tests on the 2nd and 3rd days, respectively.

Figures 6b–e show that rats in the control (solvent-injected) and drug-injected groups had similar levels of freezing in the contextual fear test, indicating that the animals had similar learning abilities.

Figure 6f shows that the freezing level of rats in the low-dose CCPA (0.03 mg/kg) group by intraperitoneal injection was similar to that of the control rats, indicating no effect on fear memory.

Figure 6g-i Intraperitoneal injection of high-dose CCPA (0.1/0.3/1 mg/kg) group significantly reduced the freezing level of rats, indicating that fear memory was significantly reduced.

Figure 6j,m are representative heatmaps showing the trajectories of rats in the control group (no fear conditioning training) and fear conditioning training groups in the open field at 3 and 24 hours after injection of vehicle or drug. Figure 6k-o shows that compared with the control group, the conditioned fear-trained rats had significantly reduced movement distance in the open field and exploration time in the center of the open field, which induced an anxiety phenotype. Adenosine was injected intraperitoneally after the fear conditioning training. The A1 receptor agonist CCPA (0.1 mg/kg) effectively reversed the anxiety phenotype: significantly increased distance traveled in the open field and exploration time in the center of the open field.

Example 8 Intraperitoneal injection of low-dose CCPA does not affect the spontaneous movement of animals

To observe the effect of intraperitoneal administration of adenosine A1 receptor agonists on rats.

Figure 7a is a representative heatmap. The movement trajectories of rats in the open field after injection of different doses of CCPA (0.1/0.3/1 mg/kg) are shown.

Figures 7b–e show that low dose CCPA (0.1 mg/kg) did not affect the spontaneous movement of animals, and high dose (0.3/1 mg/kg) significantly reduced the animal’s movement distance, but recovered 5 or 24 hours after injection, respectively to normal levels.

in conclusion

The present invention finds for the first time that the activation of astrocytes in hippocampal CA1 plays an important role in regulating the memory consolidation of fear memory in mammals and reducing fear memory and anxiety. Specifically, the inventors found that astrocyte activation can sustainably and significantly reduce fear memory without affecting the formation of new memories. The inventors have further discovered that activation of astrocytes reduces fear memory through the degradation products of ATP, adenosine and adenosine A1 receptors, and that local injection or systemic administration of formulations of astrocyte excitability can reduce fear Memory and anxiety disorders. The present invention thus provides methods and medicaments for regulating the memory consolidation of fear memory in mammals by increasing the excitability of astrocytes, and treating fear memory or its related diseases such as anxiety disorders.

The above is the description of the present invention, and should not be construed as limiting the present invention. Unless otherwise indicated, the practice of this invention will employ conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., it being apparent that the invention may be practiced otherwise than as specifically described in the foregoing specification and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which this invention pertains. Many modifications and variations are possible in light of the teachings of this invention and are therefore within the scope of this invention.

If there is no special indication, the unit "degree" of temperature appearing herein refers to degrees Celsius, ie, °C.

Claims (11)

1. A method of preventing or treating fear memory or a fear memory related disorder, such as anxiety, comprising the step of increasing astrocytic excitability in a subject, e.g. administering an agent that increases astrocytic excitability.

2. The method of claim 1, wherein the agent that increases astrocytic excitability is an optogenetic (optogenetic) agent, a chemigenetic (chemogenetic) agent, or a chemical drug,

for example, wherein the optogenetic agent is an agent that expresses a light sensing gene (e.g., ChR2, eBR, nahr3.0, Arch or OptoXR) in astrocytes;

or, wherein the chemogenetic agent is an agent that can express an astrocytic excitant protein in astrocytes, such as an agent that expresses receptors for neurotransmitters including acetylcholine (ACh), glutamate (glutamate), ATP, adenosine (adenosine), gamma-aminobutyric acid (γ -aminobutyric acid, GABA), norepinephrine (norpinephrine), dopamine (dopamine), endocannabinoids (endocannabinoids), nitric oxide (nitric oxide), histamine (histamine);

or, wherein the chemical agent is:

a. neurotransmitters, such as acetylcholine (ACh), glutamate (glutamate), ATP, adenosine (adenosine), gamma-aminobutyric acid (GABA), norepinephrine (norepinephrine), dopamine (dopamine), endocannabinoids (endocannabinoids), nitric oxide (nitric oxide) or histamine (histamine);

b. neurotransmitter receptor agonists, such as agonists at the Gq GPCR, including agonists at the endocannabinoid receptor (CB1 Rs); metabotropic glutamate I and II receptor agonists and AMPA receptor agonists; a purinergic receptor agonist; a GABAergic receptor agonist; alpha-adrenergic receptor agonists, dopaminergic receptor agonists; a histaminergic receptor agonist; an agonist of a protease-activated receptor (protease-activated receptor); or

c. Agonists of ion channels, such as agonists of transient voltage receptor cation channels, subclass V, member 1 (transdertector potential channel, subset V, TRPV 1); na (Na)⁺/Ca²⁺Agonists of the changers (NCXs).

3. The method of claim 1 or claim 2, wherein the agent that increases astrocyte excitability is administered systemically,

preferably, the dosage is from about 0.01 to about 20mg/kg, more preferably from about 0.05 to about 5mg/kg, and most preferably from about 0.01 to about 1 mg/kg.

4. The method according to claim 1 or claim 2, wherein the agent that increases the excitability of astrocytes is administered locally, such as locally to the hippocampus, in particular to the hippocampus CA1,

preferably, the dosage is from about 0.001 to about 0.1mg/kg, more preferably from about 0.005 to about 0.05mg/kg, and most preferably about 0.01 mg/kg.

5. The method of claim 1 or claim 2, wherein the agent that increases astrocytic excitability is administered to a patient after exposure to a fear stimulus to reduce or prevent the formation of fear memory by interfering with memory consolidation (memory coherence),

for example, the agent that increases astrocytic excitability is administered within about 6 hours, e.g., within about 2 hours, after exposure to the fear stimulus.

6. The method of claim 1 or claim 2, wherein the agent that increases astrocytic excitability is administered to a patient that has developed fear memory.

7. Use of an agent that increases the excitability of astrocytes, preferably an emergency pharmaceutical composition,

wherein the adenosine a1 receptor agonist is as defined in any one of claims 1 to 6.

8. A pharmaceutical composition for the prevention or treatment of fear memory or terrorist memory related diseases such as anxiety comprising an agent that increases astrocytic excitability.

9. The pharmaceutical composition of claim 8, wherein the agent that increases astrocytic excitability is an optogenetic (optogenetic) agent, a chemigenetic (chemigenetic) agent, or a chemical drug,

for example, wherein the optogenetic agent is an agent that expresses a light sensing gene (e.g., ChR2, eBR, nahr3.0, Arch or OptoXR) in astrocytes;

or, wherein the chemogenetic agent is an agent that can express an astrocytic excitant protein in astrocytes, such as an agent that expresses receptors for neurotransmitters including acetylcholine (ACh), glutamate (glutamate), ATP, adenosine (adenosine), gamma-aminobutyric acid (γ -aminobutyric acid, GABA), norepinephrine (norpinephrine), dopamine (dopamine), endocannabinoids (endocannabinoids), nitric oxide (nitric oxide), histamine (histamine);

or, wherein the chemical agent is:

a. neurotransmitters, such as acetylcholine (ACh), glutamate (glutamate), ATP, adenosine (adenosine), gamma-aminobutyric acid (GABA), norepinephrine (norepinephrine), dopamine (dopamine), endocannabinoids (endocannabinoids), nitric oxide (nitric oxide) or histamine (histamine);

b. neurotransmitter receptor agonists, such as agonists at the Gq GPCR, including agonists at the endocannabinoid receptor (CB1 Rs); metabotropic glutamate I and II receptor agonists and AMPA receptor agonists; a purinergic receptor agonist; a GABAergic receptor agonist; alpha-adrenergic receptor agonists, dopaminergic receptor agonists; a histaminergic receptor agonist; an agonist of a protease-activated receptor (protease-activated receptor);

or

c. Agonists of ion channels, such as agonists of transient voltage receptor cation channels, subclass V, member 1 (transdertector potential channel, subset V, TRPV 1); na (Na)⁺/Ca²⁺Agonists of the changers (NCXs).

10. The pharmaceutical composition according to claim 8 or claim 9, in a dosage form for systemic administration, preferably in a dose of about 0.01-20mg/kg, preferably about 0.05-5mg/kg, more preferably about 0.01-1mg/kg,

or

It is in a form for topical administration, e.g. to the hippocampus, e.g. CA1,

preferably, the dosage is from about 0.001 to about 0.1mg/kg, preferably from about 0.005 to about 0.05mg/kg, and more preferably about 0.01 mg/kg.

11. The pharmaceutical composition according to claim 8 or claim 9, which is an emergency pharmaceutical composition,

for example, it is a pharmaceutical composition that is administered within about 6 hours after exposure of the mammal to the fear stimulus, such as within about 2 hours after exposure to the fear stimulus.

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