CN111789951A

CN111789951A - Method and pharmaceutical composition for preventing fear memory and related diseases

Info

Publication number: CN111789951A
Application number: CN201910980756.XA
Authority: CN
Inventors: 段树民; 虞燕琴; 李玉兰
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2020-10-20

Abstract

The present invention provides methods for preventing and treating fear memory and its associated disorders such as anxiety by increasing astrocyte excitability. The invention also provides pharmaceutical compositions comprising agents that increase astrocytic excitability, which are useful for the prevention and treatment of fear memory and its related diseases.

Description

Method and pharmaceutical composition 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 its associated disorders such as anxiety.

Background

Astrocytes are widely present in the central nervous system and are the most abundant glial cells. Providing energy metabolism and structural support to neurons, maintaining water, ion, neurotransmitter homeostasis, regulating blood flow and blood-brain barrier, etc., are considered traditional functions of astrocytes (alaman, i., et al, Trends neurosci, 2011.). Recent years have seen increasing research in processes of astrocytes and presynaptic and postsynaptic formation of the trisynaptic loop (Zhang, j.m.et al. neuron., 2003). Astrocytes respond to neuronal synaptic activity with an increase in intracytoplasmic calcium concentration, thereby releasing glial transmitters, modulating synaptic activity and plasticity, such as long-term potentiation (LTP) and 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 dynamic changes in hippocampal-dependent scenes 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 and intermediate neurons of the hippocampus in fear memory formation has received widespread attention, and the involvement and regulation of astrocytes in fear memory continues to lack sufficient evidence.

Learning is the process of obtaining information and knowledge, and memory is the process of saving learned information. The content of the short-term memory can be converted into a long-term or even permanent storage form through the memory consolidation process, however, the memory consolidation process does not necessarily need the short-term memory as an intermediary. Long-term memory and short-term memory may exist in parallel (Mark F. bear et al, Neuroscience: expanding the Brain, 2)^ndEdition,2001)。

Memory consolidation process memory consolidation is a process in which new memory becomes stable after being acquired. Processing occurs at the molecular and cellular level, allowing an intracellular signaling cascade to occur, activating transcription factors leading to gene expression and protein synthesis, enhancing synaptic plasticity (long term potentiation, LTP) and synaptic connectivity in the hippocampus and other brain regions, a process also known as synaptic consolidation (Mednick, s.c., et al, Trends neurosci.2011). LTP is widely recognized as a molecular mechanism for learning and memory. Studies in ex vivo brain slices, anaesthesia and free-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 memory can lead to inappropriate behavioral responses and severe physiological or psychological damage (Izquierdo, i., et al., physiol.rev., 2016). In humans, traumatic fear memory (traumatic fear memory) causes a variety of mental disorders, including schizophrenia (schizophrenia), posttraumatic stress syndrome (PTSD), anxiety (anxiety disorder), phobias (phobias), and depression (depression) among others (Luthi, a., et al, nat. neurosci, 2014; Garfinkel, s., n.et al, j.neurosci, 2014). The estimated lifetime prevalence of fear and stress-related diseases is about 29% (Kessler, r.c., et al, arch.gen.psychiatry, 2005). Fear memory ablation (fear ablation), also known as exposure therapy, is an ethological method of reducing fear. Exposure therapy is a learning process that relies on contextual cues and does not interrupt or eliminate the initially generated fear memory. Studies have shown that patients undergoing exposure therapy are spontaneously restored (spontaneousreviver) or regenerated (renew) upon re-exposure to a scene similar to or identical to that at the time of trauma (Britton, j.c., et al, depress.anxiety, 2011; Tovote, p.et al, nat.rev.neurosci, 2015). It is therefore necessary to find a new strategy that specifically interrupts or prevents the formation of fear memories, or reduces or eliminates the already formed fear memories.

There is also a need in the art for safer methods and drugs that are effective in preventing and/or treating fear memory or its associated disorders, such as anxiety, without producing or reducing side effects.

Disclosure of Invention

The invention discovers for the first time that activation of astrocytes of hippocampal CA1 plays an important role in regulating and controlling memory consolidation of fear memory, and even in reducing fear memory and preventing and/or treating fear memory related diseases such as anxiety disorder. In particular, the inventors have found that astrocyte activation can continue to significantly eliminate or reduce fear memory formation without affecting the formation of new memory. The inventors have further found that activation of astrocytes reduces fear memory through adenosine, a degradation product of ATP, and adenosine a1 receptors, and that astrocyte excitability can reduce fear memory and its associated diseases such as anxiety by local injection or intraperitoneal injection. The present invention thus provides methods and medicaments for the prevention and treatment of fear memory or its associated disorders such as anxiety by increasing the excitability of astrocytes.

In one of its aspects, the invention provides a method of modulating memory consolidation of fear memory in a mammal comprising the step of increasing astrocyte excitability in said subject, thereby inhibiting the formation of fear memory in the mammal following an event (such as a fear stimulus).

In one of its aspects, the invention provides methods for preventing and treating fear memory or a disorder associated therewith in a subject comprising increasing astrocytic excitability, e.g., by light stimulation or administration of an agent that increases astrocytic excitability.

Accordingly, the present invention provides the use of an agent that increases astrocytic excitability for the manufacture of a medicament for memory consolidation to modulate fear memory in a mammal. Accordingly, the invention also provides the use of an agent that increases astrocytic excitability for the manufacture of a medicament for the prevention and treatment of fear memory or a disorder related thereto in a subject. Wherein the agent that increases astrocyte excitability inhibits the formation of fear memory in a mammal following an event (such as fear stimulation).

The invention also provides a pharmaceutical composition for the prevention and treatment of fear memory or a disease associated therewith comprising an agent that increases astrocytic excitability.

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

The manifestation of astrocyte excitability includes calcium signaling response of astrocytes. In the absence of neuronal activity, astrocytes can develop a spontaneous calcium elevation; during synaptic activity, the released neurotransmitter also triggers an increase in calcium in the astrocytes. The synaptic regulation of astrocytic calcium signaling is based on astrocytes expressing a number of functional neurotransmitter receptors. These receptors are mostly metabotropic and G-proteinsCoupled to stimulate phospholipase C to form IP upon activation₃Further via intracellular IP₃Sensitive calcium stores release calcium ions, thereby increasing intracellular calcium ion concentrations.

Agents that increase astrocyte excitability include optogenetic (optogenetic) agents, chemogenetic (chemogenetic) agents, or chemical drugs.

In the present invention, the optogenetic preparation is an agent that can express a light sensing gene (e.g., ChR2, eBR, nahr3.0, Arch or OptoXR) in astrocytes.

In the present invention, the chemogenetic agent is an agent that can express an astrocyte excitable protein in astrocytes, for example, an agent that expresses a receptor of a neurotransmitter, including acetylcholine (ACh), glutamate (glutamate), ATP, adenosine (adenosine), gamma-aminobutyric acid (GABA), norepinephrine (norepinephrine), dopamine (dopamine), endocannabinoid (endocannabinoids), nitric oxide (nitrooxide), histamine (histamine).

In the present invention, the chemical agents capable of increasing the excitability of astrocytes include, but are not limited to:

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, e.g. transient voltage receptorsAn agonist of the cation channel, subclass V, member 1 (transdertectrirecterportentional capture channel, subfamily V, TRPV 1); na (Na)⁺/Ca²⁺Agonists of the changers (NCXs).

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

In the present invention, the fear memory may be a fear memory mediated in different brain regions, and may be particularly referred to as "hippocampal CA 1-mediated fear memory". The inventors of the present application found and demonstrated that the activity of astrocytes of hippocampal CA1 plays an important role in fear memory and anxiety formation: astrocyte activation consistently reduced fear memory significantly without affecting the formation of new memory. In addition, the inventors have found that astrocyte activation achieves reduced fear memory and anxiety by increasing the activity of the pathway in which adenosine a1 receptors participate.

The method of the present invention has been shown to act more effectively in the consolidation phase of fear memory. When the brain obtains external information, a Short Term Memory (STM) is formed, and when the short term memory enters a Long Term Memory (LTM), the information is strengthened and integrated and is stored in a specific brain area to be stable. This is the consolidation phase of memory. The memory consolidation means that under the condition of similar induction, consolidated memory is activated and modified again, and the original stable memory becomes fragile and easy to change; the reactivated memory is again secured.

Memory consolidation (memory consolidation) is the process by which new memory becomes stable after acquisition. Processing occurs at the molecular and cellular level, allowing an intracellular signaling cascade to occur, activating transcription factors leading to gene expression and protein synthesis, enhancing synaptic plasticity (long term potentiation, LTP) and synaptic connectivity in the hippocampus and other brain regions, a process also known as synaptic consolidation (s.c.), which typically occurs within 6 hours after memory acquisition (Mednick, s.c., et al, Trends neurosci, 2011). LTP is widely recognized as a molecular mechanism for learning and memory. Studies in ex vivo brain slices, anaesthesia and free-moving animals have shown that LTP can be divided into two phases, early LTP independent of protein synthesis, lasting approximately 2 hours, and LTP dependent on late protein synthesis. LTP maintenance is hardly affected with prolonged post-LTP induction time, such as after 1-2 hours by drug treatment or other intervention (Fujii et al, BrainResearch.1991; Abraham, W.C.et al, Neurobiol.Learn.Mem.2008;). Animal behavioural studies have shown that intervention is more likely to block memory formation when it occurs within a relatively short time after learning (say 1 hour) and is less likely to interfere with memory formation if it occurs within a longer time after learning (say greater than 6 hours) (Mednick, s.c., et al, Trends neurosci, 2011; Dudai, y.rev.psychol.2004). Therefore, once LTP or memory enters a protein synthesis-dependent stage, memory is difficult to erase due to consolidation, and therefore intervention should be taken to block fear memory formation prior to the onset of synaptic consolidation.

The methods of the invention are useful for the prevention and treatment of fear memory or disorders related thereto. Intervention within a certain time window after a particular fear-memory causing event occurs can significantly block and reduce the fear-memory and associated anxiety associated with that event without recurrence. The method of the invention is therefore suitable for administration within about 6 hours of exposure to the fear stimulus, preferably within about 2 hours of exposure to the fear stimulus.

In another of its aspects, the methods or pharmaceutical compositions of the invention are particularly useful for the emergency prevention and treatment of fear memory or its related diseases. The methods or pharmaceutical compositions of the invention may be used for emergency treatment, such as prophylactic treatment of a subject within a relatively short period of time following a sudden accident or sudden bulk injury. In another of its aspects, the invention is useful for preventing people, particularly people susceptible to fear (e.g., people colloquially referred to as "choledochus"), from acquiring a fear memory or even developing a related disorder such as anxiety or the like after experiencing an event that typically triggers fear.

In one aspect of the invention, the methods and pharmaceutical compositions of the invention for treating fear memory or a disorder associated therewith are suitable for use in other patients for whom the methods and medicaments of the invention are not effective. The inventors of the present application have for the first time discovered and demonstrated that astrocytes of the hippocampus, particularly hippocampal CA1, have an important role in fear memory, and thus provide methods and medicaments for treating (inhibiting) fear memory or its associated diseases by stimulating astrocytes of hippocampal CA 1. This is a mechanism known in the art to treat fear memory or its associated diseases and a pathological mechanism to which the drug fails to address and a target at the level of the brain target tissue or molecule to be treated.

In one embodiment of the invention, the agent that increases astrocyte excitability may be administered systemically. The agent that increases 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 chosen agent that increases astrocyte excitability. In a more preferred embodiment, the agent that increases astrocyte excitability is used in an amount of 0.05-50 mg/kg.

If desired, the formulations for increasing astrocyte excitability may be formulated for local administration to the brain, particularly the hippocampal region. For example, formulations prepared for local injection through a cannula. Topical formulations have the advantage that any possible systemic side effects of the drug can be avoided.

In one aspect of the present invention, the method of the present invention for the treatment and prevention of fear memory and its associated diseases and the medicament (pharmaceutical composition) of the present invention for the treatment and prevention of fear memory and its associated diseases are methods and medicaments which are effective locally in the hippocampus, particularly hippocampal CA1, i.e. are administered in the hippocampus, particularly hippocampal CA 1. For drugs for nerve tissue, in particular brain nerve tissue, such as hippocampus, it is beneficial to confine the effect of the drug to the target tissue. The use of topical administration in the hippocampus is a limiting feature of both therapeutic methods and the preparation of medicaments. The method or medicament for use in hippocampal region, particularly hippocampal CA1, requires consideration of whether the method or medicament is able to exert its effectiveness in hippocampal region, particularly hippocampal CA1, including whether the medicament is able to reach hippocampal region, particularly hippocampal CA1, and whether an effective concentration is achieved in hippocampal region, particularly hippocampal CA 1. In the present invention, the drug or pharmaceutical composition is in a dosage form for local administration in the hippocampus, particularly in the hippocampus CA 1. The targeting of the drug effect to the target tissue can be achieved by local administration, for example by making the agent that increases astrocytic excitability into a form that can be administered locally by cannula implantation into the hippocampus, particularly the hippocampal CA 1. For example, the drug is prepared into a sustained release formulation after being implanted into a tissue. The above drugs can also be made into the form of a tissue-specific targeted drug delivery system. For example, by encapsulating an agent that increases astrocytic excitability in hippocampal tissue-targeted exosomes.

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

The pharmaceutical compositions of the present invention may be administered by any convenient route appropriate to the desired therapy. Preferred routes of administration include oral administration, particularly in the form of tablets, capsules, lozenges, powders and liquids; and parenteral administration, especially cutaneous, subcutaneous, intramuscular and intravenous injection. The pharmaceutical compositions of the present invention may be prepared by those skilled in the art using standard methods and conventional techniques appropriate for the desired formulation. If desired, compositions suitable for sustained release of the active ingredient may 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, intraarterial, intracerebral, intraocular injection or infusion) administration or those in a form suitable for administration by inhalation or insufflation (including powder and liquid aerosol administration) or for administration by sustained release systems. Examples of suitable sustained release systems include semipermeable matrices of solid hydrophobic polymers containing a compound of the invention, which matrices may be in the form of shaped articles, e.g., films, or microcapsules.

The active ingredients in the pharmaceutical compositions of the present invention may thus be formulated together with conventional adjuvants, carriers or diluents into pharmaceutical compositions and unit dosage forms thereof. Such forms include solid, especially tablet, filled capsule, powder and pellet forms, as well as liquid, especially aqueous or non-aqueous solutions, suspensions, emulsions, elixirs and capsules filled with the above forms, 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 comprise conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the desired daily dosage range to be employed.

For preparing a pharmaceutical composition from the active ingredients in the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier may be 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 also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

If desired, compositions suitable for providing sustained release of the active ingredient may be employed.

The pharmaceutical preparation is preferably in unit dosage form. In such forms, 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 the preparation, such as packeted tablets, capsules, and powders in vials or ampoules. In addition, 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 an amount of active ingredient that alleviates 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/ED 50.

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

The actual dosage will depend upon the nature and severity of the condition being treated, the exact mode of administration and the form of administration, and may be varied within the judgment of the practitioner according to the particular circumstances of the invention by increasing the dosage to produce the desired therapeutic effect.

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

In yet another aspect of the invention, the methods and pharmaceutical compositions of the invention, the agent that increases astrocytic excitability is administered topically to the hippocampus, particularly to hippocampal CA 1. The dosage is about 0.001-0.1mg/kg, preferably about 0.005-0.05mg/kg, and more preferably about 0.01 mg/kg.

In another aspect of the invention, the use of the preparation for increasing astrocyte excitability provided by the invention in the preparation of a medicament for regulating memory consolidation of fear memory in a mammal, or preventing and treating fear memory or a disease related thereto in a subject, wherein the medicament is prepared in a form that the preparation for increasing astrocyte excitability is administered systemically. Preferably, the dose of the drug is about 0.01-20mg/kg, more preferably about 0.05-5mg/kg, for example about 0.01-1 mg/kg.

In another aspect of the invention, in the application of the preparation for increasing the excitability of the astrocytes in preparing a medicament for regulating the memory consolidation of the fear memory of a mammal or preventing and treating the fear memory or the related diseases in a subject, the medicament is prepared in a form that the preparation for increasing the excitability of the astrocytes is locally applied. For example, topically to the hippocampus, particularly to hippocampal CA 1. Preferably, the dose of the drug is about 0.001-0.1mg/kg, more preferably about 0.005-0.05mg/kg, for example about 0.01 mg/kg.

In another aspect of the invention, the use of the preparation for increasing astrocytic excitability provided by the invention in the preparation of a medicament for regulating memory consolidation of fear memory in a mammal, or preventing and treating fear memory or a disease related thereto in a subject, is an emergency pharmaceutical composition.

Drawings

Figure 1 optogenetically activating astrocytes of hippocampal CA1 reduces fear memory and fear-related anxiety. a, b rat hippocampal CA1 optic fiber implantation scheme, white columns indicating optic fiber implantation position, and below dotted line CA1 position. c, experimental design and process of fear of condition, light activation paradigm, memory and open field test. Since the experiment was intended to explore the effect of light-activated astrocytes on fear memory during the memory consolidation phase, astrocytes were light-activated immediately after conditioned fear. d in conditioned fear training, rats in the control and photoactivated groups had similar levels of freezing, indicating similar learning capacity. e, in the scene fear memory test, levels of freezing in the light activated astrocyte group rats immediately after conditioned fear training were significantly reduced, indicating that light activated astrocytes significantly reduced scene fear memory. f in the Withania phobia memory test, the levels of freezing in the rats in the light activated astrocyte group were similar to those in the control group, indicating that light activated astrocytes did not affect clue memory. g representative heatmaps show the movement trajectories in the open field of rats in the control group (no conditioned fear training), conditioned fear training group, light activated astrocyte group after conditioned fear training. h, histogram shows that conditioned fear training elicits an anxiety phenotype compared to control; light-activated astrocytes effectively reversed the anxiety phenotype after fear training: the distance of movement in the open field (h) and the exploration time in the center of the open field (i) are significantly increased. All data are expressed 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 scene fear memory and do not affect new memory formation. a, experimental design and flow of conditioned fear training, light activation paradigm and memory testing. b in the scene fear test, the level of freezing in the light activated astrocyte group rats immediately after the conditioned fear training was significantly reduced and could last for 26 days after the light activation, indicating that the light activated astrocytes continue and significantly reduce the scene fear memory. c in the conditioned fear training, the level of freezing of the photoactivated group rats subjected to the conditioned fear training again is similar to that of the control group rats subjected to the conditioned fear training for the first time, which shows that the photoactivated group rats have similar learning ability and the activation of astrocytes does not influence new learning. d in the fear memory test, the level of freezing in the conditioned fear trained light activated group rats was similar to that in the control group rats, indicating that similar fear memory levels were present, and also suggesting that astrocyte activation did not affect new memory formation.

Figure 3. light activation of astrocytes over a time window significantly reduced scene fear memory. and (3) experimental design and process of a, d and g conditional fear training, light activation paradigm and memory testing. b, e, h in conditioned fear training, rats in the control and photoactivated groups had similar levels of freezing, indicating similar learning capacity. c 1h after conditioned fear training, astrocytes were light activated and rat levels of freezing were significantly reduced, indicating a significant reduction in fear memory. f, i light-activated

astrocytes

2 or 3 hours after conditioned fear training, rat levels of freezing were similar to controls, indicating no effect on fear memory.

Figure 4 adenosine and adenosine a1 receptor activation mediate a decrease in contextual fear memory. a schematic representation of bilateral cannula implantation of rat hippocampus CA 1. b experimental design and procedure for conditioned fear training, drug injection and memory testing. c-e rats in the conditioned fear training, control (solvent injection) and drug injection groups had similar learning abilities. The levels of freezing in rats of the topical bilateral injection ATPrS (2mM,9mM,1 μ l per side) and the MesADP (5mM,1 μ l per side) groups were similar to those of the control group rats (injected with the corresponding solvents), indicating that ATPrS and MesADP did not affect the contextual fear memory.g. the levels of freezing in rats of the topical bilateral injection NECA (2mM,1 μ l per side) and CCPA (5mM,1 μ l per side) groups were significantly reduced, indicating that the contextual fear memory was significantly reduced.h the levels of freezing in rats of the topical bilateral injection CGS 21680(5mM,1 μ l per side) group were similar to those of the control group, indicating that CGS 21680 did not affect the contextual fear memory.

FIG. 5 local bilateral injection of NECA to the hippocampal CA1 brain region, CCPA did not affect spontaneous locomotion in animals. a experimental flow chart of drug injection and open field testing. b, d regional bilateral NECA injection (2mM, 1. mu.l/side) and CCPA (5mM, 1. mu.l/side) did not affect the distance of movement of the animals in the open field. c, e local bilateral injection of NECA in brain area, CCPA did not affect the time of animal exploration in the center of the open field.

Figure 6 intraperitoneal injection of adenosine a1 receptor agonist significantly reduced fear memory and anxiety. a experimental design and flow chart of conditioned fear training, drug injection and memory testing. Drug was injected immediately after conditioned fear and a scene fear memory test was performed on day 2 and day 3, respectively. b-e in the scene fear test, rats in the control (solvent injection) and drug injection groups had similar levels of freezing, indicating that the animals had similar learning abilities. F intraperitoneal injection of CCPA (0.03mg/kg) in the rats in the low dose group and the rats in the control group have similar levels of freezing, which shows no influence on fear memory. J, m representative thermograms show the kinetic trajectories of rats in the control group (without conditional fear training), the conditioned fear training group at 3 hours and 24 hours after solvent or drug injection in open field. k-o compared to the control group, the rats trained by conditioned fear had significantly reduced distance to movement in the open field and exploration time in the center of the open field, inducing an anxiety phenotype, and the abdominal cavity was injected with the adenosine a1 receptor agonist CCPA (0.1mg/kg) after conditioned fear training effectively reversed the anxiety phenotype: the distance of movement in the open field and the exploration time in the center of the open field are significantly increased.

Figure 7 intraperitoneal injection of low dose CCPA did not affect spontaneous locomotion in animals. a representative heatmap shows the movement trajectory of rats in the open field after injection of different doses of CCPA (0.1/0.3/1 mg/kg). The low b-e dose of CCPA (0.1mg/kg) did not affect spontaneous locomotion in the animals, and the high dose (0.3/1mg/kg) significantly reduced the locomotion distance of the animals, but returned to normal levels after 5 hours or 24 hours post-injection, respectively.

Detailed Description

The spirit and advantages of the present invention will be further illustrated by the following examples, which are provided by way of illustration and are not intended to be limiting.

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). The GFAP-ChR2-EYFP rat was produced by the institute of neuroscience, Chinese academy of sciences. Rats, 4-5/cage, were able to ingest water and food freely and were housed in a 12 hour light-dark cycle (7 o 'clock-19 o' clock light). The rats were raised in a single cage after surgery. All animal experiments were approved by the animal protection and use committee of the university of zhejiang.

Optical fiber sleeve embedded for three-dimensional positioning operation

The rats are anesthetized by intraperitoneal injection of pentobarbital sodium (1%, wt/vol) and then fixed on a stereotaxic apparatus. The body temperature is controlled by the heating pad in the operation process, so that the body temperature is kept stable. The scalp is cut open with ophthalmic scissors. The target site is drilled with a cranial drill. The fiber-optic cannulae (core diameter 200 μm, NA 0.37, Newdoon, China) are embedded on both sides of the hippocampal CA1 brain region (anterior-posterior distance bregma: 3.75mm (AP), left-right side opening + -2.46 Mm (ML), and the surface of the skull vertically downwards-2.63 mm (DV)). The fiber optic ferrule is connected to the laser (China) by a fiber optic jumper. The output power of the fiber tip was measured with an optical power meter (LP1, sanwa, Japan) before each experiment.

Three-dimensional positioning operation embedded drug administration sleeve

For pharmacological experiments, two administration cannulas (RWD Life Science, China) were embedded in bilateral hippocampus CA1 (anterior-posterior distance bregma: 3.75mm (AP), left-right side ± 2.46Mm (ML), skull surface down-2.63 mm (DV)) respectively. The optical fiber and the administration cannula are fixed to the surface of the skull with dental cement. After the dental cement is completely solidified, an inner core with the same length as the sleeve is inserted into the sleeve, and the nut is screwed to prevent the inner core from falling off. The animals recovered 7 days after surgery and the behavioural test started. After the experiment was completed, the position of the fiber and the ferrule was identified histologically. Animals with incorrect fiber and ferrule positions were excluded.

Conditioned fear experiment

The conditioned fear experiments were performed in a 25X 25cm fear box (Panlab Harvard Apparatus, Spain). The conditioned fear experiment contains 3 stages in total: conditioned fear training (fear conditioning), hippocampal-dependent contextual fear test (Kim, J.J et al, Science 256, 675-containing 677,1992) and hippocampal-independent cue fear test (Phillips, r.g. et al, behav. neurosci.106,274-285,1992). Rodents exhibit a characteristic immobility response upon fear. If the animal is immobile for more than 2 seconds, it is defined as stiff (freezing). Animals were placed in the experimental environment 3 days before the start of the experiment and were subjected to handling once a day. The first day of the experiment, the conditioned fear box was wiped with 70% alcohol and the box contained a stainless steel floor grid that could be used to deliver an electric shock. The rats were gently placed in the chamber to acclimate for 2 minutes and the basal levels of freezing recorded during this period. Followed by a 30 second sound stimulus (2kHz,85dB), and a2 second foot shock (0.6mA) to the rat when the sound lasts until 29 seconds, with the sound and shock ending simultaneously. A total of 3 sound-shock paired trains were given, each time 60 seconds apart. The rats remained in the training chamber for 90 seconds after the end of the last shock and were then returned to their cages. Conditioned fear training lasted for a total of 7 minutes, and the entire process was completed in a relatively dark box. After the conditioned fear training is finished for 24 hours, a scene fear memory test is carried out, and the rats are placed in the same training box for 5 minutes to detect the fleezing time. The following 2 hours, a cue fear memory test was performed. The cue fear test was conducted in a brighter box with an environment and odor different from the training box, wiped with 1% acetic acid. After 1 minute of acclimation, rats were given 3 acoustic stimuli (2kHz,85dB) for 30 seconds, with 30 second intervals between each acoustic stimulus, and the level of freezing induced by the acoustic stimuli was recorded. The entire recording process was automatically recorded using commercial software (FREEZING, Panlab harvard apparatus, Spain). The level of fear is expressed as a percentage of the rigor time. In the scene fear memory test, the freezing level is calculated as the percentage of the freezing time in the 5 minute scene test. In the chordal fear memory test, the freezing level was calculated as the percentage of the freezing time under 3 acoustic stimuli.

To explore the effect of astrocyte activation on fear memory, rats were given optogenetic blue light stimuli for 15 minutes (473nm,10Hz, fiber tip light intensity 1-3mW, 30s light on, 30s light off) immediately after conditioned fear training or at 1 hour, 2 hour, 3 hour intervals, respectively.

To explore the effect of drug injection on fear memory, rats were injected with drug and corresponding control solvent in the hippocampal brain region or the abdominal cavity immediately after conditioned fear training.

Anxiety-like behavior testing

Open field testing: open field testing is the classical method of detecting anxiety-like behavior and spontaneous activity in rodents (Cai, Y.Q et al, j.neurosci.2018). Animals are less active in the central area and mainly active in the peripheral area due to fear of a new open environment, but the animals spontaneously develop the characteristic of going to the central area for exploration due to curiosity. Animals with anxiety-like behavior have less time to explore in the center of the open field than normal animals. The length of the rat open field, width and height are 100X 40(cm), and the inner wall is black. The region of 50cm x 50cm at the center of the open field is defined as the central region of the open field, and the remaining regions are defined as the peripheral regions. The rats were placed in an open field corner at the start of the experiment, allowed to explore freely for 5 minutes and recorded with the top camera. The behavioral activity of rats in the open field was analyzed using an automated behavioral tracking software (ANY-maze, stoeltingco., USA). After each animal was tested, the open field apparatus was wiped with paper towels and 75% alcohol and the next animal was tested again in order to avoid residual odors affecting the behavior of the next animal. The distance traveled (total distance) of the rat within 5 minutes of the open field was defined as the total distance traveled and the time of exploration in the central zone as the central zone time.

Medicine injection

The experimental drugs ATP-. gamma. -S, NECA, CCPA, CPT, SCH58261 and ARL67156 trisodium salt were purchased from Sigma-Aldrich. MesADP and CGS 21680 were purchased from Tocris corporation. NECA, CCPA, CPT, SCH58261, CGS 21680 were dissolved in DMSO to give stock solutions, and diluted with sterile saline to final concentrations at the time of the experiment. ATP-gamma-S, MesADP and ARL67156 were dissolved in sterile physiological saline to give stock solutions, and diluted with sterile physiological saline to give final concentrations at the time of the experiment. The injection of the drug into the brain area is through a cannula that has been previously embedded. During the experiment, an injection inner tube is inserted into a guide sleeve, the inner tube is 2mm longer than the sleeve, and each side of the core mass is injected with 1 microliter of medicine. The control group was injected with an equal amount of solvent. For intraperitoneal injections, animals were injected with different doses of CCPA and corresponding solvents.

Statistical analysis

All data statistics were done using GraphPad Prism (Version 6.01). The data analysis adopts single-factor variation analysis and two-factor variation analysis, and then Bonferroni or Newman-Keuls test and double-tail unpaired t test are carried out. Experimental animals were randomly grouped. Data are presented as mean ± sem. P <0.05 was set as statistically significant.

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

GFAP-ChR2-EYFP and WT rats were stereotactically surgically embedded in fiber optic cannulae according to the method described in example 1 and observed for the effect of different treatments on the fear memory and fear-related anxiety of the rats after fear-stimulated training.

The experimental methods and results are shown in figure 1.

Wherein, fig. 1a and fig. 1b are schematic diagrams of rat hippocampal CA1 fiber implantation, wherein white pillar indicates the fiber implantation position, and the dotted line below indicates the CA1 position. FIG. 1c is the experimental design and procedure for conditioned fear, light activation paradigm and memory, open field testing of rats. Rats were individually conditioned fear trained, hippocampal-dependent contextual fear testing and hippocampal-independent clue fear testing according to the methods described in example 1. Since the experiment explored the effect of light-activated astrocytes on fear memory during the memory consolidation phase, astrocytes were light-activated immediately or 1 hour, 2 hours, 3 hours after conditioned fear.

FIG. 1 d-FIG. 1i show the results of the experiments. Wherein FIG. 1d shows that rats in the control and the light activated groups have similar learning abilities in the conditioned fear training. Figure 1e shows that in the scene fear memory test, the levels of freezing in the light activated astrocyte group of rats immediately after conditioned fear training were significantly reduced, indicating that light activated astrocytes significantly reduced scene fear memory. FIG. 1f shows that in the Leptophobia memory test, the levels of freezing in the light activated astrocyte group rats were similar to the control group, indicating that light activated astrocytes did not affect clue memory. Fig. 1g is a representative heatmap showing the movement trajectories in the open field of rats in the control group (no conditioned fear training), conditioned fear training group, light activated astrocyte group after conditioned fear training. FIG. 1h is a bar graph showing that conditioned fear training elicits an anxiety phenotype compared to the control group; light-activated astrocytes effectively reversed the anxiety phenotype after fear training: the distance of movement in the open field (h) and the exploration time in the center of the open field (i) are significantly increased.

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

Example 3 light-activated astrocytes have a long-term reducing effect on scene fear memory and do not affect new memory formation

GFAP-ChR2-EYFP and WT rats were stereotactically surgically embedded with fiber optic cannulae according to the procedure described in example 1, and after fear-stimulated training, the effect of different treatments on the fear memory of the rat scene was observed.

Figure 2a is the experimental design and procedure for conditioned fear, light activation paradigm and memory, open field testing in rats. Rats were subjected to conditioned fear training and retraining, respectively, and hippocampal-dependent contextual fear testing according to the method described in example 1.

Figure 2b shows that in the scene fear test, levels of freezing in the light activated astrocyte group of rats immediately after conditioned fear training were significantly reduced, which could persist up to 26 days after light activation, indicating that light activated astrocytes persist and significantly reduce scene fear memory. Figure 2c shows that the photoactivated group of rats re-conditioned for fear training had similar levels of freezing as the control group of rats first conditioned for fear training, indicating similar learning capacity, and also suggesting that astrocyte activation did not affect new learning. Figure 2d shows that conditioned fear trained photoactivated and control rats had similar levels of greezing in the scene fear memory test, indicating similar levels of fear memory, and also suggesting that astrocyte activation did not affect new memory formation.

Example 4 light activation of astrocytes over a time window significantly reduces scene fear memory

GFAP-ChR2-EYFP and WT rats were subjected to stereotactic surgery using the method described in example 1 to embed fiber optic cannulae, and after fear-stimulated training, the effect of treatment of rats at different times on the fear memory of the rat scene was observed.

Fig. 3a, d, g are experimental designs and procedures for photoactivation paradigm and memory test at 1 hour, 2 hours and 3 hours after conditioned fear training, respectively. The results in fig. 3b, e, h show that rats in the control and photoactivated groups have similar levels of freezing during conditioned fear training, indicating similar learning capacity. Figure 3c shows that 1 hour after conditioned fear training light activates astrocytes, the level of rat freesing is significantly reduced, indicating a significant reduction in fear memory. Fig. 3f and 3i show that light activated

astrocytes

2 or 3 hours after conditioned fear training, the level of rat freesing was similar to the control group, indicating no effect on fear memory. These results suggest that activation of astrocytes at the early stage of memory consolidation after the occurrence of a traumatic event can significantly reduce fear memory.

Example 5 adenosine and adenosine A1 receptor activation mediated reduction of contextual fear memory

GFAP-ChR2-EYFP and WT rat rats were surgically embedded with drug-coated cannulae according to the method described in example 1, and after fear-stimulation training, the effect of rat treatment on the rat's fear memory of the scene was observed by administering different adenosine A1 receptor agonists through the cannulae in the hippocampal CA 1.

The ATP receptor agonists tested included the non-hydrolyzed analog of ATP, ATPrS, the agonist of the P2Y receptor, MesADP, the non-hydrolyzed analog of adenosine, NECA, the adenosine a1 receptor agonist CCPA, and the adenosine A2A receptor agonist CGS 21680.

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

The results in fig. 4c-e show that rats in the control group (solvent injection) and drug injection groups have similar learning abilities in the conditioned fear training.

Figure 4f shows that rats in the local bilateral ATPrS (2mM,9mM,1 μ l per side) and MesADP (5mM,1 μ l per side) groups and rats in the control group (injected with the corresponding solvent) had similar levels of freezing, indicating that ATPrS and MesADP did not affect the scene fear memory.

Figure 4g shows a significant reduction in the level of freesing in rats of the non-hydrolysed analogue of local bilateral injection of adenosine, NECA (2mM,1 μ l per side) and the adenosine a1 receptor agonist, CCPA (5mM,1 μ l per side), indicating a significant reduction in contextual fear memory.

Figure 4h shows that rat levels of freezing in the local bilateral adenosine A2a receptor agonist CGS 21680(5mM,1 μ l per side) group are similar to the control group, indicating that CGS 21680 does not affect the scene fear memory.

Example 6 local bilateral injection of NECA to the hippocampal CA1 brain region, CCPA did not affect spontaneous locomotion in animals

GFAP-ChR2-EYFP and WT rats were surgically implanted with a cannula according to the method described in example 1 and the effect on spontaneous activity of the rats was observed by treatment of the rats with different adenosine A1 receptor agonists administered through the cannula in the hippocampus CA 1.

Figure 5a is a flow chart of the experiment for drug injection and open field testing in rats.

FIG. 5b, d shows that local bilateral injection of NECA (2mM, 1. mu.l per side) in rat brain regions, CCPA (5mM, 1. mu.l per side) did not affect the distance of movement of the animals in the open field. Fig. 5c, e local bilateral injection of NECA in brain area, CCPA did not affect the time of exploration of animals in the center of the open field.

Example 7 intraperitoneal injection of adenosine A1 receptor agonist significantly reduces fear memory and anxiety

After fear-stimulated training of GFAP-ChR2-EYFP and WT rats, the effect of treatment of rats with an intraperitoneal adenosine A1 receptor agonist on the memory of the rats was observed.

Figure 6a is an experimental design and flow chart for conditioned fear training, drug injection and memory testing in rats. Experiments explored the effect of injection of the adenosine a1 receptor agonist CCPA on fear memory during the memory consolidation phase, with the drug injected immediately after conditioned fear, and with the scene fear memory test performed on day 2 and day 3, respectively.

Fig. 6b-e show that in the scene fear test, rats in the control (solvent injection) and drug injection groups had similar levels of freezing, indicating that the animals were similar in learning ability.

FIG. 6f shows that the levels of freezing in the i.p. low dose CCPA (0.03mg/kg) group rats were similar to the control group rats, indicating no effect on fear memory.

FIG. 6g-i rats in the group given intraperitoneal injections of high dose CCPA (0.1/0.3/1mg/kg) had significantly reduced levels of freezing, indicating a significant reduction in fear memory.

Figure 6j, m is a representative heat map showing the movement traces of rats in the control group (without conditioned fear training), conditioned fear training group at 3 hours and 24 hours after solvent or drug injection in the open field. Figure 6k-o shows that conditioned fear-trained rats have significantly reduced locomotor distance in the open field and exploration time in the centre of the open field, inducing an anxiety phenotype, and that abdominal injection of the adenosine a1 receptor agonist CCPA (0.1mg/kg) effectively reversed the anxiety phenotype: the distance of movement in the open field and the exploration time in the center of the open field are significantly increased.

Example 8 intraperitoneal injection of Low dose CCPA did not affect spontaneous locomotion in animals

The effect of intraperitoneal administration of an adenosine a1 receptor agonist on rats was observed.

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

Figures 7b-e show that low doses of CCPA (0.1mg/kg) did not affect spontaneous locomotion in animals, and high doses (0.3/1mg/kg) significantly reduced locomotion distance in animals, but returned to normal levels after 5 hours or 24 hours post-injection, respectively.

Conclusion

The invention discovers for the first time that activation of astrocytes of hippocampal CA1 plays an important role in regulating and controlling memory consolidation of fear memory of mammals and reducing fear memory and anxiety. In particular, the inventors have found that astrocyte activation can continue to significantly reduce fear memory without affecting the formation of new memory. The inventors have further discovered that activation of astrocytes reduces fear memory through adenosine, a degradation product of ATP, and adenosine a1 receptors, and that formulations of astrocyte excitability can reduce fear memory and anxiety disorders by local injection or systemic administration. The present invention thus provides methods and medicaments for modulating memory consolidation of fear memory in mammals by increasing the excitability of astrocytes, for treating fear memory or disorders related thereto such as anxiety.

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

The unit "degree" of temperature as used herein refers to degrees celsius, i.e., degrees celsius, unless otherwise indicated.

Claims

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:

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,

or, wherein the chemical agent is:

or

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.