CN108853502A

CN108853502A - The method and pharmaceutical composition for treating depression

Info

Publication number: CN108853502A
Application number: CN201710322647.XA
Authority: CN
Inventors: 胡海岚; 杨艳; 崔卉; 崔一卉
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-05-09
Filing date: 2017-05-09
Publication date: 2018-11-23
Anticipated expiration: 2037-05-09
Also published as: CN108853502B

Abstract

The present invention is to treat the method and pharmaceutical composition of depression, the purposes for inhibiting the reagent of burst discharge in the habenular nucleus of outside in the drug of preparation treatment depression is provided, wherein the reagent of burst discharge is N-methyl-D-aspartate acceptor inhibitor and/or T-type calcium channel inhibitor in habenular nucleus on the outside of the inhibition.The present invention also provides the pharmaceutical compositions for the treatment of depression, wherein the reagent comprising inhibiting burst discharge in the habenular nucleus of outside.The present invention also provides the methods of the potential substance of screening treatment depression, include the steps that applying tester to be screened to animal models of depression, provide wherein the animal models of depression has the abnormal of outside Habenular Neurons burst discharge.

Description

The method and pharmaceutical composition for treating depression

Technical field

The present invention relates to disease treatments and drug field.Specifically, the present invention relates to the treatment of depression and for treating The pharmaceutical composition and preparation method of depression.

Background technique

Depression has very high proportion in crowd.If patients with depression is without suitably treating, in feelings It all can be by significant impact in thread and body.According to American National mental health research institute (US National Institute Of Mental Health, NIMH) definition and description, depression includes following symptom：" persistent sad, anxiety or inanition " Mood；Feeling of despair, pessimistic sense；Sense of guilt, valueless sense, helplessness；Lose to the hobby and movable interest enjoyed and It is happy；Energy reduces, is tired；It is difficult to concentrate on, memory difficulty, is difficult to make a decision；More dynamic, irritabilities etc..

Outside habenular nucleus (lateral habenula, LHb) is the component part of habenular nucleus, is located at epithalamus.Outside habenular nucleus is The Main Tissues of information are transmitted between limbic forebrain and midbrain.In recent years, discovery outside habenular nucleus and dopaminergic and serotonin energy Nerve fibre connection and regulation so that outside habenular nucleus takes part in a variety of physiological activities, influence body function, with drug habit, The state of mind such as award-detest, pain, sleep are related to illness.

It has been found that outside habenular nucleus has with the generation of depression be associated with.Studies have shown that in depression rat, outside habenular nucleus Frequency to the miniature excited postsynaptic current mEPSC of VTA projection neuron significantly increases relative to normal rat, this prompt In habenular nucleus, the relevant state of being overexcited of this depression is likely to be (Li, the B.et mediated by synaptic plasticity mechanism Al.Nature 470,535-539,2011).Under normal circumstances, outside habenular nucleus has low-level inhibition to make VTA and DRN With.In depression, stress causes β CaMKII expression quantity to significantly rise, on the GluR1 for leading to outside Habenular Neurons Film, neuronal excitability and cynapse transmission efficiency significantly increase.The fever enhancing of outside habenular nucleus inhibits VTA and DRN, Lead to the desperate performance (Li et al., Science 341,1016-1020,2013) on anhedonia and behavior..

This field has had some common antidepressants, but these drugs are usually after long a period of time It can just take effect.And the pathomechanism of depression is caused not to be realized completely also.This field also need it is new, or work faster The method and drug of the safer treatment depression of speed, dosage.

Summary of the invention

The present invention for the first time and have been surprisingly found that outside habenular nucleus neuron burst discharge (burst) in the generation of depression Play a significant role, and have found the key factor for influencing the burst discharge of outside habenular nucleus, thus provides by inhibiting outside The burst discharge of habenular nucleus treats the method and drug of (inhibition) depression, especially the quick method for the treatment of (inhibition) depression And drug.

Specifically, the present invention provides the methods for treating depression by inhibiting the burst discharge of outside habenular nucleus.This hair It is bright to provide the purposes for the drug for inhibiting the reagent of burst discharge in the habenular nucleus of outside to be used to treat depression, it is especially applied in outer The drug for being used to treat depression of side habenular nucleus.The present invention also provides the pharmaceutical compositions for the treatment of depression, especially apply The pharmaceutical composition for being used to treat depression of habenular nucleus on the outside contains the reagent for inhibiting burst discharge in the habenular nucleus of outside.

Term " burst discharge ", or " burst discharge " refer to neuron during discharge while generating two or two The discharge mode of the above spike.

Burst discharge is inhibited to refer to the granting degree for inhibiting burst discharge, frequency or tufted including reducing burst discharge are put In electric process in cluster spike potential number, reduce the intensity of burst discharge, even eliminate the generation of burst discharge.

Term " single to provide ", or " single electric discharge ", are that neuron provides a spike every time during discharge Discharge mode.

The reagent of inhibition burst discharge includes can be in compound, compound or the mixing for playing inhibiting effect to burst discharge Object, and the preparation used in the method (containing surgical method) for inhibiting burst discharge etc..The reagent includes small molecule Compound or the Large molecule actives ingredient such as compound or albumen, nucleic acid, for example, with the albumen knot in burst discharge physiological pathway The antagonist of conjunction such as antibody, or influence the nucleic acid etc. of the expression of these albumen.

In the present invention, " treatment " includes：Improvement mitigates, reduces or prevents the ongoing of symptom relevant to depression Process or result；Improve the ongoing process or result of symptom relevant to depression；Make to be in and leads to specific body function The ongoing process or result of the disease of damage or the body function normalization in illness；Or cause one kind or more of disease The ongoing process or result that the clinical measurable parameter of kind improves.In one embodiment, therapeutic purposes be prevention or Slow down (mitigation) undesirable physiological conditions, conditions or diseases, or obtains beneficial or desired result.The result can be, Such as medicine, physiology, clinical, physiotherapy, occupational therapy, towards health worker or patient；Or this field understands For " quality of the life " or the parameter of number of storage tanks produced per day.In the present invention, beneficial or desired clinical effectiveness includes but is not limited to, Mitigate symptom；Reduction/reduce the situation, the degree of conditions or diseases；Stablize the shape of (the i.e. non-deterioration) situation, conditions or diseases State；Postpone the situation, conditions or diseases beginning or slow down its progress；Improve or mitigate the situation, conditions or diseases；And mitigation (no matter partially or totally), no matter it is detectable or it is undetectable go out；Or enhance or improve the situation, conditions or diseases. In one embodiment, treatment includes causing clinical significant response without the side effect of excessive level.In an embodiment party In case, if treatment also includes extending survival period compared with the expected survival period for not receiving treatment.In one embodiment, Treatment refers to administration medicine or executes medical procedure to patient.In the present invention, treatment can be prevention (preventing), cure weakness or Disease, or the clinical setting of improvement patient, including the course of disease or disease severity are reduced, or subjectivity improves the quality of the life of patient or prolongs The survival period of long patient.

It is of the invention to treat depression by inhibiting the burst discharge of outside habenular nucleus in one aspect of the invention Method, the reagent of burst discharge is used to treat the purposes of the drug of depression, Yi Jiben in habenular nucleus on the outside of inhibition of the invention In the pharmaceutical composition of the treatment depression of invention, the reagent for inhibiting burst discharge is N-methyl-D-aspartate receptor Inhibitor.

N-methyl-D-aspartate is a kind of excitatory amino acid (excitatory amino acids, EAA), is The excitatory neurotransmitter of pivot nervous system.N-methyl-D-aspartate receptor (nmda receptor or NMDAR) is a kind of ion Receptor participates in excitatory synapse transmitting.The nerve mediated to the adjustable glutamatergic neurotransmission of the adjusting of nmda receptor is made With.

N-methyl-D-aspartate acceptor inhibitor for use in the present invention includes but is not limited to：

1) competitive inhibitor (inhibitor with glutamate-binding site competition) of nmda receptor：AP5, AP7, CPPene, Selfotel (Selfotel)；

2) noncompetitive inhibitor (inhibitor for blocking allosteric binding site) of nmda receptor：Aptiganel (Aptiganel), ketamine, Memantine (memantine), Huperzine A, ibogaine (Ibogaine), HU-211 add Bar spray fourth (Gabapentin), PD-137889 etc.；

3) channel blocker (channel blocker) of nmda receptor uncompetitive：Amantadine (Amantadine), atropic west Spit of fland (Atomoxetine), AZD6765, dextromethorphan (Dextromethorphan), hydrochloric acid magnesium amantadine, MK801 (Dizocilpine)；

4) glycine binding site point inhibitor：TK-40, kynurenine (Kynurenic acid) etc..

It is of the invention to treat depression by inhibiting the burst discharge of outside habenular nucleus in one aspect of the invention Method, the reagent of burst discharge is used to treat the purposes of the drug of depression, Yi Jiben in habenular nucleus on the outside of inhibition of the invention In the pharmaceutical composition of the treatment depression of invention, the reagent for inhibiting burst discharge is T-type calcium channel inhibitor.

T-type calcium channel or T-type calcium channel (transient calcium channel) also known as low-voltage activation calcium are logical Road (Low voltage activate calcium channel, T-type calcium channel).T type calcium channel exists The excitability of maincenter and peripheral neverous system has important role in adjusting.In vertebrate, T-type calcium channel family includes 3 different 1 subunit genes of α：CACNA1G, CACNA1H, CACAN1I are separately encoded α 1G, α 1H and α 1I, to constitute Cav3.1, Cav3.2 and Cav3.3,3 kinds of T-type calcium channel hypotypes.T-type calciphorin is the structure of tetramer composition, each 1 subunit of monomer, that is, α contains four homology regions.Channel protein duct is made of aforementioned four homology region.Duct spiral and thin The end connection of extracellular S6 segment is enough to pass through filter at calcium ion selective.The S4 segment of each homeodomain is every three Amino acid has positively charged amino acid residue, forms the voltage sensor in channel, is sent out based on this structure in film potential It can control the open and close of channel when changing.

T-type calcium channel inhibitor for use in the present invention includes but is not limited to：

Succinimide class (Succinimides), such as ethymal (ethosuximide), mesuximide (methsuximide)；Hydantoins (hydantoins)；Zonisamide (zonisamide)；valproate； phenytoin；Mibefradil；Phenytoinum naticum (Phenytoin)；sipatrigine；Piperazine analog such as Flunarizine, Z941；Piperidines analog such as Z944 and Fluoropiperidine； TTA-P1；TTA-P2；Quinazolinone (quinazolinone)；TTA-Q4；ML218 etc..

It is of the invention to treat depression by inhibiting the burst discharge of outside habenular nucleus in one aspect of the invention Method, the reagent of burst discharge is used to treat the purposes of the drug of depression, Yi Jiben in habenular nucleus on the outside of inhibition of the invention In the pharmaceutical composition of the treatment depression of invention, the reagent for inhibiting burst discharge is N-methyl-D-aspartate receptor The combination of inhibitor and T-type calcium channel inhibitor.

In the present invention, depression can refer in particular to " depression that outside habenular nucleus mediates ", refer in particular to " outside habenular nucleus cluster The depression that shape electric discharge mediates ".The inventor of the present application discovered that and demonstrate the abnormal of neuron of outside habenular nucleus and provide, especially It is that abnormal provide of burst discharge plays a significant role in the generation of depression.It is outer that influence is also found in present inventor The key factor of the burst discharge of side habenular nucleus is able to suppress or eliminates depression by the adjusting to these key factors.

It is of the invention to treat depression by inhibiting the burst discharge of outside habenular nucleus in one aspect of the invention Method, the reagent of burst discharge is used to treat the purposes of the drug of depression, Yi Jiben in habenular nucleus on the outside of inhibition of the invention In the pharmaceutical composition of the treatment depression of invention, this method or drug are suitable for not rising in other antidepression methods and drug It is used in the patients with depression of effect.

The antidepressants that this field has used, the mechanism for inhibiting depression according to it are classified, it may include：

● melatonin agonists；

● selective serotonin reuptake inhibitor (SSRIs)；

● 5-HT and norepinephrine reuptake double inhibitor (SNRIs)；

● monoamine oxidase inhibitors (MAOIs)；

● tricyclic anti-depressants (TCAs)；

● triple monoamine uptake blocking agents；

● metabotropic glutamate receptor (mGluRs)；

● GABA antagonist；

● NK1 antagonist；

● NK2 antagonist；

● CRF1 antagonist；

● Argipressin V1b antagonist；

● MCH receptor antagonist；

● NT-3, NT-4 antagonist；

● CREB antagonist etc..

The antidepressants of the above type and its specific drug are listed in WO2007/137247.It is introduced herein by full text.

Present inventor has found for the first time and demonstrates the abnormal of the neuron of outside habenular nucleus and provides, and especially tufted is put Abnormal provide of electricity plays a significant role in the generation of depression, thus provides the neuron by inhibiting outside habenular nucleus Abnormal to provide, especially the abnormal of burst discharge is provided to treat the method and drug of (inhibition) depression.This be this field The brain target tissue or its molecular water that the mechanism and drug for the treatment depression known fail the pathomechanism being directed to and treated Target on flat.Therefore, method provided by the invention and drug or pharmaceutical composition are particularly suitable in above-mentioned antidepression It is used in the patients with depression that method and drug do not work.

Certain nmda receptor antagonists known in the art can be used for treating depression.But in these reports, discovery Or the antidepression mechanism speculated with present invention discover that mechanism, i.e., by inhibiting the abnormal of outside Habenular Neurons to provide, especially It is that the abnormal of burst discharge is provided to inhibit depression, it is entirely different.In the case where not destroying novelty of the invention, at this The one aspect of invention, the method for the invention for treating depression by inhibiting the burst discharge of outside habenular nucleus, this hair The reagent of burst discharge is used to treat the purposes and treatment suppression of the invention of the drug of depression in habenular nucleus on the outside of bright inhibition In the pharmaceutical composition of strongly fragrant disease, the reagent for inhibiting burst discharge does not include these NMDA receptor antagonists, such as AP5, CPPene, MK801, Memantine (memantine), ketamine, Fei Er ammoniacum (felbamate), glycine, D-Ser, D- Seromycin, Pidolidone ifenprodil etc..

There is an other report and find that certain compounds can inhibit T-type calcium channel in this field, it may have depression The phenomenon that.For example, Prozac, Trazodone and ethymal.But in these reports, the antidepression mechanism and sheet that propose or assume The mechanism of discovery is invented, i.e., inhibits depression by inhibiting the exception of burst discharge in the Habenular Neurons of outside to provide, completely It is different.It is of the invention by inhibiting outer in one aspect of the invention in the case where not destroying novelty of the invention The burst discharge of side habenular nucleus come the method for the treatment of depression, on the outside of inhibition of the invention in habenular nucleus the reagent of burst discharge for controlling In the purposes of drug and the pharmaceutical composition for the treatment of depression of the invention for treating depression, the inhibition burst discharge Reagent does not include Prozac, Trazodone and ethymal.

It is of the invention to treat depression by inhibiting the burst discharge of outside habenular nucleus in one aspect of the invention Method and inhibition of the invention on the outside of in habenular nucleus the reagent of burst discharge be used to treat depression drug use on the way, institute The reagent for the inhibition burst discharge stated does not inhibit individually to discharge (tonic pulse).

It is of the invention to treat depression by inhibiting the burst discharge of outside habenular nucleus in one aspect of the invention Method, the reagent of burst discharge is used to treat the purposes of the drug of depression, Yi Jiben in habenular nucleus on the outside of inhibition of the invention In the pharmaceutical composition of the treatment depression of invention, the method, drug or pharmaceutical composition are locally to rise in habenular nucleus on the outside Effect is applied in the method and drug or pharmaceutical composition of outside habenular nucleus.For the method and drug for nerve fiber, especially It is brain neuroblastoma tissue, such as the habenular nucleus of outside, it is beneficial that the effect of drug, which is limited to destination organization,.For on the outside Administration is to treatment method in habenular nucleus and to prepare drug all be restrictive technical characteristic.Method or drug needs for LHb are examined Consider whether this method or drug can play validity in LHb, including whether drug can reach LHb, and whether can in LHb Reach the concentration etc. of action.In the present invention, the drug or pharmaceutical composition are the dosage form of habenular nucleus local administration on the outside.It can Reach in a manner of through local administration and drug effect is limited to destination organization, such as set can be passed through by the way that drug to be made The dosage form of pipe implantation outside habenular nucleus local administration.In another example drug to be made to the dosage form etc. being sustained after implanting tissue.In addition may be used also Said medicine is made to the form of the targeted drug delivery system of tissue specificity.Such as tufted can be inhibited to put by that will have The small molecule compound or bioactive molecule (nucleic acid such as encoding histone DNA or mRNA molecule, albumen such as antibody etc.) of Electricity Functional The protein bound antibody or antibody fragment specific expressed with that can specifically bind habenular nucleus on the outside connect to be formed and can be known The compound molecule of cell that is other and combining outside habenular nucleus.

Locally work in the habenular nucleus on the outside provided for aforementioned present invention (be applied in outside habenular nucleus) method, prepare medicine In the purposes or pharmaceutical composition of object, the reagent for inhibiting burst discharge includes nmda receptor antagonist.Specifically, described Nmda receptor antagonist may include but be not limited to：

2) noncompetitive inhibitor (inhibitor for blocking allosteric binding site) of nmda receptor：Aptiganel (Aptiganel), ketamine, Huperzine A, ibogaine (Ibogaine), HU-211, Gabapentin (Gabapentin), PD-137889 etc.；

Locally work in the habenular nucleus on the outside provided for aforementioned present invention (be applied in outside habenular nucleus) method, prepare medicine The purposes or pharmaceutical composition of object, the reagent for inhibiting burst discharge includes T-type calcium channel inhibitor.This field exists A other report finds that certain compounds can inhibit T-type calcium channel in habenular nucleus on the outside, it may have the phenomenon that depression. For example, ethymal.But in these reports, discovery or hypothesis antidepressant mechanism with present invention discover that mechanism, i.e., Inhibit depression by inhibiting the abnormal granting of burst discharge in the Habenular Neurons of outside, it is entirely different.Do not destroying this hair It is provided by the invention to treat depression method above by the burst discharge of outside habenular nucleus is inhibited in the case where bright novelty Or inhibit the reagent of burst discharge in the habenular nucleus of outside be used to treat depression drug use on the way, it is described to inhibit burst discharge Reagent does not include ethymal.

It is of the invention to treat depression by inhibiting the burst discharge of outside habenular nucleus in one aspect of the invention Method, the reagent of burst discharge is used to treat the purposes of the drug of depression, Yi Jiben in habenular nucleus on the outside of inhibition of the invention The pharmaceutical composition of the treatment depression of invention, particularly suitable for quickly treating (inhibition) depression.Medicine provided by the invention Object is suitable as treatment (inhibition) depression of quick acting.The most of antidepressants in this field generally require one week to several weeks The antidepressant effect of time competence exertion, such as common 5-HT reuptaking inhibitor (SSRI) is usually in 2-3 weeks effective, the 5- of HT and norepinephrine reuptake double inhibitor are usually just effective at 1 week.Antidepression method provided by the invention and drug Or the onset time of pharmaceutical composition is lower than one week, preferably shorter than three days, more preferably less than one day are for example, lower than 12 hours. Drug provided by the invention is also adaptable as quick acting and with middle effect or long-acting treatment (inhibition) depression, single dose The antidepressant effect of amount can continue one day or more, preferably last for three days or more, more preferably continue one week or more.

The pharmaceutical composition for the treatment of depression provided by the invention, it includes clusters in habenular nucleus on the outside of the inhibition of therapeutically effective amount The reagent of shape electric discharge.

Active constituent in pharmaceutical composition provided by the invention is the reagent for inhibiting burst discharge in the habenular nucleus of outside.Although Active constituent suitable for the pharmaceutical composition of the invention for the treatment of can be administered in the form of raw material compound, but preferably will Active constituent, optionally in the form of physiologically acceptable salt, with one or more adjuvants, excipient, carrier, buffer, Diluent and/or other conventional excipient substances are concomitantly introduced into pharmaceutical composition.

It can be by being arbitrarily easily suitable for it is expected that the approach of therapy gives pharmaceutical composition of the invention.Preferably give Medicine approach includes oral administration, especially with tablet, capsule, pastille, powder and liquid form；And parenteral, especially Skin, subcutaneous, intramuscular and intravenous injection.Pharmaceutical composition of the invention can be by those skilled in the art by using suitable It is prepared in the standard method of desired preparation and routine techniques.It is suitable for making active constituent sustained release if it is required, then can be used Composition.

Pharmaceutical composition of the invention can be those be suitable for taking orally, rectum, bronchus, nose, lung, part (including cheek With it is sublingual), transdermal, vagina or parenteral (including in skin, subcutaneous, intramuscular, peritonaeum, intravenous, intra-arterial, intracerebral, intraocular note Penetrate or be transfused) pharmaceutical composition of administration or those be suitable for by sucking or being blown into administration (including powder and Liquid Aerosol Administration) or be suitable for slow-released system be administered by way of pharmaceutical composition.The example of suitable slow-released system includes containing The semi-permeable matrix of the solid hydrophobic polymers of the compounds of this invention, the matrix can be shaped article form, such as thin Film or micro-capsule.

Therefore the active constituent in pharmaceutical composition of the invention can be made together with conventional adjuvant, carrier or diluent At pharmaceutical composition and its form of unit dose.Such form includes solid and especially tablet, filling capsule, powder With the form and liquid of pellet, especially aqueous solution or non-aqueous solution, suspension, emulsion, elixir and the above-mentioned form of filling Capsule, all these forms are used to take orally, for rectally suppository and for parenteral sterile injection it is molten Liquid.Such pharmaceutical composition and its unit dosage forms may include the conventional ingredient of conventional ratio, with or without other activity Compound or ingredient, and such unit dosage forms contain and required daily application dose range is comparable any suitable has The active constituent of effect amount.

To prepare pharmaceutical composition from the active constituent in pharmaceutical composition of the present invention, pharmaceutically acceptable carrier can be with It is solid or liquid.The preparation of solid form includes powder, tablet, pill, capsule, cachet, suppository and dispersible Granule.Solid carrier can be one or more diluents, corrigent, solubilizer, lubricant, suspending agent, viscous of can also act as Mixture, preservative, tablet disintegrant or coating material substance.

Being suitable for the aqueous suspensions being administered orally can be by being dispersed in finely divided active constituent containing emplastic, such as Natural or synthetic natural gum, resin, methylcellulose, sodium carboxymethylcellulose or other well-known suspending agents water in And it prepares.

It further include being intended to facing before the Solid form preparations for being converted into the liquid form preparation for oral administration.In this way Liquid form include solution, suspension and emulsion.In addition to the active ingredient (s), such preparation also may include colorant, flavoring Agent, stabilizer, buffer, artificial and natural sweetener, dispersing agent, thickener, solubilizer etc..

In order to locally apply to epidermis, the compounds of this invention can be configured to ointment, creme or lotion or transdermal patch Agent.For example, ointment and creme can be formulated with water or oily matrix additional suitable thickener and/or gelling agent.It washes Agent can be formulated with water or oily matrix, and usually also containing one or more emulsifying agents, stabilizer, dispersing agent, suspending agent, Thickener or colorant.

Respiratory tract administration can also realize that wherein active constituent is together with suitable propellant in compression package by aerosol It is provided in dress, suitable propellant such as chlorofluorocarbon (CFC), such as dicholorodifluoromethane, trichlorofluoromethane or dichloro-tetrafluoro second Alkane, carbon dioxide or other suitable gases.Aerosol can also suitably contain surfactant, such as lecithin.The agent of drug Amount can be by being equipped with metering valve control.

Alternatively, the active constituent in pharmaceutical composition of the present invention can provide in dry powder form, such as compound is being suitble to In powdered substrate (such as lactose, starch, starch derivatives (such as hydroxypropyl methyl cellulose) and polyvinylpyrrolidone (PVP)) Mixture of powders.Expediently, dust carrier will form gel in nasal cavity.Powder composition can be presented with unit dosage forms, example Such as in the form of capsule or cylindrantherae (capsule or cylindrantherae of such as gelatin), or the blister package that can be therefrom administered by inhalator with powder Form.

It, can be using the composition for being adapted to provide for active constituent sustained release when needing.

Pharmaceutical preparation is preferably unit dosage forms.In this kind of form, preparation is subdivided into the unit containing appropriate active component Dosage.Unit dosage forms can be the preparation of packaging, preparation of the packaging containing discrete magnitude, the tablet, capsule of such as packaging, Yi Ji little Powder in bottle or ampoule.In addition, unit dosage forms can be capsule, tablet, cachet or pastille itself, or can be suitable The packaged form of any of these dosage forms of quantity.

Tablet for oral administration or capsule and liquid for intravenous administration and continuous infusion are preferably to combine Object.

In one embodiment, when being intended to that there is abuse liability and because of nicotine using medicine composite for curing of the invention Caused by habituation when withdrawal symptom, concern such as natural gum, patch, spray, inhalant, aerosol as preparation.

Treatment effective dose means to alleviate the amount of the active constituent of symptom or the patient's condition.Therapeutic efficiency and toxicity, such as ED50 And LD50, it can be measured by the Standard pharmacological program in cell culture or experimental animal.Therapeutic and toxicity effect Dose ratio between fruit is therapeutic index, can be expressed by the ratio of LD50/ED50.

The dosage given must be directed to age, weight and the illness and administration route, dosage form of individual treated certainly And dosage regimen and desired result and carefully adjust, and exact dosage should be determined certainly by doctor.

The present invention also provides a kind of animal models of depression, preferably rat or mouse.Depression of the present invention is dynamic Object model has characteristics of depression, there is the abnormal of outside Habenular Neurons burst discharge and provides.

The present invention also provides the methods screened using above-mentioned animal model for treating the potential substance of depression, including Step：

(1) tester to be screened is applied to animal models of depression；With

(2) related symptoms and/or index of the depression in the animal models of depression are observed, and are carried out with control group Compare.

Wherein, if the related symptoms of depression are significantly improved in the animal models of depression, then it represents that the test Object is to can be used for treating the potential substance of depression.

Further include in the method for wherein another aspect of the invention, potential substance of the screening for treating depression Following one or more steps：

To the potential substance that previous step filters out, its influence to neuron burst discharge is further tested；

To the potential substance that previous step filters out, it is applied to animal model, observes its influence to symptoms of depression；

When testing its influence to neuron burst discharge, if compared with negative control group (or blank control group), Being added or apply neuron burst discharge ratio in the test group of the tester significantly reduces, then it represents that the tester is treatment The potential substance of depression.

Detailed description of the invention

Habenular nucleus locally blocks nmda receptor to be enough to generate quick antidepressant effect on the outside of Fig. 1.(A) cLH Lateral Habenular Nucleus Bilateral casing is implanted into schematic diagram.White dashed line indicates habenular nucleus position.(B-G) local bilateral applies ketamine (the 25 every sides μ g, B- D) and AP5 (the every side 40nmol, E-G) arrives LHb, and (0.5 or 1 hour) can effectively reverse the depressed table of cLH rat in a short time Type：It significantly reduces the dead time (C and F) in forced swimming and significantly increases depressed animal to the Preference (D and G) of syrup. (H-I) antidepressant effect that LHb bilateral applies ketamine can continue to after administration the 14th day.All data are represented as average Value ± SEM.*P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001 compared with the control group.N.S. difference is indicated not Significantly.Other icons are identical.

In Fig. 2 rat and mouse depression animal model outside Habenular Neurons burst discharge enhancing and can by ketamine institute instead Turn.(A) the record site of Whole-cell recording is indicated, record site is distributed in the different subprovinces of outside habenular nucleus.(B-D) Three kinds of neuron are do not discharge (silent) respectively from Firing Patterns typical figure in the habenular nucleus of outside, it is single discharge (tonic) and Burst discharge (burst).Centre is reaction of the same neuron to TTX, and TTX can block the peak of single electric discharge and burst discharge Current potential.The right electric discharge trajectory diagram is the enlarged drawing in left side shadow region.(E-F) scatter plot (E) and accumulation curve (F) show tranquillization film The mean value and distribution of current potential (RMPs).(G-H) frequency is provided in cluster rather than frequency and resting membrane electric potential hyperpolarization journey are provided between cluster Degree is positively correlated.(I-N) in the depression model mice of congenital depressed (cLH) rat and chronic restricted stress induction, burst discharge Neuron ratio significantly increase.(I, L) pie statistical chart is shown in rat and depression model mice, burst discharge neuron Number increases.(J, M) column figure shows the cell proportion individually to discharge with burst discharge in all granting cells.(K, N) cylindricality system Meter figure shows the distribution of neuron interspike interval in motionless depressed animal habenular nucleus.

Fig. 3 shows that ketamine inhibits chronic restricted stress mouse Habenular Neurons burst discharge activity in body electrophysiological recording With the synchronization of θ wave band.(A) the record site in body recording electrode in control and CRS mouse LHb.(B) it is recorded in body Control, the representative example (left side) of the mouse LHb Neural spike train of CRS and CRS+ ketamine and averagely granting waveform (right side), leads to Analysis interspike interval (ISI) is crossed to separate burst discharge.(C-D) CRS mouse LHb neuron burst discharge ratio and per minute The number of burst discharge is all significantly higher than control mice, and can be inverted by ketamine.(E) control mice and CRS mouse exist Integral distribution curve (the control group of ketamine injection front and back interspike interval：143ms, CRS group：33ms, CRS+ ketamine group： 121ms).Dotted line indicates the point that spike potential 50% changes.(F) control group and CRS group the mouse neuron before and after to ketamine are sent out Put related field potential, the time interval between the adjacent trough of CRS group be 140ms or so (period is about 7hz) (G) control mice and The correlation (SFC) of CRS mouse single unit discharge and field potential before and after ketamine injection.The SFC of each electric discharge unit (left side), average SFC (in), SFC percentage in θ wave band (4-10Hz).All data are represented as average value ± SEM. *P< 0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001 compared with the control group.N.S. indicate that difference is not significant.Others figure It marks identical.

The burst discharge of Fig. 4 LHb needs to activate nmda receptor.(A) neuron is clamped down on to the excitement generated in -80mV Property postsynaptic currents typical figure.By the way that GABA acceptor inhibitor is added in the artificial cerebrospinal fluid (ACSF) of no Mg2+ (picrotoxin) and ampa receptor blocking agent (NBQX) come separate nmda receptor mediation excitatory postsynaptic currents (NMDAR-EPSCS), and with nmda receptor blocking agent AP5 confirm electric current.(B) LHb neuron is clamped down on remembers under different voltages The NMDAR-EPSCs recorded, the electric current can be blocked completely by AP5.(C-H) ketamine (C-D) in LHb, AP5 (E-F) and NBQX (G-H) to the influence of spontaneous burst discharge.Left side is typical figure, and right side is statistical chart.(I-J) NMDA perfusion can make not provide Cell generates burst discharge, and the burst discharge of this induction can be inhibited by ketamine.NMDA can induce big excitatory synapse Current potential and burst discharge afterwards.All data are represented as average value ± SEM.*P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001 compared with the control group.N.S. indicate that difference is not significant.Other icons are identical.

Fig. 5 LHb burst discharge needs the participation of neuron membrane hyperpolarization and T-type voltage-sensitive dry passage.(A) slope electricity Streamer penetrates induction LHb neuron from burst discharge to the typical figure of single electric discharge conversion, state of the neuron in opposite hyperpolarization Under be easy to produce burst discharge, and single electric discharge is generated in the state of opposite depolarising.(B) statistical chart is shown in rats and mice LHb neuron can induce the neuron ratio for generating burst discharge after injecting super galvanic current.(C-E) current clamp, which adds, is recorded Electric discharge number (E) is related to neuron resting membrane electric potential in burst discharge frequency (C), burst discharge duration (D) and cluster Property.(F) the typical case figure that spontaneous single electric discharge neuron is converted to burst discharge under hyperpolarization.(G) spontaneous cluster under depolarizing The typical case figure that shape discharges to single electric discharge conversion.(H, I) T-VSCC blocking agent Mibefradil (H) and HCN carrier frequency channel break Influence of the agent ZD7288 (I) to the spontaneous burst discharge of LHb neuron.Left side is typical figure, and right side is statistical chart.(J) one It provides exemplary diagram and summarizes required various ions and channel in LHb neuron burst discharge.The T-VSCC of activation to hinder The magnesium ion of disconnected nmda receptor is removed, and the opening driving membrane potential of neurons super cluster shape of T-VSCC and nmda receptor channel is put Electric threshold value direction change.As rapid deactivation T-VSCC and nmda receptor channel, neuron resting membrane electric potential is restored to -55mV Once, another burst discharge period is originated.All data are represented as average value ± SEM. *P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001 compared with the control group.N.S. indicate that difference is not significant.Other icons are identical.

Fig. 6 T-VSCC antagonist shows quick antidepressant effect.(A-C) locally injecting mibefradil is to bilateral LHb shows quick antidepressant effect in FST (B) and SPT (C) behavior.(A) CTB is injected for LHb determine casing Injection site figure.All data are represented as average value ± SEM.*P<0.05, * * P<0.01, * * * P<0.001, * * * * P< 0.0001 compared with the control group.N.S. indicate that difference is not significant.Other icons are identical.

Fig. 7 eNpHR photoactivation induction rebounding type burst discharge make animal show can by ketamine invert detest and Depressed phenotype.(A) eNpHR virus expression carrier constructs schematic diagram (above), photoelectricity and record schematic diagram (following figure).(B, C) exists In the mouse LHb of AAV2/9-eNpHR expressing viral, the brain piece of yellow light activation neuron (B) and the neuron that is recorded in body (C) typical figure of rebound burst discharge.The cell percentages for successfully inducing burst discharge are shown in right side statistical chart.(D) point The system of battle formations and post-stimulus time column figure are shown in a representative LHb neuron in body optoelectronic pole record and stimulate 100ms yellow light Reaction.(E) provide in the cluster of rebounding type burst discharge caused by eNpHR photoactivation provided in frequency and cluster the distribution of number with It is recorded in CRS mouse habenular nucleus suitable.The right-angled intersection in center represents average value.(F) rebounding type caused by eNpHR photoactivation The real time position that burst discharge induces detests (RTPA).Left side shows the representative thermal map of RTPA, and right side shows quantitative analysis Detest and avoids parameter.(G) the FST depression phenotype that rebounding type burst discharge caused by eNpHR photoactivation induces.(H) eNpHR light swashs The SPT depression phenotype that rebounding type burst discharge caused by living induces.All data are represented as average value ± SEM.*P< 0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001 compared with the control group.N.S. indicate that difference is not significant.It is other Icon is identical.

The single electric discharge of Fig. 8 and burst discharge identical frequency cannot cause depressed phenotype.(A) in vitro electrophysiological recording is shown The photoactivation oChIEF optical channel of 5Hz generates the single electric discharge of 5Hz.(B) compared with expression compares the eGFP mouse of non-optical channel, Photoactivation does not change the locomitivity of animal.(C) the single electric discharge that the photoactivation of 5Hz generates cannot induce depressed phenotype.

Specific embodiment

Further illustrate that substantive content and beneficial effect of the invention, the embodiment are only used for below in conjunction with embodiment Bright of the invention rather than limitation of the present invention.

1 material of embodiment and method

Animal material

Male cLH rat (4-12 week old), Sprague Dawley rat (4-12 week old).CLH rat is a selection Property cultivate with congenital Xi Zezong depression phenotype animal models of depression (D.Schulz, M.M.Mirrione, F.A.Henn, Neurobiol Learn Mem 93,291, Feb, 2010).The cLH rat of this experiment is from U.S.'s Cold SpringHarbor It introduces in the laboratory Malinow.It is described in cLH rat feeding and breeding such as aforementioned D.Schulz, et al, Feb, 2010.Rat 4 Only/cage, 12 hours light and shade periods (7am-7pm has light).1/cage of cLH rat raising for casing experiment.Grow up (8-12 Week old) C57BL/6 mouse is used for performance testing：4/cage, 12 hours light and shade periods (5am-5pm has light).Rat and mouse Stable water and food can be freely absorbed, all zooperies are by Zhejiang University's animal protection and use the committee Approval.

Virus formulation

AAV9-CaMKII-eNpHR3.0-eYFP, plasmid are purchased from Addgene, Cat#26971, and virus is by the upper Haitai court of a feudal ruler Biotechnology Co., Ltd's coating；AAV9-Ubi-eGFP is presented by the laboratory Gao Guangping of UMass；AAV9-hSyn- OChIEF-tdTomato, plasmid are purchased from Addgene, Cat#50977, and virus is wrapped by upper Haitai court of a feudal ruler Biotechnology Co., Ltd Quilt.

Stereotaxical injection and histology

Mouse injecting virus:Mouse peritoneal injects ketamine (100mg/kg weight) and Xylazine (8mg/kg) mixed liquor (Stoelting instruments) after anesthesia, is fixed on stereotaxic instrument.The every side LHb of every mouse injects 0.1- The AAV virus (~10 of 0.2ul purifying concentration¹³Infectious unit/ml), LHb stereotactic coordinates (longitudinal separation Bregma：- 1.7mm (AP), left and right is other to open ± 0.46mm (ML), cortical surface -2.56mm (DV) down).It is micro- using the glass voluntarily drawn Electrode is slowly injected into (~100-150nl/min), and injection terminates let the acupuncture needle remain at a certain point 5min, then slowly removes injection electrodes in 5min again.

Postoperative at least 14 days, carry out behavioral experiment or electro physiology experiment.Injection position is checked after behavioral experiment, only Use those of correct injection animal data.

The brain section for having injected AAV virus checks under fluorescence microscope or other viruses that GFP is marked are in micro- inspection GFP albumen is checked with antibody before looking into.The habenular nucleus area of each brain be cut into 6 groups continuously slice (slice of mouse 30um, every group 6；Rat 40um slice, every group of 8-9 piece).All slices use Hoechst counterstain before being installed to fixinig plate.

The embedding casing of rats with bilateral LHb：After 4% amobarbital of rats by intraperitoneal injection that (60mg/kg weight) anesthesia, Gu Due on rat stereotaxic instrument.Set effective LHb stereotactic coordinates (longitudinal separation Bregma：- 3.7mm (AP), left and right are other It opens ± 0.7mm (ML), cortical surface -4.1mm (DV) down).Above the corresponding skull of LHb, cranium bores drilling, later in skull On fix three screws.Bilateral casing (purchased from U.S. plastics one company) is set to enter habenular nucleus by pipe LHb coordinate is set Top, and be inserted into casing stifled with the isometric flush end of casing after denture fixing device cement solidifies completely with denture fixing device cement fixed sleeving Core, and nut is screwed on to prevent stifled core from falling off.Postoperative 7 days, after rat after restoring in operation wound, it can be used for detecting drug Behaviouristics effect.After the anesthesia of rat using gas (isoflurane) Anesthesia machine, with the injection needle of sleeve kit, slowly infused from casing Enter 1ul drug to be tested (about 100nl/min), then injection terminates that let the acupuncture needle remain at a certain point 10min removes injection inner core.According to having for drug It imitates time-histories and carries out Behavior test.After behavioral experiment, CTB-488or 555 is injected by casing and checks sleeve position, LHb The accurate Animal Behavior Science data of injection site are just used to statistically analyze.

In vitro electrophysiological recording

40-50 days rats or the mouse being born 8 weeks are after isoflurane is anaesthetized after birth, with the slice of the ice-cold oxygenation of 20ml Liquid carries out perfusion.Quickly broken end takes out brain, puts into the slice liquid of oxygenation.It is being oxygenated followed by Leica vibratome Ice-cold slice liquid in, carry out 350um tubulose section slice.34 DEG C of ACSF (118mM of the habenular nucleus brain piece in oxygenation NaCl,2.5mM KCl,26 mM NaHCO₃,1mM NaH₂PO₄,10mM glucose,1.3mM MgCl₂and 2.5mM CaCl₂, gassed with 95%O₂And 5%CO₂) in, room temperature, which is transferred to, after restoring at least 1 hour is recorded.Inject chlorine The rat and mouse of amine ketone group 1h before animal takes brain are carried out.

The Patch-clamp techniques of outside habenular nucleus brain piece use Axon Multiclamp 700B amplifier, in 32 ± 1 DEG C of environment Under, it is recorded under the Olympus microscope of assembly infrared videomicroscopy optical lens.All cells are in full cell It is recorded under mode.Neuron recording electrode impedance is 4-6M Ω, and liquid ingredient is (mM) in electrode：105K-Gluconate, 30KCl, 4Mg-ATP, 0.3 Na-GTP, 0.3EGTA, 10HEPES and 10Na-phosphocreatine, pH 7.35.Note Recording artificial cerebrospinal fluid (ACSF) ingredient used in external solution is (mM)：125NaCl,2.5KCl,25NaHCO₃, 1.25NaH₂PO₄, 1MgCl₂25 glucose of and.Data are recorded using Digidata 1322A in 10kHz down-sampling after 2kHz is filtered.Data It is analyzed using 10 software of pClamp.

Action potential from frequency is provided, is carried out under I=0 current clamp logging mode in rats and mice LHb neuron, is continued 60 seconds, averagely granting frequency was provided by this 60s to count.Definition to the different discharge modes generated in LHb is：It does not provide Cell, refer to does not have the cell of action potential granting in entire recording process；The cell individually to discharge refers to discharge frequency in 0.1- 10Hz, rarely 10-20Hz；Burst discharge cell refers to the granting that can generate cluster, and provides very high frequency in cluster, but table Reveal gradually decline trend, sending out frequency rigid between cluster is up to 200Hz.

The excitatory postsynaptic currents that the nmda receptor of induction mediates are at the ACSF of zero magnesium, cell is pinned in- It is recorded under 50mV to -80mV.The T-type voltage-sensitive Calcium Current of induction, is to clamp down on cell at -50mV, Then cell is clamped down on into -100mV again, continues 1 second.Stimulation is provided according to nominal frequencies 0.1Hz.Calcium current is by linearly seeping Leakage subtracts each other to obtain.

In body electrophysiological recording

After adult male mice intraperitoneal injection of ketamine (100mg/kg weight) and the anesthesia of Xylazine (8mg/kg) mixed liquor, (Stoelting instruments) is fixed on stereotaxic instrument.It (will be made of four wire electrodes) 8 tetrode The travelling electrode array of (resistance is 250-500K Ω, California fine wire) composition is implanted to LHb (AP：- 1.72mm；ML：±0.46mm；DV： -2.44mm).Stainless steel wire is wound on two screws being fixed on skull for connecing Ground.Electrode is fixed on skull surface with denture fixing device cement.Animal restore 5-7 days after, start adapt to record used in adapter, one day It 10 minutes, adapts to 2-3 days altogether.Record using 64 channels OmniPlex-D Neural Signal Collecting system (Plexon Inc., Dallas, TX), spontaneous firing activities (the sample frequency 40kHz, 300- of 30 minutes animal LHb are recorded in rearging cage 6000Hz bandpass filtering)) and field potential (LFP, sample rate 1kHz, 250Hz low-pass filtering), gain 5000.To may not be used The channel of the Neural spike train signal of discrimination is as reference electrode.Every time record after tetrode with 70 μm of depth steppings down, and Restore 2 days to start to record next time.For chronic restricted stress mouse, record before ketamine administration after 30min and administration 1h Discharge activities.The animal of record used finally all determines electrode site with the mode of Electricity damage.

Action potential sorting：All electric signals being recorded are imported into Offline Sorter V3 (Plexon Inc.), then single unit discharge is sorted manually using threshold method and principal component analysis (PCA).Interspike interval The neuron that granting less than refractory period (1.4ms) is left out, and ensures not sub-elect by cross-correlation analysis is not sent out It is raw to repeat.The signal that can not be separated with background noise is excluded.Data analysis：The partial data analyzes software used Neuroexplorer4 (Plexon Inc.) and MATLAB.Data analysis：The partial data analyzes software used Neuroexplorer4 (Plexon Inc.) and MATLAB.

Behaviors survey

Forced swimming tests (forced swim test, FST)

Experiment carries out under normal daylight lamp.The diameter of mouse forced swimming test hydrostatic column is 12cm, high 25cm.Test The depth of water is 14cm, 23-24 DEG C of water temperature.Swimming situation of the camera from side record mouse in 6min.It is united using double-blind fashion The dead time (prone float or four limbs of animal absolutely not movable time) of 4min after counting in mouse swimming 6min.

Syrup preference tests (sucrose reference test, SPT)

Experiment mice is individually raised 1 week, then gives within continuous 2 days mouse two bottles of light waters, water is changed to two in two days later The sucrose water of bottle 2% is trained.After training, gives one bottle of light water of animal and one bottle 2% of sucrose water is tested, The consumption (weighing to water bottle) of the position of the water bottle of exchange in every 12 hours, water of every 24 hour record and syrup, remembers altogether Record 48 hours.

The building of chronic restricted stress (chronic restraint stress, CRS) depression model mice

Animal is randomly divided into two groups, one group daily 11:00 to 14:50ml centrifuge tube constraint 2h is put between 00, it is continuous to fetter 14 days, in order to be conducive to animal breath, several apertures of 2mm diameter are drilled on the centrifuge tube of 50ml used；Another group of control-animal Do not receive Restraint Stress then.After daily constraint, animal puts back to the cage of oneself and raises with control animals same Receptacle.Forced swimming was carried out at the 15th day and the depressed phenotype of assessment animal is gone in the test of syrup preference.

It move freely the test of conscious mouse light genetic behavior

The part all Animal Behavior Science detection is all the dark period that animal is in biological rhythm, and expressing viral extremely It is few to carry out after three weeks.The optical fiber of implantation by porcelain bushing be connected to patch cables (porcelain bushing be purchased from Hangzhou China NEWDOON company), patch cables were connected a FC/PC adapter and were connected to a swivel (purchased from Canada Doric, Qu é bec) on, allow animal unlimitedly free movement.Another patch cables are connected by FC/PC adapter It is connected to the DPSS laser (Aurora-220-473) or 589nm DPSS laser (Aurora-220- of computer and 473nm 589), laser is purchased from Hangzhou China NEWDOON company.

Real time position avoids (Real-time place aversion, RTPA) test

Based on forefathers experimental method (Matthews et al., 2016；Zhu et al., 2016), an intermediate connection The open chests of 23 centimetres of 52x 26x, be divided to or so the chest (23 centimetres of 26x 26x) of two same sizes for behaviouristics survey Examination.Mouse, which is placed in case, to be move freely 20 minutes, horizontal to the basic preference of two sides chest for assessing mouse.Next 20 minutes test phases, mouse is evenly distributed to left and right sides chest, and using this side as stimulation case, the other side is then every The corresponding non-stimulated case of mouse.Mouse is put into non-stimulated case to start to test.Mouse just can activate Huang once entering stimulation case Light stimulus (eNpHR3.0:The interval 589nm, 1Hz, 16mW, 100ms), terminate until mouse returns to non-stimulated side.Avoid chest Surface is equipped with camera, records the activity condition of each experimental animal.And with Any-maze software (U.S. Stoelting Company) analyzing animal corelation behaviour index.Avoid score=rear 20min stimulated side and non-stimulated side residence time difference-is preceding 20min stimulated side and non-stimulated side residence time difference.

Spacious field test：Based on the experimental method (Matthews et al., 2016) of forefathers' research, spacious field test box used Size is：45 centimetres of 45x 45x, four walls and bottom are white resin material.Animal is first freely explored altogether in spacious field 9 minutes, laser stimulation (eNpHR3.0 was given in 3 minutes in centre:The interval 589nm, 1Hz, 16mW, 100ms).It is taken the photograph right above spacious field As head records the motion conditions of each experimental animal, with the corelation behaviour index of Any-maze software analyzing animal.

Forced swimming test：Based on existing forced swimming test method (Li et al., 2013).Experiment is in normal light According to being carried out under (about 100lux).The diameter of mouse forced swimming test cylindrical transparent containers is 12cm, high 25cm.Testing the depth of water is 14cm, 22.5-23 DEG C of water temperature.When mouse into the water after, laser stimulation i.e. start, continue 6 minutes (eNpHR3.0:589nm, The interval 1Hz, 16mW, 100ms).Swimming situation of the camera from side record mouse in 6min.After experiment, using double The dead time that blind mode counts rear 4min in mouse swimming 6min in video, (prone float or four limbs of animal were absolutely not The movable time).

Statistical analysis

All data are all with average value ± SEM.For all behavioral datas, using two-tailed Student's t-tests。

Embodiment 2 can produce quick antidepressant effect in the outside habenular nucleus local administration nmda receptor inhibitor of rat

By observing depressed phenotype to ketamine is given after cLH depression the rat on the outside implantation of habenular nucleus bilateral casing Variation.The A of Fig. 1 is cLH Lateral Habenular Nucleus bilateral casing implantation schematic diagram, and white dashed line indicates habenular nucleus position.The B-G of Fig. 1 Provide the result of experiment：Local bilateral applies different NMDAR inhibitor ketamine (the 25 every sides μ g, the B-D of Fig. 1) and AP5 (the every side 40nmol, the E-G of Fig. 1) arrives LHb, and (0.5 or 1 hour) can effectively reverse the depressed table of cLH rat in a short time Type significantly increases depressed animal to the Preference of syrup including significantly reducing the dead time (C and F of Fig. 1) in forced swimming (D and G of Fig. 1).

Be also observed in an experiment, LHb bilateral apply NMDAR inhibitor ketamine antidepressant effect can continue to The 14th day (as shown in the H-I of Fig. 1) after medicine.

Above it is demonstrated experimentally that the outside habenular nucleus in rat locally carries out inhibition processing to nmda receptor, can produce quickly and Lasting antidepressant effect.

This is that nmda receptor inhibitor, such as chloramines are given in discovery in the local organization of brain for the first time in this field Ketone can generate quick and lasting antidepressant effect.

Three kinds of Spontaneous Discharges of Neurons modes (not discharging, single electric discharge and burst discharge) exist in 3 outside habenular nucleus of embodiment Granting characteristic in depressed animal

With the electric discharge mould of whole-cell patch-clamp recording technique Habenular Neurons on the outside of in vitro brain section observation depression animal Formula.The A of Fig. 2 shows that the record site of Whole-cell recording, record site are distributed in the different subprovinces of outside habenular nucleus.It was found that Neuron is not discharge (silent) respectively (shown in the B of Fig. 2) there are three kinds from Firing Patterns typical figure in the habenular nucleus of outside, single A electric discharge (tonic) (shown in the C of Fig. 2) and burst discharge (burst) (shown in the D of Fig. 2).

The E (scatter plot) and F (accumulation curve) of Fig. 2 shows the mean value and distribution of resting membrane electric potential (RMPs).The results show that Compared to the cell that do not provide, single electric discharge cell resting membrane electric potential shows depolarization and burst discharge cells show is super Change.

In addition the experiment has found that in rat and mouse depression animal model, the neuron of the spontaneous burst discharge of outside habenular nucleus Ratio is significantly higher than control intact animal, and NMDAR inhibitor ketamine can significantly reduce the spontaneous burst discharge of depressed animal Neuron ratio.Prompt Habenular Neurons burst discharge enhancing in outside in depression.I and L (pie statistical chart) such as Fig. 2 is aobvious Show, in rat and depression model mice, burst discharge neuron number increases.J and M (column statistical chart) display of Fig. 2 is all Provide the cell proportion individually to discharge with burst discharge in cell.The motionless depressed animal of K and N (column statistical chart) display of Fig. 2 The distribution of neuron interspike interval in habenular nucleus.

4 experiment in vivo of embodiment observes the discharge mode of Habenular Neurons on the outside of depression animal

In order to further confirm effect of the outside Habenular Neurons burst discharge to depression, it is raw to be used in body multichannel electricity Recording method is managed, a kind of recording method of more preferable simulation animal physiological state, habenular nucleus is placed in multichannel on the outside of awake mouse Electrophysiological recording electrode, the granting of record outside Habenular Neurons, the synchronization including mouse burst discharge activity and θ wave band are living It moves.As a result as shown in Figure 3.

The A of Fig. 3 is shown in body recording electrode in control and CRS depression mouse (CRS, chronic restrain Stress the record site in LHb).The B of Fig. 3 is to record to compare in body, the mouse LHb neuron of CRS and CRS+ ketamine The representative example (left side) of electric discharge and averagely granting waveform (right side) are put by analysis interspike interval (ISI) to separate tufted Electricity.The C-D of Fig. 3 shows that the number of CRS mouse LHb neuron burst discharge ratio and burst discharge per minute is all significantly higher than Control mice, and can be inverted by ketamine.The E of Fig. 3 shows, control mice and CRS the mouse peak before and after ketamine injection Integral distribution curve (the control group of interpotential interval：143ms, CRS group：33ms, CRS+ ketamine group：121ms).Dotted line instruction The point that spike potential 50% changes.

The synchronization granting of neuroid can be enhanced in known burst discharge.We provide correlation by calculating neuron Field potential (spike-triggered averages, STAs) goes detection to provide the synchronization effect vibrated between field potential.Figure 3 F show control mice to CRS mouse the related field potential of neuron grantings before and after ketamine injection, control mice granting phase Closing field potential distribution is in more gentle trend, and no neuron is prompted to synchronize effect.The granting that 7Hz then occurs in CRS group is related Field potential distribution prompts Habenular Neurons network electric discharge on the outside of CRS depression mouse to show θ wave band (4-10Hz) rhythm and pace of moving things, and this Kind synchronizes effect and can be blocked by ketamine.

The G of Fig. 3 is average by analyzing the Neural spike train of each electric discharge unit and the correlation (SFC, left) of field potential SFC (in) and θ wave band (4-10Hz) in SFC percentage (right side), further demonstrate Habenular Neurons on the outside of CRS depression mouse Network electric discharge shows can be by θ wave band (4-10Hz) rhythm and pace of moving things that ketamine is blocked.

The above results show that in the depressed mouse model of chronic restricted stress induction, outside Habenular Neurons burst discharge Number is provided in frequency and cluster and is all significantly higher than control normal mouse, and this increase can be anti-by NMDAR inhibitor ketamine institute Turn.

5 isolated experiment of embodiment proves that the activation of nmda receptor is the abundant necessary item that LHb neuron generates burst discharge Part

The result of study having in other brain areas shows that the generation of burst discharge needs the calcium ion of nmda receptor mediation Interior stream.The present invention further studies to illustrate effect of nmda receptor during burst discharge generates in the habenular nucleus of outside, first First confirm the expression that LHb has NMDAR：Patch-clamp techniques NMDA electric current into LHb brain piece.The A of Fig. 4 is to clamp down on neuron The excitatory postsynaptic currents typical figure generated in -80mV.By in no Mg²⁺Artificial cerebrospinal fluid (ACSF) in be added GABA acceptor inhibitor (picrotoxin) and ampa receptor blocking agent (NBQX) are prominent come the excitability for separating nmda receptor mediation Electric current (NMDAR-EPSCS) after touch, and electric current is confirmed with NMDA receptor blocking pharmacon AP5.

The B of Fig. 4 shows that LHb neuron clamps down on the NMDAR-EPSCs being recorded under different voltages, which can quilt NMDAR inhibitor AP5 is blocked completely.This confirms the expression that LHb has NMDAR.

Ketamine (C-D) in the C-H display outside habenular nucleus of Fig. 4, AP5 (E-F) and NBQX (G-H) are to spontaneous burst discharge Influence.Left side is typical figure, and right side is statistical chart.By taking the C-D of Fig. 4 as an example, it can be seen that ketamine does not influence neuron Resting membrane electric potential (resting membrane potentials, RMPs), but almost blocked spontaneous burst discharge. C such as 4 is shown, 10 seconds after being handled with ketamine, the burst discharge of LHb is converted to individually discharge.The results show that NMDA by The frequency of spontaneous burst discharge is effectively reduced in body inhibitor ketamine and AP5, and blocks another glutamate receptor AMPA, right The influence of spontaneous burst discharge is weak more than nmda receptor inhibitor.

Further experiment direct perfusion NMDA in brain piece, discovery can be such that the cell that do not provide in LHb generation tufted puts Electricity, and this burst discharge can be blocked by ketamine.As shown in the I and J of Fig. 4, NMDA perfusion can make the cell that do not provide Burst discharge is generated, the burst discharge of this induction can be inhibited by ketamine.NMDA can induce electricity after big excitatory synapse Position and burst discharge.

The results show that the activation of nmda receptor is the sufficient and necessary condition that LHb neuron generates burst discharge in LHb.

Embodiment 6LHb burst discharge needs the participation of neuron membrane hyperpolarization and T-type voltage-sensitive dry passage

The phenomenon that found in embodiment 3, i.e. the neuron of different Firing Patterns has different resting membrane electric potentials in LHb (RMP), the neuron of spontaneous burst discharge shows the resting membrane electric potential of hyperpolarization.

Inventor further confirms that the relationship between resting membrane electric potential and neuron Firing Patterns.As shown in figure 5, firstly, giving The neuron of record injects an incremental slope current, make the resting membrane electric potential of cell from -80 to -40mV change.It is super Galvanic current injection, makes cell generate burst discharge.The peak value of number is between -56~-60mV in the cluster of burst discharge, and spontaneous The resting membrane electric potential of burst discharge cell is close.The electric current for giving hyperpolarization or depolarising to the cell of electric discharge certainly simultaneously injects Cell can also be made to mutually convert between burst discharge in single provide.

A in Fig. 5 is slope current injection induction LHb neuron from burst discharge to the typical figure of single electric discharge conversion. As shown, neuron is easy to produce burst discharge in the state of opposite hyperpolarization, and produced in the state of opposite depolarising Raw single electric discharge.B in Fig. 5 is statistical chart, shows that LHb neuron can induce generation after injecting super galvanic current in rats and mice The neuron ratio of burst discharge.C-E in Fig. 5 is：Current clamp adds the burst discharge frequency (C) being recorded, and burst discharge is held The correlation of electric discharge number (E) and neuron resting membrane electric potential in continuous time (D) and cluster.

Since nmda receptor is the channel just activated under the conditions of depolarizing, and generate the neuron tranquillization of burst discharge Film potential is hyperpolarization, and nmda receptor is how to be activated under the conditions of present inventor so further studies hyperpolarization And it participates in burst discharge.

The inventor of the present application discovered that being activated in neuron hyperpolarization and the ion that neuron can be made to depolarize is logical Road：The calcium channel of T-type voltage-sensitive.The calcium channel of T-type voltage-sensitive is the calcium channel activated under a kind of hyperpolarization, Make flow of calcium ions after the activation of channel and neuron is caused to depolarize.The channel there are three hypotype, Cav3.1, Cav3.2 and Cav3.3, three hypotypes have expression in LHb.

The activation energy that inventor has been experimentally confirmed the calcium channel of T-type voltage-sensitive in LHb, which causes tufted, puts Electricity.F in Fig. 5 is the typical case figure that spontaneous single electric discharge neuron is converted to burst discharge under hyperpolarization.G in Fig. 5 is Typical case figure of the spontaneous burst discharge to single electric discharge conversion under depolarising.

Inventors tested a large T-VSCC it is spontaneous to LHb or induce burst discharge influence.In addition, testing another ion (hyperpolarization-activated cyclic nucleotide-gated channel) is right in the channel channel-HCN LHb it is spontaneous or induce burst discharge influence.Experiment is spontaneous to LHb by the blocking agent of test T-VSCC blocking agent and HCN Or the influence of the burst discharge induced carries out.H in Fig. 5, I show that T-VSCC blocking agent Mibefradil (H) and HCN are logical Influence of road blocking agent ZD7288 (I) to the spontaneous burst discharge of LHb neuron.Left side is typical figure, and right side is statistical chart.Knot Fruit proves that T-VSCC blocking agent can significantly inhibit the burst discharge frequency that LHb is spontaneous or induces.And HCN channel blocker pair LHb it is spontaneous or induce burst discharge influence be much smaller than T-VSCC blocking agent.

Inventor has found that nmda receptor and T-VSCCs collaboration cause the spontaneous tufted in the habenular nucleus of outside to be put for the first time as a result, Electricity.The burst discharge physiology course is as shown in the J in Fig. 5：The T-VSCC of activation moves the magnesium ion for blocking nmda receptor It opens, the opening of T-VSCC and nmda receptor channel drives membrane potential of neurons super cluster shape discharge threshold direction change.It is lost when quick When T-VSCC and NMDA receptor channel living, neuron resting membrane electric potential is restored to -55mV once, originates another burst discharge Period.

The electrophysiological recording of experiment and the data of model all demonstrate T-VSCC and nmda receptor collaboration mediates LHb neuron Burst discharge.

Embodiment 7 blocks the T-VSCC of outside habenular nucleus in the outside habenular nucleus local administration of animal model, eliminates depression Symptom

Inventor's habenular nucleus bilateral on the outside of congenital depression rat is placed in casing, gives the blocking agent Mibefradil of T-VSCC (side 10nmol/ul/), as shown in Figure 6.The A of Fig. 6 is the injection site figure that LHb injects that CTB determines casing.It observes Mibefradil effect 1h can have quick antidepressant effect：It is showed in FST (B of Fig. 6) and SPT (C of Fig. 6) behavior Quick antidepressant effect out.Demonstrating part blocks LHb T-VSCC can quick antidepression.

8 burst discharge of embodiment rather than the increase for entirely providing frequency contributes to the generation of depression

Inventor has found for the first time, and burst discharge mode rather than the increase for entirely providing frequency contribute to the production of depression It is raw.

Inventor can detect the cluster of rebound by the photaesthesia channel eNpHR3.0 of activation inhibition on brain slice in vitro Shape electric discharge.Meanwhile inventor, by experiment in vivo, the eNpHR3.0 photaesthesia that habenular nucleus is expressed on the outside of photoactivation in animal is logical Road, discovery can also quickly mediate the generation of aversion and depressed phenotype.

The A of Fig. 7 gives eNpHR virus expression carrier building schematic diagram (above), photoelectricity and record schematic diagram (following figure).

The B of Fig. 7, C be in the mouse LHb of AAV2/9-eNpHR expressing viral, yellow light activation brain piece neuron (B) and In the typical figure for neuron (C) the rebound burst discharge that body is recorded.The cell percentages for successfully inducing burst discharge are shown In the right side statistical chart of figure C.(D) dot chart and post-stimulus time column figure be shown in body optoelectronic pole record in one it is representative The reaction that LHb neuron stimulates 100ms yellow light.Neuron has the discharge frequency of a rebounding type to increase after yellow light. The frequency being recorded in the granting frequency of action potential and CRS depression animal LHb in cluster caused by 1Hz yellow light eNpHR3.0 Rate is suitable, prompts the burst discharge under 1Hz yellow light eNpHR3.0 analog depressive state horizontal.Praxiology research knot simultaneously Fruit is, it was also found that the rebounding type burst discharge of eNpHR photoactivation induction makes animal show to detest and depressed phenotype.And the F of Fig. 7, G, H show that rebounding type burst discharge caused by eNpHR photoactivation can induce real time position and detest (RTPA) and depressed phenotype.With Upper result explanation, increases LHb burst discharge and is enough to generate depressed sample phenotype.

In contrast, the single electric discharge (Fig. 8 A) of 5Hz is generated with the photoactivation oChIEF optical channel of 5Hz, and in electric discharge (one burst discharge of generation per second has 5 in every cluster to total discharge frequency that the photoactivation eNpHR3.0 of frequency and 1Hz activation generate A granting, total discharge frequency are 5Hz) quite (Fig. 7 B), but depressed phenotype (Fig. 8 C) cannot be induced.Non- light is compareed with expression The eGFP mouse in channel is compared, and photoactivation does not change the locomitivity (Fig. 8 B) of animal.

Result above proves, burst discharge mode rather than the increase for entirely providing frequency contribute to the generation of depression.

Conclusion

Burst discharge of the present invention for the first time with the neuron for having been surprisingly found that outside habenular nucleus has weight in the generation of depression It acts on, and has found the key factor for influencing the burst discharge of outside habenular nucleus, the activation including nmda receptor is LHb neuron The sufficient and necessary condition and LHb burst discharge for generating burst discharge need neuron membrane hyperpolarization and T-type voltage-sensitive The participation of dry passage.Especially had been surprisingly found that be burst discharge rather than entirely provide frequency increase contribute to depression It generates.Thus it inventor provides by inhibiting the burst discharge of outside habenular nucleus and treats the method and medicine of (inhibition) depression Object especially quickly treats the method and drug of (inhibition) depression.

The above is the explanation carried out to the present invention, cannot be regarded as the limitation carried out to the present invention.Unless in addition referring to Out, practice of the invention will use the routine techniques of organic chemistry, polymer chemistry, biotechnology etc., it is clear that except stating upper Except being particularly described in bright and embodiment, the present invention can also be realized otherwise.Other aspects within the scope of the present invention It will be apparent to those skilled in the art in the invention with improving.Introduction according to the present invention, many changes and variation are It is feasible, therefore it is within the scope of the present invention.

DEG C if without particularly showing, the unit " degree " of herein presented temperature refers to degree Celsius, i.e.,.

Claims

1. inhibiting purposes of the reagent of burst discharge in the habenular nucleus of outside in the drug of preparation treatment depression.

2. the purposes of claim 1, wherein the reagent of burst discharge is N-methyl-D-aspartate in habenular nucleus on the outside of the inhibition Acceptor inhibitor, it is preferred that the N-methyl-D-aspartate acceptor inhibitor is：

1) competitive inhibitor of nmda receptor, such as AP5, AP7, CPPene, Selfotel (Selfotel)；

2) noncompetitive inhibitor of nmda receptor, such as Aptiganel (Aptiganel), ketamine, Memantine (memantine), Huperzine A, ibogaine (Ibogaine), HU-211, Gabapentin (Gabapentin), PD- 137889；

3) channel blocker of nmda receptor uncompetitive, such as amantadine (Amantadine), atomoxetine (Atomoxetine), AZD6765, dextromethorphan (Dextromethorphan), hydrochloric acid magnesium amantadine, MK801 (Dizocilpine)；Or

4) glycine binding site point inhibitor, such as TK-40, kynurenine (Kynurenic acid).

3. the purposes of claim 1, wherein the reagent of burst discharge is the inhibition of T-type calcium channel in habenular nucleus on the outside of the inhibition Agent, it is preferred that the T-type calcium channel inhibitor is：

Succinimide class (Succinimides), such as ethymal (ethosuximide), mesuximide (methsuximide)； Hydantoins (hydantoins)；Zonisamide (zonisamide)；valproate；phenytoin；Mibefradil；Benzene Appropriate English (Phenytoin)；sipatrigine；Piperazine analog such as Flunarizine, Z941；Piperidines analog such as Z944 and Fluoropiperidine；TTA-P1；TTA-P2；Quinazolinone (quinazolinone)；TTA-Q4 or ML218.

4. the purposes of claim 1, wherein the reagent of burst discharge is N-methyl-D-aspartate in habenular nucleus on the outside of the inhibition The combination of acceptor inhibitor and T-type calcium channel inhibitor.

5. the purposes of any one of claim 1-4, wherein the reagent for inhibiting burst discharge does not inhibit individually to discharge (tonic pulse)。

6. the purposes of any one of claim 1-5, wherein the drug is the dosage form of habenular nucleus local administration on the outside.

7. the purposes of any one of claim 1-6, wherein the drug is the drug of fast-acting treatment depression, preferably , the drug is the drug of middle effect and long-acting treatment depression.

8. the pharmaceutical composition of depression is treated, wherein the reagent comprising inhibiting burst discharge in the habenular nucleus of outside, the inhibition cluster The reagent of shape electric discharge is as defined in claim 1-5.

9. the pharmaceutical composition of claim 8, for the dosage form of habenular nucleus local administration on the outside.

It is the pharmaceutical composition of fast-acting treatment depression 10. the pharmaceutical composition of claim 8, it is preferred that during it is The pharmaceutical composition of effect and long-acting treatment depression.