CN1450922A - Method and apparatus for the detection of agents that affect cognition - Google Patents

Abstract

This invention provides assays and devices for the effective screening and characterization of agents (e.g. drugs) for psychoactive properties. The assays utilize intact neural circuitries exhibiting functional network properties to provide effective amplification of subtle alternations of neurologic function. In a preferred embodiment, the assays utilize novel multi-element electrode arrays that interfaced with a portion of a mammalian brain (.e.g. a hippocampal tissue slice, dissociated hippocampal neuron culture, or co-culture of eptum and hippocampus slice). The methods involve providing a portion of a mammalian brain in culture contacted with a multi-electrode array; stimulating the mammalian brain with a time-varying input signal through two or more electrodes comprising the multi-electrode array in the presence of at least two different concentrations of the agent.

Description

Be used to detect the analytical method and the device of the cognitive medicament of influence

The rights statement of this invention under the research and development that federal government sponsors

This work has obtained the part support (grant number: N-00014-89-J-1255) of research office of naval.U.S. government enjoys certain right to the present invention.

Technical field

The present invention relates to novel biosensor, this biosensor is used to detect and characterize various chemical agents and the change of the cognitive function that causes.Therefore this biosensor provides the analytical tool that is used for the chemical compound that rapid screening has influences the ergasia effect (as drugs) and characterizes these compounds properties.

Background technology

So far, also do not exist sensitive quick repeatably analytical method to be used to detect of the influence of various medicaments to cognitive function.Being used for detecting the main analytical method that neural activity is had an active medicament (as drugs) depends on (living animal) behavior change usually or utilizes biosensor based on tissue.Yet behavior analysis is expensive consuming timely to be difficult for quantitatively, and has only limited repeatability.Biosensor based on tissue can overcome some above-mentioned restriction usually and can provide than quantitative results.Yet these biosensor susceptivenesss are limited, and can not detect the delicate variation of cognitive function.Tissue biological's pick off that can detect the medicament of damage or change function of nervous system generally includes the neuron of artificial culture, and this neuron is kept on row's electrode, the passive performance (for example input impedance) or the spontaneous action potential activity of this electrode monitoring film.At most, the biosensor of these types can only detect the serious consequence that has toxicant to cause that is exposed to suitable high concentration, can cause the situation of tangible cell death.In addition, most of biosensors only can provide the data of short-term.

Though some specimen have a quite long survival period, almost can not get about its stability, reproductive performance, sensitivity or data optionally.Though found several method maximize the artificial culture neuron and the record site spatial distribution between correspondence, up to the present, also do not have system to provide a best solution to writing down localized dynamic restructuring.Finally, there is not culture systems to be proved to be energy Presentation Function network performance (i.e. dynamic group), this functional network performance is that complete brain is peculiar, and is necessary to generality reduction or other the change that detects the higher cognitive function aspect as memory, expressive ability and logical reasoning.

Therefore, the pick off that is used to detect the medicament that influences ergasia has three kinds of major defects below one or more: 1) though there is the detector that is used to detect known particular agent or medicament class, they can not detect the existence of medicament the unknown or new that may influence ergasia.2) though can detect the medicament that works rapidly, can not detect the medicament that needed a plurality of hours or just worked for more time usually with known method.At last, 3) can detect its movable medicament that influences all neuron behaviors with the mononeuron checkout gear, yet, be affected iff some neurons, will be difficult to maybe can not detect the effect of medicament in not comprising complete mature neuron circuit analysis.

Summary of the invention

The invention provides the analytical method and the device that are used to effectively screen and characterize medicament (as drugs) with the characteristic that influences ergasia.This analytical method is utilized the complete neuron circuit of Presentation Function network performance, and the delicate variation of function of nervous system is effectively amplified.In a preferred embodiment, this analytical method has been utilized novel multiple-unit electrod-array, this multiple-unit electrod-array with contact to the part of mammal brain (as the co-cultivation thing of a hippocampal tissue slice, isolating digitation of hippocamps neuron culture or barrier film and digitation of hippocamps section).

Therefore, in one embodiment, the invention provides the method that screening can change the medicament of (as weakening or strengthening) brain function.This method relates generally to the part that the mammal brain that contacts with multiple electrode array is provided.When at least two kinds of variable concentrations of detected " test " medicament exist, with one or more electrodes (preferred 2 or more, 4 or more, or even 8,16, or 32 or more) subregion by a kind of time-varying input signal stimulus mammal brain.Two or more electrodes by forming multiple electrode array measure from this mammal brain quality award from the ministry Selected Inspection and become output signal when a kind of.This output signal is the function of this input signal normally.The difference of the output signal that is produced by given input signal under at least two kinds of variable concentrations of this medicament shows that this medicament works and changes the brain function.Two or more concentration of this medicament can comprise a negative control (drug concentration is zero).Needn't measure this negative control in the identical time or in identical nervous tissue's sample.Like this, in a preferred embodiment,, need this output signal and an above-mentioned signal " storehouse " contrast in order to analyze and/or to discern and/or classifying.

This part of mammal brain can be that the part of the mammal brain of cultivation (as has or do not have the digitation of hippocamps brain section of barrier film input, isolating digitation of hippocamps neuron specimen, barrier film and hippocampal co-cultivation thing, the neopallium section, the section of thalamus cortex, ganglion basal (striatal) section and/or the section of cortex striatum.)。This part of mammal brain also can be acute specimen.Preferred digitation of hippocamps brain section can show that myelin forms and/or dendron shape ridge and/or long-term enhanced ability.The preferred part of mammal brain can represent the functional network performance of mammal brain.This part of mammal brain can be transplanted one of them or how additional nervous tissue.This nervous tissue can be allogenic or from body on origin, and desirable from sophisticated animal, the animal that is growing, germling or animal embryo.Position in neural graft, this neural graft self just can be medicament (being to organize inductive variation in the cognitive activities) maybe this tissue can discharge a kind of medicament (as neurotransmitter, somatomedin or the like.)。

This time dependent input signal also can be a kind of input signal with spatial variations.Preferred input signal has a kind of known spatiotemporal mode that can induce synapse viscosity.Special preferred input signal has a kind of θ pattern.This input signal preferably is sent to following one or more positions: the cellular layer of dentate gyrus, the cellular layer of CA3, the cellular layer of CA1, the superficial cell layer of entorhinal cortex, the deep cellular layer of entorhinal cortex, the cellular layer of subiculum, the cellular layer of presubiculum, the cellular layer of side subiculum, the dendron shape zone of dentate gyrus, the dendron shape zone of CA3, the dendron shape zone of CA1, the dendron shape zone of entorhinal cortex, the dendron shape zone of subiculum, the dendron shape zone of presubiculum, or the dendron shape zone of side subiculum.

In a preferred embodiment, from following this output signal of one or more regional records (preferred a kind of θ activity pattern), these zones, include but are not limited to, the pyramidal cell of granular cell, CA3, in the CA1 deep layer cell smelt pyramidal cell, subiculum cell, presubiculum cell, side subiculum cell, CA3 dendron shape zone, CA1 dendron shape zone, in smell the dendron shape zone of deep layer cell, the dendron shape zone of subiculum, the dendron shape zone of presubiculum or the dendron shape zone of side subiculum.

The characteristic that influences ergasia of in fact any medicament can be measured with analytical method of the present invention.In a preferred embodiment, the previous the unknown of this medicament has the characteristic that influences ergasia.In another embodiment, the characteristic of the influential ergasia of known this medicament, and also there is a threshold concentration (as less than about 100mM, more preferably less than about 10mM, most preferably less than about 1mM) in this medicament at this position of a mammal brain.Special preferred agents comprises the cholinolytic medicament.

This method also relates to a kind of active signature of this medicament of identification, and should the activity signature compares with member in the active signature storehouse.In one embodiment, this multiple electrode array preferably includes at least 64 electrodes, these 64 electrodes are preferably having spacing between an extreme electrode between the adjacent electrode, between this extreme electrode spacing less than about 300 μ M, more preferably less than 200 μ m, most preferably less than about 100 μ m.Particularly preferred array is made with siliceous.This siliceous going up is electroplated a kind of metal (as gold, platinum, copper or silver).

This signal detection and/or analysis relate to the neural net method in a kind of hidden Markov model (HMM) or small echo or the signal processing simulates relation between this input signal and this output signal.

In another embodiment, the invention provides a kind of blended biology-electronic sensor, this biology-electronic sensor is used to handle one or more analytical methods as herein described (changing the medicament of brain function as screening).Preferred sensor comprises the part (as In vitro culture thing or acute specimen) of mammal brain as herein described, and the part of this mammal brain contacts with multiple electrode array as herein described; Device with this mammal brain of time-varying input signal stimulus of the one or more electrodes by forming this multiple electrode array; By forming the one or more electrodes of this multiple electrode array, when detecting, this mammal brain becomes the device of output signal, and wherein this output signal is the function of described input signal.This device also can comprise the storage medium (as computer and readable media) with active signature storehouse.In certain embodiments, in this electrod-array, can provide time-varying input advertiser (as signal generator), the input signal with spatial variations also can be provided.This device also can provide the input signal with spatiotemporal mode, and this spatiotemporal mode is known can induce synapse viscosity.In certain embodiments, provide the device of time-varying input signal that a kind of θ pattern also is provided.

This electrod-array in this device can be put in position, and described like this time-varying input signal can be sent to the cellular layer of dentate gyrus, the cellular layer of CA3, the cellular layer of CA1, the superficial cell layer of entorhinal cortex, the deep cellular layer of entorhinal cortex, the cellular layer of subiculum, the cellular layer of presubiculum, the cellular layer of side subiculum, the dendron shape zone of dentate gyrus, the dendron shape zone of CA3, the dendron shape zone of CA1, the dendron shape zone of entorhinal cortex, the dendron shape zone of subiculum, the dendron shape zone of presubiculum or the dendron shape zone of side subiculum.

In a special preferred embodiment, this device that becomes output signal during detection can be discerned a kind of θ activity pattern.This device can be set to make to write down this output signal from following one or more positions, these positions comprise, but be not limited only to, the pyramidal cell of granular cell, CA3, in the CA1 deep layer cell smelt pyramidal cell, subiculum cell, presubiculum cell, side subiculum cell, CA3 a dendron shape zone, CA1 a dendron shape zone, in smell a dendron shape zone of a dendron shape zone, presubiculum of a dendron shape zone, the subiculum of deep layer cell or a dendron shape zone of side subiculum.The present invention also provides a kind of biosensor, in this biosensor, this nervous tissue (part of a mammal brain) or contact with medicament that whether a kind of the unknown influences ergasia, or contact with the medicament of known effect ergasia, wherein this medicament exists with threshold concentration.

In yet another embodiment, the invention provides an active signature storehouse.This storehouse is made up of a kind of storage medium of the active signature of most compounds that comprises, in this storage medium, every kind of chemical compound in this storehouse is described and discerned to each active signature uniquely.In a preferred embodiment, when a cultivation part of a mammal brain contacts with the chemical compound that produces this signature, cultivate four or more a plurality of position of part at this, each activity signature is made up of an electrogram of at least 0.5 second.When lacking this chemical compound that produces this signature, this activity signature also can comprise an electrogram of at least 0.5 second in identical four or more a plurality of location of a mammiferous part.Sign nervous tissue's specimen or acute specimen of available any cultivation as herein described of this activity obtains.

This signal library is an ingredient of a computer system preferably, and this computer system allows the one or more active signature member in the storehouse is sorted, searches for and retrieves.The download of the active signature section of this nervous tissue of this biosensor of the uploading or drive of the on-line analysis of various medicaments for convenience, new activity signature/stimulate, this storehouse also can comprise and being connected an of biosensor as herein described.

Definition

Term " medicament " or " test agents " are represented a kind of detected in the method for the invention active chemical compound of ergasia that whether influences in this article.This test agents can be the chemical compound that a kind of the unknown has the characteristic that influences ergasia, and in this test, the performance of this chemical compound is not only discerned but also characterized to this analytical method.On the other hand, this test agents can be a kind of known chemical compound that influences the ergasia performance that has, and in this test, this analytical method can further describe the activity signature of this medicament.In fact this medicament can be any element, molecule or chemical constituent.Preferred agents or biological molecule (as protein, glycoprotein, lipid, saccharide, nucleotide etc.) or little organic molecule.This medicament can be taken from chemosynthesis (as denitrogenation) medicine or the known drug that environment (as polluter, damaged article, refuse etc.), various animals and plants, people and/or animal are used.Test agents will preferably not comprise known cytotoxin, common material as tissue culture's based component, buffer agent etc.

A kind of " influencing the medicament or the chemical compound of ergasia " or a kind ofly have the medicament or the chemical compound that influence the ergasia performance and be meant a kind of when contacting with nervous tissue, when particularly contacting, can change the neururgic medicament or the chemical compound of this nervous tissue with the part of a mammal brain.The preferred medicament of ergasia or the cognitive activities that chemical compound can change this nervous tissue of influencing.

Term " cognitive activities ", as used herein, include but are not limited in complete and healthy brain circuit or the spatiotemporal mode of the part that this circuit of conduct of producing when passing this circuit turns round naturally.

A kind of damage of term " cognitive disorders " expression cognitive function or weak.This damage or weak be to produce with the form that normal spatiotemporal mode changes, and this variation is outside the normal variation seen in the normal cognitive function.Preferred cognitive function is a kind of network performance.

Term " functional network performance " refers to the ability that a kind of nervous tissue specimen is showed a kind of electrical activity (response), and this electrical activity is peculiar by the nervous tissue of a mammal brain complete, preferred maturity.Therefore network performance is the feature of the dynamic population in complete circuit, rather than discrete neuronic feature.

" complete or completely or fully completely neuron circuit " refers to the nervous tissue of the cultivation of display function network performance.Preferred neuron circuit can show synapse viscosity, as illustrated by long-term potentiation.

Term " synapse viscosity " refers to the variation of the effectiveness of iuntercellular connection, in this connects, and the neuronic ability of the target that one or more stimulations are connected with its synapse or increase or minimizing.

Term " long-term potentiation " (" LTP ") refers to the mechanism as the basis of the synapse viscosity of particular types, normally the permanent and approaching permanent synapse viscosity that changes can take place and keep as in normal longterm memory to the synapse viscosity of this particular types in fast.Exist different LTP patterns as dissimilar and memory mechanism basis various durations.

Phrase " part of the mammal brain of a cultivation " refers to the nervous tissue's (in body of earlier external back) that derives from brain in tissue culture.The preferred part of mammal brain is the part that represents network performance and synapse viscosity ability is more preferably arranged in specific long-term potentiation.In a most preferred embodiment, this part of a mammal brain is brain section (as the digitation of hippocamps section preparation) or a barrier film and a hippocampal co-cultivation thing.

When animal energetically when exploring or learn, but when usually not being the state non-exploration or that do not wake up resembling sleep, a kind of spontaneous brain rhythm and pace of moving things occurs in a lot of akrencephalon district that comprises digitation of hippocamps, term " θ " just is meant the brain rhythm and pace of moving things of a kind of so spontaneous about 4-8Hz.It should be noted that the rhythm and pace of moving things that has been found that this dependent learning is the optimum physiological stimulation rate of inducing LTP.

When relating to a kind of test agents, when itself being a kind of medicament of drugs especially, term " threshold concentration " is meant under a kind of treatment instructions about how to take medicine of minimum (promptly as a kind of composition of materia medica, be under the common minimum therapeutic dose of opening of animal or human), in nervous tissue, the drug concentration of in central nervous tissue (brain), finding most preferably.Preferred threshold concentration is less than about 100mM, preferably less than about 10 μ M,, more preferably less than about 1 μ M, and most preferably less than about 0.1 μ M.

A kind of " biological standard " concentration is meant in the organism (animal or human) of a normal healthy, or under a kind of treatment instructions about how to take medicine of standard, in nervous tissue, most preferably in central nervous tissue (brain), (as a kind of test agents) concentration of discovery.

Term " input signal " and " stimulation " are interchangeable, all refer to be added in a kind of signal of telecommunication in the nervous tissue.This signal of telecommunication (as a voltage and/or an electric current) can change and/or in time with spatial variations." time change " signal is meant the time dependent signal of the amplitude of this signal (voltage) therein.The variation of sort signal can be continuous function or jump function.Similarly, the signal with spatial variations is meant therein with one or more positions and the time dependent signal of signal application in this nervous tissue.

" output signal " or " response " be meant one or more position probing to the electrical activity in a nervous tissue.This output signal also can be time dependent or with spatial variations.

When the variation and/or the existence of output signal response input signal or do not exist and when changing, a be known as function of input signal of output signal.

" active signature " of a kind of medicament of term, " cognitive signature ", " activity summary " be meant by nervous tissue and the sort of medicament contact generation in a kind of nervous tissue, preferably in the variation of the electrical activity of the part of brain.This variation can be the one or more stimulations of response (as chemistry or electronics) the active variation of nervous tissue or the variation of the endogenous or spontaneous electrical activity of this nervous tissue.Preferred active signature a kind ofly measure relatively down (as under two kinds of one or more medicaments different concentration, or this medicament exist with non-existent contrast in).This medicament is described or discerned to special preferred active signature uniquely in the electronic signature of one group of various medicament.These signatures may be the combinations of the signature that produces under different location and different situations in a tissue sample, so the evaluation of the performance completely of medicament may be depended on a plurality of tests of carrying out under conditions such as different stimulated, different medium.

One " active signature storehouse " be multiple (as two or more, preferably more than 10, most preferably more than 100, most preferably more than 1000,10,000 or even more than 1,000,000) set of the activity signature of various medicaments.In a preferred active signature storehouse, the activity signature of any two kinds of various medicaments is diacritic, and therefore discerns this medicament with respect to other medicament in this activity signature storehouse uniquely.

When being used for active signature or active signature storehouse, term " storage medium " is meant any information storage medium that can store one or more active signatures.Storage medium comprises, handwriting device, printing equipment, electronics and/or magnetic memory apparatus (as memorizer, disk), optical memory (as holographic memory and/or CD ROMS and/or DVD storage medium), logic device is (as programmable array (array) logic, flash memory, or other is based on the storage medium form of chip), or any other energy long preservation signature also can read the medium of signature at any time.

" multi-electrode " array is meant with the set of permission as the interconnective electrode of mode of the co-operate of all electrodes in the array of a group.Siliceous multiple electrode array is meant an array that utilizes the distinctive micro-fabrication technique of microelectric technique industry (as miniature carving, little spraying plating etc.) to make on substrate.Though can on siliceous material, produce siliceous array, not need this restriction.Other material (as known material in gallium, quartz, various polymer and other microelectronics industry) also can be used for doing substrate.Except imprint lithography, also can comprise the array that uses other to produce such as electric crystal technology such as vacuum coating, little spraying plating, ray etching, laser-induced thermal etching arts.

During the test agents of variable concentrations, phrase " at least two kinds of different concentration " also comprises zero-dose (promptly this test agents does not exist) in relating to analysis of the present invention.Like this, exist with two kinds of concentration and/or wherein a kind of concentration when being a negative contrast (this test agents does not exist), can detect with the experimental pharmacy of two or more variable concentrations in this test agents.People's test agents of two or more variable concentrations of also thinking it unnecessary to measure simultaneously, even also needn't in identical single nervous tissue, measure.Therefore, in certain embodiments, these two kinds of different concentration can be formed a measurement and the early stage comparison of doing measurement, the i.e. Discrepancy Description of finding between its (current measurement) and a kind of known or pattern of having write down in the past, the pattern that the past has write down must be to measure in certain time with analysis of the present invention.

Term " acute specimen " is meant a kind of sample that has just exsomatized of nerve (preferred brain) tissue, and this sample is placed in the cerebrospinal fluid usually, and preferably is subjected to Oxidation.Acute specimen can be survived 8 to 10 hours usually.Term " acute specimen " is used for distinguishing specimen of newly doing and the specimen that is kept at culture medium.

Brief Description Of Drawings

Fig. 1 illustrates assay device of the present invention with sketch map.Put it briefly, this device comprises a signal generator, and this signal generator is with one or more time dependent and/or with the signal application of the spatial variations part (as cultivating) in brain.This part of brain contacts with a kind of test agents, thus one of this part of brain or preferred a plurality of location detection to signal.Owing to use one or more to influence the medicament of ergasia, can detect the variation of signal.

Fig. 2 mainly is illustrated in available complex network nerve (input and output) circuit in the common digitation of hippocamps section.Dg: dentate gyrus, sub: subiculum, pre: presubiculum, para: side subiculum, I, II, III, IV, V: the equivalent layer of entorhinal cortex.

Fig. 3 illustrates with sketch map and comprises the stratified digitation of hippocamps section of having of a complicated neutral net.

Fig. 4 illustrates an experiment, in this experiment, manipulator on a kind of ampa receptor is significantly contrasted the effect in the multisynaptic reaction in single synapse, shows that multisynaptic reflex has very high susceptiveness.The intermediary curve chart in top shows at CA3 stimulates this tissue, when CA1 writes down this tissue, the reaction of manipulator medicine on two kinds of ampa receptors.A single synapse circuit when this expression separates stimulation and output by a single synapse.In this illustration, come down to identical to the reaction of two kinds of drugs.Although stimulate in the porous path of entorhinal cortex, and to measure be to be obtained by identical CA1 cell, and right vertical curve is shown in the record that the identical moment obtains from identical specimen.This has formed one three synapse circuit, and, as shown in the figure, the reaction of these two kinds of drugs is differed widely.Therefore, the performance of Internet disclosed in fact in simple neuron circuit, detect less than difference.

Fig. 5 illustrates a complete culture apparatus that is used for method of the present invention with sketch map.This device generally includes one and a plurality of control rooms (1) acute nervous tissue of maintenance or cultivation nervous tissue.Be used for this nervous tissue is carried out the I/O circuit that circuit connects.Gas is (as O ₂, CO ₂And air (ATMOS)) input port of gateway, liquid (as test agents solution) gateway, the gateway of preserving liquid (as cerebrospinal fluid (CSF), artificial cerebrospinal fluid (ACSF) or culture medium etc.) and fixative is used for preserving the tissue after the use.Though signal source and data acquisition unit illustrate respectively that in the drawings these two functions also can be incorporated an one device in fact.

Fig. 6 A and 6B are illustrated in the position of an electrod-array on the digitation of hippocamps section preparation.Fig. 6 A is illustrated in a cultured rat hippocampal toe section on the electrod-array of a kind of low-density (electrode spacings of 450 μ m), and Fig. 6 B is illustrated in a cultured rat hippocampal toe section on the electrod-array of a kind of high density (electrode spacings of 150 μ m).

Fig. 7 illustrates kainate and stabilizes the influence on the network performance in digitation of hippocamps section: shown in the time point of picture in picture target bottom righthand side, measured the standard deviation of 20 successive reactions, and also to have analyzed the persistent period be the time period of 300-400 millisecond.This figure also shows the determination of activity on eight passages.

Fig. 8 A, Fig. 8 B and Fig. 8 C illustrate the ingredient of analytical system of the present invention.Fig. 8 A illustrates a multi-electrode dish (MED), one and comprises the measuring device of the switch enclosure that is used for electrode selection, amplifier and isolator, a control device that comprises computer, A/D and D/A converter.In a preferred embodiment, an edge connector is provided in an incubator, for this multiple electrode array, with the electronic installation (Fig. 8 B) of join dependency.Fig. 8 C provides the detailed view of the microelectrode of forming this multiple electrode array.

Fig. 9 illustrates the very fine and close associating feedback system of digitation of hippocamps zone C A3, and this feedback system allows to be delivered to other neuron (left figure) rapidly since the activity at a discontinuous position.The right side illustrates the different groups effect of feedforward and/or feedback γ-An Jidingsuan neurokyme relay cell, and the activity of these relay cells may influence the mix of activities with respect to the variation increase of single synapse.

Figure 10 illustrates in 10 μ m are stable when having (lower tracer) and not having (upper tracer) 64 electrodes wherein 8 record.

The specific embodiment

I. detect the medicament that influences ergasia that changes cognitive function.

A) screening and sign influence the medicament of ergasia.

The invention provides and detect and/or characterize pharmacological medicament, environment medicament and other acts on the analysis that brain can change the material of brain activity.In one embodiment, the invention provides new method and identify the existence that to induce the medicament that the running of brain circuit gets muddled, also do not have the relevant method of identifying before this.

This method is preferably used circuit complete in the mammal brain, and the ruuning situation of the material that can observe detection these circuit when existing and not existing, and this circuit is operation automatically not only, also corresponding artificial stimulation operation.In these methods, can be owing to induce the circuit-mode and the normal non-disorderly pattern that show cognitive disorders to differentiate.In other words, various medicaments can be levied and/or comparison by the change list of normal mode.This not only provides a kind of active signature of this test agents, and has disclosed the details about this medicament activity pattern.Can further illustrate the specific activities pattern that influences the different chemical of ergasia medicine by the data that method of the present invention provides.For example, as described in example 2, the stable resulting activity pattern of response or " signature " show, stabile effect be by stimulate and feedback suppression between interaction make the response synchronization, thereby so that when stabilizing less stimuli responsive still causes more consistent bigger field potential when existing.

In one embodiment, can by from mammal brain part from complete circuit and be kept at and obtain analysis of the present invention the culture medium, thereby make these circuit obtain the performance of normal matures, and keep survival (as up to the several months) for a long time.Screen the medicament that may have cognitive disorders or cognitive activities " signature ", must realize by complete (preferred maturity) circuit usually, since above-mentioned reason, the basis that independent neuron or other jejune neuron circuit are not normal cognitive functions.

Other advantage also has, and method and system of the present invention allows to detect various medicaments to the delicate of functional network performance and secular relatively influence (as if such higher cognitive performance of finding) in complete brain.The major advantage of these methods of the present invention include but not limited to, and 1) behavior prediction; 2) raising of susceptiveness; 3) detection of the side effect of detected medicament; With 4) the former medicament activity pattern that be can not get of detailed description.

About the prediction of behavior, be noted that not to be individual unit but network generation behavior.Therefore, the analysis of decision neutral net response is being inferred various medicaments to aspect the influence of main body brain behavior, and nature provides more information than one synapse.

About sensitivity, have recognized that the neural activity pattern is produced by a large amount of neuronic coactions, thereby easily is conditioned and disturbs.Even therefore drugs effect very in a small amount also may produce huge consequence.Yet, use method as herein described, under the concentration of permitting, can identify the effect of the medicament that influences ergasia far below previous method, this is that what a surprise of the present invention is found.

Because network comprises some kinds of cells, sensor and pick off, so can be for a long time in different complexity level operations.This complexity provides the chance of abundant demonstration for the undesirable potential side effect of various medicaments.Therefore analysis of the present invention also can carry out responsive detection to the side effect of long-term appearance.

In addition, a plurality of elements that exist in the network provide a lot of positions, to show the multiple difference in relevant drugs or other chemical compound.The synchronous recording at a plurality of positions makes the possibility that is combined into of this network complexity and dense sampling, and " drugs summary " or " active signature " that this combination makes acquisition have new features becomes possibility.Compare with previously possible medicament classification, this permission is carried out more accurate classification to medicament.

B) the preferred analysis

Method of the present invention generally includes a blended biology-electronic sensor, this pick off comprises a tissue culture's model with memory function, this tissue culture's model and a multi-electrode input source and/or output combine, to detect and to determine weak or other variation of the cognitive function that the acute or low-level effect by the medicament that is applied to culture causes.This assay device usually as shown in Figure 1.In brief, the present invention utilizes a kind of signal source, this signal source will import (as a kind of time dependent and/or with the current potential of spatial variations, promptly a kind of stimulation) supply with a kind of nervous tissue's (as comprising and a plurality of complete neuron circuits) with cultivation of functional network performance.In a preferred embodiment, this nervous tissue can be the part of mammal brain.Thereby can also can detect of the response of this nervous tissue one and a plurality of location detection to the response (being the signal of telecommunication) of nervous tissue to this input signal to varying input signal.This nervous tissue will produce response particular stimulation input pattern the characteristic response pattern (as time dependent and/or with the current potential of spatial variations).

This nervous tissue can contact with one or more medicaments (as drugs, the chemical compound that the treatment function is arranged, chemical reagent, potential toxin, environmental contaminants, food pollution thing etc.), thereby measures the influence that this medicament responds input signal at this tissue.As the result that medicament uses, this nervous tissue shows a kind of variation of the electronic response pattern of input signal, the performance of the influential ergasia of this medicament.

It was gratifying, always do not require to have a kind of input signal or signal mode.This mammal brain loop that is used for the present invention's test also shows spontaneous and/or endogenic activity.Also can measure medicament to this spontaneous and/or endogenic active effect.

1) screening of potential drugs

Analysis of the present invention can be used for very wide scope.A main uses is to screen potential drugs fast and delicately.Pharmaceutical factory is actually the medicament storehouse of potential treatment usefulness, usually needs could screen the medicament for the treatment of usefulness through the behavior test of arduous (and not being very revealing), so " rapid screening " had permanent demand.A discovery of the present invention is that in test, a kind of drugs (as stable) physiological related concentrations (as under a kind of dosage instructions about how to take medicine of prescribing, the quantity that occurs in particular organization) demonstrates a kind of discernible or feature " signature ".This is former result from never getting.There is not the medicament effect that the outer test of organism can detect can influence ergasia stable when resembling normal low dosage.

Therefore, in one embodiment, analytical method of the present invention is used to identify a kind of " activity summary " or " cognitive effect signature ", also refers to a kind of signature of medicament of known or potential treatment usefulness in this article.This method generally include make irriate and/or unprovoked nervous tissue with medicament detected or that characterized contact, thereby determine that this medicament is to the active effect of nervous tissue (as endogenous active variation and/or respond the variation of particular stimulation).The overall variation (as the response to particular stimulation) of neural activity provides a signature for the physiologically active of this medicament (as referring to example 2).

Like this, the compilation (" storehouse ") of this medicament signature with the medicament effect signature (" circuit vestige ") of a known existence can be compared.This permission is included into a class or multiclass sensitivity or potential drug with this medicament.In addition, similar medicine is (as stable ^TMThe Autograph Session of (stabilizing), versus Halcion (P14, triazole benzene phenodiazine) demonstrate small or great difference.Thereby this treatment regime that makes a technical staff in this area can discern the optimization certain drug becomes possibility.In addition, this analyzes the potential side effect (i.e. signature section outside conceivable summary) that also allows identification novel drugs or known drug.

2. be used to screen the standard signature of chemical compound treatment usefulness or treatment guiding usefulness

In another embodiment, method of the present invention can be used for setting up active signature storehouse.This summary library mainly is the set that the multiple activity that influences the medicament of ergasia is signed.

With member in the above-mentioned storehouse relatively will disclose the activity pattern of variety classes medicament, and/or be used for medicament is included into the chemical compound bunch with similar signature or different signatures (activity patterns).

Medicament signature with needed physiologically active is taken as benchmark then, is used to screen the medicament that other has similar or different activity summary.

In addition, can produce a desirable signature again, search demonstrate this activity summary chemical compound the time can be with it as benchmark.

3. screen deleterious medicament

In one embodiment, this analysis is used to detect the cognitive disorders (as the weak, dazed and confused of normal cognitive function or damage) that is caused by various medicaments.In these are analyzed, the ability of the normal cognitive function of main check and analysis chemical compound (as the chemical substance of in a kind of specific environment, finding) infringement.Yet, itself not causing damage even underproof this medicament is movable, people will recognize that also identifying any active existence of nervous tissue or lacking also is a useful achievement.

4. screen cognitive enhancer

Analysis of the present invention also can be used for screening cognitive enhancer.This enhancer can show multiple effect, and this effect includes but are not limited to and is the stimulation of the long-term necessary minimizing of potentiation, the synapse viscosity of increase etc.

5. estimate neural graft's therapy

In another embodiment, the invention provides analysis, be used to estimate the effect of the effect of neural transplantation and/or various medicament neural transplantation.Neural transplantation provides the possible clinical treatment of impaired brain for the first time.Especially, the autotransplantation of nervous tissue and xenogenesis (body) are transplanted and have been used for the treatment of Parkinson's disease.Yet Parkinson's disease is mainly as " by the cell transplantation reparation " a kind of so more common strategic model.Parkinson's disease is considered to that loss by small amounts of cells causes, this cell is produced as most of necessary neuromodulator, the dopamine of brain.Initial studies show that, the allosome of central nervous tissue and peripheral nerve tissue and autograft can, at least temporarily, the minimizing of additional dopamine.Yet above-mentioned transplanting is not analyzed on material particular the influence of cognitive activities.

Therefore, in one embodiment, brain culture of the present invention also can comprise the nervous tissue of transplanting.This nervous tissue can obtain from central nervous tissue or peripheral nerve tissue, and on the source can be consubstantiality or allosome.The source of preferred nervous tissue is the embryo.The cultivation part (as the section of cultivating) of brain can keep the sufficiently long time-to-live easily, so that tissue transplantation shows a kind of effect (as changing the electrical activity of brain) of brain.The method of carrying out neural graft is known (as referring to Redmond, etal 1993) Ann N Y Acad Sci 695:258-266, Zager and Black (1998) Surg.Neurol.29 (5): 350-366 and list of references of wherein quoting by those skilled in the art).

Therefore, in one embodiment, cultivate part with the brain under neural graft's condition and carry out test as herein described.The cultivation of brain part can be the part of healthy brain, or from the part of (as if Parkinson's disease or the such degeneration disease of the Alzheimer disease) brain that shows one or more pathology.

With a kind of control culture that lacks an above-mentioned graft, can determine with respect to the active variation of identical culture before this neural graft, this graft has only been measured absolute change (as without comparison), or only with the one or more summaries in an active signature storehouse relatively.

In addition, above-mentioned transplanting culture can be used for estimating the influence activity of various medicaments (as influencing the drugs of the mental status) to neural graft's thing.

C) other advantage of this analysis

This analysis is sustainable exist considerable time (as up to 3 to 6 hours, preferably up to one day, more preferably from one day to a week, most preferably up to 3 weeks, 4 weeks or even 5 weeks or 6 weeks or even up to some months).The medicament that the activity that this makes it possible to detect needs a lot of hours just can be worked.In addition, because movable in a nerve net, measure, so can detect the medicament that only influences some neurons and mainly act on complete sophisticated neuron circuit.Therefore, for instance, analytical method of the present invention can clearly be identified in the activity pattern of stabilizing under the physiology related concentrations with Halcion.

II. biological sample

A) preferred specimen

As mentioned above, in a preferred embodiment, the present invention utilizes the complete circuit of mammal brain.Thereby this biological sample comprises neurocyte and/or nervous tissue's (as part of a mammal brain).In fact can obtain neurocyte and/or nervous tissue from any mammal, this animal includes but are not limited to rodent, lagomorph, ungulate, Bears class animal, Bovidae (family) animal, the primate that comprises the people, Canis animals, felid etc.On the other hand, in a preferred embodiment, this neurocyte and/or nervous tissue can be from primates, and lagomorph, felid, Canis animals or inhuman primate obtain.

The cortex system of this mammal brain, for example digitation of hippocamps has a kind of big extension expressive ability, and this ability is very strong for noise, variation and part completion.Hippocampal cognitive function is encoded the content (about the information of environmental stimulus and/or behavior) of impermanent memory, thus so that the mode of the minimum interference of already present longterm memory layer, with the content stores of impermanent memory in longterm memory.Hippocampal input is come from senior neopallium brain area, and form by many groups characteristic of ongoing environmental stimulus of expression and/or behavior.This digitation of hippocamps changes the performance of many characteristics, and this performance is the output of neopallium, and by synapse viscosity mechanism, as long-term potentiation (LTP), the different sub-features of these characteristics is fused into a new performance (forming an one object).Thereby the digitation of hippocamps specimen provides a particularly suitable neural substrate for method of the present invention.

Therefore preferred biological sample include but not limited to 1) cell and the section of cultured rat hippocampal toe; 2) cultured rat hippocampal toe neuron; With 3) barrier film and hippocampal co-cultivation thing.Each culture systems all has the advantage of oneself.

B) from digitation of hippocamps isolated cells culture

In a preferred embodiment, method therefor of the present invention utilizes from the digitation of hippocamps isolated cells.This isolating digitation of hippocamps neuron specimen has several advantages.One of them advantage is in localizing electrode array more than, and boot process is neuronic to output to discernible location.Though isolating neuron specimen has lost in the body and visible inherent cell arrangement in the digitation of hippocamps slice culture specimen, synapse contact and neuron circuit are cultivated in the specimen at isolating neuron and are developed rapidly.

The data of our front shows that high density cultures shows electrode and the very strong adhesion characteristics of many siliceous substrates to electrogilding and aluminum.In addition, spreading of neuron process is on a large scale, forms neutral net clearly.In the low-density culture, observe cyton and neuron trend easily.Wherein, in other low-density culture, mononeuric a large amount of aixs cylinders are limited in a single electrode surface.(Kleinfeid et al. (1988) .J.Neurosci. 8:4098-4120) shows, can adjust neuron circuit by the adhesion characteristics of control neuron number, hardware component and silicon and electrode substrate for the data of our front and other people data.Preferred culturing room is that ((1994) Neurosci., improved model 52:815-827) allow after improving to carry out microcosmic monitoring and electrophysiological recording from monolayer neural networks to Gross and Schwalm.

1. the preparation of isolated cells culture

According to people such as Brinton ((1997) Neurochem.Res., 22:1339-1351) described method preferred for preparation digitation of hippocamps neuron culture.Fascia Tarin's is scaled off from the brain of (E18:E0 is breeding day) at 18 days initial stages of the Mus tire of Sprague-Dawley.Then when 37C, this organizer usefulness is contained 0.05% tryptic Hank balanced salt solution (KCI of 50mM, the KH of 3mM ₂PO ₄, the NaCl of 80mM, the NaH of 0.9mM ₂PO ₄(7H ₂The glucose of O, 10mM, the HEPES of 0.3M)) handled 5 minutes.Behind the incubation, improve Eagle's medium (DMEM with the cold phenol red Dulbecco that do not contain; Gibco), the NaHCO that contains 10mM ₃, 10% Ox blood serum, the penicillin of 5 μ g/ml, the streptomycin of 5 μ g/ml and 10% F12 Nutrient medium, handled three minutes, trypsin is lost activity, then wash this tissue with Hank balanced salt solution (2x), then make this tissue repeatedly by a series of (a lot) thus fire slick narrow pasteur pipet and separate this tissue.

As described below will be in high density 1 * 10 ⁶Cell/ml or low-density 20,000-30, the cell of 000 cell/ml are taped against on many localizing electrodes, and these many localizing electrodes are potted in the silicon dioxide.(this culture medium is not impelled neurogliocyte propagation when existence contains the Neurobasal culture medium; Gibco), the streptomycin of the penicillin of the glutamine of the glutamic acid of B27 medium additives (Gibco), 25 μ m, 0.5mM, 5 μ g/ml, 5 μ g/ml phenol red the time, neuron is cultivated and is kept in the incubator of 37 ℃ of 5% carbon dioxide.

C) digitation of hippocamps section preparation

1. digitation of hippocamps circuit

This digitation of hippocamps section preparation preferably includes a complicated neutral net, and this neutral net has about 90% input by the fiber transmission that comes from entorhinal cortex.This digitation of hippocamps section preparation comprises a well-bedded complicated neutral net (Fig. 3).These digitation of hippocamps section preparations can be acute specimen or cultivate specimen; The latter may keep the several weeks of surviving, and does not have a large amount of neurone loss, and keeps neuronic connectedness effectively.

This network complexity that obtains in the digitation of hippocamps circuit that a common brain section is provided as shown in Figure 2.Even to Fig. 2's) rough detection shows that also the integrated of network is complicated.For example, each granular cell nearly 10 in the dentate gyrus (dg), 000 synapse contact, and (as from the entorhinal cortex layer) the space-time activity pattern (" input signal ") in the subclass of porous passage aixs cylinder produces the space-time activity pattern the granular cell subclass.This granular cell then produces an aixs cylinder system, and tongue Portugal fiber causes the activity of the pyramidal cell nearside of digitation of hippocamps zone C A3.In addition, the polymorphic neuron activity that the granular cell aixs cylinder is also impelled the entorhinal cortex door throws back on the granular cell again then, thereby produces an excited loop of powerful circulation.

Fig. 4 provides the sketch map of one three synapse lattice network.By the fibroplastic end in tongue Portugal on the CA3 pyramidal cell is very large, and activates number tongue Portugal fiber ends relatively seldom synchronously and enough excite a CA3 cone neurone.Therefore the space-time activity pattern of granular cell produces the space-time activity pattern in sub-fraction CA3 cone neurone.CA3 cone neurone aixs cylinder or Schaffer pleurapophysis not only project digitation of hippocamps CA1 cone neurone, and form an excited network of circulation on a large scale in CA3.Like this, the space-time activity pattern of CA3 pyramidal cell changes into the space-time activity pattern of new CA1 cone neurone.In addition, porous passage aixs cylinder also projects the tip top zone of CA3 and CA1 cone neurone; Like this, the same input signal to granular cell is transferred into successive three synapse Internets.

Generally speaking, illustrated digitation of hippocamps circuit will carry out 3 continuous variations from the input signal to the output signal, and wherein, each circuit layer carries out multi-form calculating and storage by the modification of synapse to the signal in the process.Common cultivation section preparation approximately is hippocampal 1/40, thereby adds that all local circuit neurons have about 15,000 granular cells, 5,000 CA3 cone neurones and 10,000 CA1 cone neurones.These specimen keep the several weeks of surviving, and not a large amount of loss neurons, and kept effectively neuronic connectedness (people (1995) Hippocampus such as Bahr, 5:425-439).In addition, above-mentioned specimen is fit to the pharmacological action of research various medicaments very much, because :) a large amount of various synapses guarantee that a lot of paths, neurotransmitter sensor and capturing system are arranged in this culture; 2) the continuous variation of input signal occurs on the different electric loop layers, causes the very big enhancing of drug effect, thereby increases the susceptiveness that detects; With 3) many viscosity mechanism in not homo-synapse operation also can detect the illeffects (vide infra) to cognitive process.

2. preferred digitation of hippocamps section preparation

In special preferred embodiment, can use two kinds of different cultured rat hippocampal toe section preparations, a specimen does not have barrier film output (H specimen), and a specimen has barrier film output (H/S specimen).In two specimen, input signal preferably is sent to the granular cell of dentate gyrus.In both cases, output signal preferably is recorded on the different Internets, that is, and and the pyramidal cell of granular cell, CA3 and the pyramidal cell of CA1.

Other sampling specimen includes but are not limited to: comprise the acute digitation of hippocamps section preparation of dentate gyrus, CA3 and CA1 and cultivate the digitation of hippocamps section preparation, until comprising projection plane from dentation to CA3 tongue Portugal fiber people (1990) Synapse5:333-335 such as () Staubli; Comprise the digitation of hippocamps that resembles subiculum, presubiculum and side subiculum and the entorhinal cortex and the cultivation section preparation in side digitation of hippocamps zone; Or cortex section preparation (as neopallium or olfactory sensation cortex (referring to as people such as Jung (1990) Synapse 6:279-283)).The method for preparing brain slice culture thing be technology well known in the art (referring to as, people (1990) supra. such as people such as Staubli (1990) supra. and Jung).

3. prepare brain slice culture thing

((1991) Neurosci.Meth., 37:173-182) described method prepares the organotypic culture thing of digitation of hippocamps section according to people such as Stoppini.In brief, brain from 11-22 days Sprague-Dawley Mus under aseptic situation obtains digitation of hippocamps, this brain is kept in the ice-cold culture medium (MEM) (Gibco Corp.no.61100-061), and this culture medium comprises HEPES (25mM), Tris-base (10mM), D-glucose (10mM) and MgCl ₂(3mM), and be placed on one and have on the polytetrafluoroethylene platform that McIllwain organizes chopper.Brain is cut into slices (as, about 100 μ m are to 600 μ m, more preferably about 200 μ m are to about 500 μ m, most preferably about 400 μ m) cut down and transfer on multiple electrode array (as, siliceous multiple electrode array as herein described), this multiple electrode array is in a good medium that contains tissue culture medium (TCM).

Can use any suitable culture medium to deposit nervous tissue.Above-mentioned culture medium is known in the art (referring to as people such as Bahr (1995) Hippocampus, 5:425-439).Special preferred culture medium comprises MEM culture medium (Gibco Corp.no.41200-072), and this MEM culture medium comprises glutamine (3mM), HEPES (30mM), NaHCO ₃(5mM), D-glucose (30mM), L-ascorbic acid (0.5mM), CaCl ₂(2mM), MgSO ₄(2.5mM), the insulin of 1 μ g, 20% horse serum, contain penicillin, pH7.2.

Then can according to standard method (referring to, as, United States Patent (USP) 5,766,948, people such as Bahr (1995) supra.) cultivate this brain tissue.In a preferred embodiment, the brain section is stored in and contains 5% carbon dioxide (CO in the air ₂) 35 ℃ constant incubator in, each week of culture medium changes twice.In initial ten days of incubation, section forms many sophisticated characteristics gradually, comprise myelin form, form the good ability of dendritic spine and long-term potentiation.Before analyzing beginning, cut into slices and be preferably kept in the culture at least 5 days, more preferably at least 10 days and most preferably at least 14 days.

D) barrier film and hippocampal co-cultivation thing

Barrier film and hippocampal co-cultivation thing allow to detect a kind of forebrain activity pattern (as the θ pattern), and it is of crucial importance to the information coding that this pattern is considered to usually, and known synapse viscosity are had connection.In addition, the co-cultivation thing provides one to be the evaluation that how to influence the brain rhythm and pace of moving things to test agents, and this is a possible primary and foremost purpose to the required understanding of any destructive chemicals.In addition, the positive role about the rhythm and pace of moving things of section can be used for predicting the effect in complete animal; The 3rd, this co-cultivation thing adopts undiscovered sensing system in this digitation of hippocamps, and known this sensing system is sensitive to the toxin of certain kind.For instance, most outstanding every digitation of hippocamps be cholinergic, and be responsive to the inhibitor of cholinesterase.This connection also merges to the γ-An Jidingsuan cell one type γ-An Jidingsuan cell connection, and finds that this connection spreads all over the motor system of brain, but does not exist in this digitation of hippocamps.Generally speaking, this culture has increased the similarity of biosensor and original position brain, and the physiological activity that directly relates to senior akrencephalon operation is provided.

1. every the digitation of hippocamps loop

Should be one every the digitation of hippocamps system is how to regulate cortex network the most intelligible physiological example about up regulating system.The vertical lobule (DBB) of central diaphragm and the little band of twill that is closely related is cholinergic outstanding and neurodynamic outstanding hippocampal three subprovinces of sending into of γ-An Jidingsuan.In considering these fiber roles, it is important to point out that though these fibers are numerous, the glutamic acid that comes from cortex connects and still quantitatively surpasses them widely, and these fibers are produced by the passage in the digitation of hippocamps.The path follow-up study is the result show first, by the relay cell that target suppresses, the barrier film input obtains the out-of-proportion influence of the relative size with them, and each relay cell stimulates hundreds of pyramidal cells, (people (1984) Brain Res. such as Baisden, 290:146-151; People such as Brashear. (1986) Neurosci., 17:439-451; Freund (1989) Brain Res., 478:375-381; People such as Kiss (1990) J.Comp.Neurol., 298:362-372; People such as Nyakas (1987) Brain Res.Bull., 18:533-545).Also contact cone neurone though cholinergic barrier film is outstanding, this γ-An Jidingsuan neurokyme cell presents selectivity in this respect.Suppose that the relay cell--as other place at the cortex akrencephalon--in the digitation of hippocamps is the γ-An Jidingsuan neurokyme, an outstanding part of barrier film set up one for the inhibition of the common type of ganglion basal to suppressing circuit (γ-An Jidingsuan cell-＞γ-An Jidingsuan cell).By this way, central diaphragm/DBB system can make the pyramidal cell group eliminate (disinthibiting) strong inhibitory action.The effect of cholinergic fiber is unconspicuous.Evidence suggests that they act on body and dendron (Valentino and dingledine (1981) J.Neurosci., the 1:784-792 of digitation of hippocamps pre-synapse relay cell tip and relay cell and pyramidal cell; People such as Ben-Ari (1981) Neurosci., 6:2475-2484; People such as krnjevic (1981) Can.J.Phyusiol.Pharmacol., 59:911-914).Someone thinks, the role of network of these arrangements be reduce the release of γ-An Jidingsuan and directly excite (by the inhibition that potassium is conducted) cone neurone (people such as Dodd. (1981) Brain Res., 207:109-127; Bemardo and Prince (1982) BrainRes.249:333-344; Cole and Nicoll (1984) Brain Res., 283-290).These two kinds of effects are collaborative mutually, again with the same with the neural exsertile synergism of barrier film γ-An Jidingsuan, can strengthen the irritability of cone neurone.

2. preparation is every the section of-digitation of hippocamps

The verified barrier film section of gathering from postnatal mouse of research in the past can stimulate digitation of hippocamps, also confirms that cholinergic fiber package is contained in this collection thing (people (1993) Neurosci. such as heimrich, 52:815-827 simultaneously; People such as Baratta (1996) Brain Res.Dev.BrainRes., 97:143-147; People such as Baratta (1996) Neurosci., 72:1117-1132).Preferred cultural method derives from method cited in these publications.

Dissecting in preparing successful slicing processes is an important step, and these methods are well narrated in document (referring to people such as Bahr (1995) supra.).The existing narration of the co-cultivation thing of barrier film and hippocampal section (referring to as, people such as Baratta (1996) BrainRes.Dev.Brain Res., 97:143-147; People such as Baratta (1996) Neurosci., 72:1117-1132).

The purpose that should be noted in the discussion above that the co-cultivation thing is to obtain to show the co-cultivation thing of maturation every-digitation of hippocamps system performance.Therefore, compare with the barrier film section, it is possible gathering the digitation of hippocamps section from a different postnatal age.

E) other cultivation specimen

The other parts that are fit to the brain tissue of analysis of the present invention include but not limited to acute specimen as described below and cultivate specimen: neopallium specimen, this specimen comprise five primary cell layers and epidermin layer, primary visual cortex section; Primary audition cortex section; Acute or cultivate elementary somatesthesia section; Acute or cultivate middle rank and cortical areas of association; Comprise the acute section in preliminary area and middle rank district or cultivate section; The section of thalamus cortex, the co-cultivation thing of thalamus and cortex, the co-cultivation thing of a plurality of cortical areass, striatal, comprise tail body and nuclear; Comprise nuclear such as the crust amine energy of black substance and ventral tegmental area; Comprise the pallidum district (GPe, GPi); Comprise the thalamus target nucleus; The section of cortex striatum, cortex and striatal co-cultivation thing; The co-cultivation thing of striatum and dopaminergic input, or the like.

F) complete culture apparatus

In a particularly preferred embodiment, the biological part retinal diseases of above-mentioned fundamental analysis device is a complete culture apparatus.So a culture apparatus preferably includes one or more electrodes or electrod-array, and this electrod-array can be fixed in this cultured tissue muchly.This device will seal, so that culture medium can be preserved therein indefinitely.In a particularly preferred embodiment, this culture apparatus will be equipped with electric connector so that connect stimulating apparatus fast and checkout gear and/or port, and this port is used for fixing quickly and easily and removes charge pump, gas tank etc.

That this culture device preferably makes up and reasonably, so can be fast easily separate storage (as in a culture device) and easily be transported to the use of remote place with the remainder of this analytical equipment.Fig. 5 is used for the complete culture apparatus of the inventive method with one of schematic view illustrating.

III. the output of Fen Xiing

Though can be with spontaneous or endogenous movable this neuron circuit (as brain slice culture thing) of measuring,, in a preferred embodiment, with a signal application in nervous tissue.This signal source is the one or more current potentials that are preferably applied to the different parts in this nervous tissue.This current potential can be constant (being constant voltage), yet in a preferred embodiment, this current potential is time dependent.In addition, this current potential can be applied to fixed position or variable position (by selecting standby electrode) so that a kind of signal with spatial variations to be provided.In addition, this signal can be time dependent and/or with spatial variations.

A) input signal source

Any signal source all can be used as the input signal source in the method for the present invention.In fact can transmit from 1nV to 20mV, more preferably from about 10nV to 5mV, all be suitable for from any equipment of the voltage of the about 200 μ A of 1V.Preferred input signal source can provide clocklike or complete programmable time varying signal.Most novel signal generators utilize computer to produce one stimulates summary, and this summary uses an analog-to-digital converter to change into analogue signal at random, and then this analogue signal is transferred into electrode.The signal generating ability is usually made electrophysiological data collecting system.The very suitable method herein of commercial signal generator that is used for electrophysiology work.Above-mentioned signal generator has been the known technology of this area, and can obtain (as the Grass apparatus, Matsushita electronics corporation etc.) from a lot of suppliers.This signal generator can be used hand control, computerizeds control, and utilizes this computer, and the output signal of this computer control response nervous tissue changes frequency input signal and/or amplitude and/or location.

In one embodiment, a plurality of input signals are transferred into nervous tissue in analysis of the present invention.In this embodiment, each signal can provide with an independent signal generator.On the other hand, a mono signal can be separated or stack, and the superposed signal that produces can be applied to different inputs.On the other hand, can to transmit the signal generator (as the multi channel signals generator) of a plurality of independent signals be more convenient in utilization.Moreover above-mentioned signal generator is the known technology of this area.

In fact any amount of input signal all can be transferred into this nervous tissue.The quantity of input signal is only limited by the electrode number that is used to handle this signal.In a preferred embodiment, the number of input signal changes between 1 to 8, more preferably change between about 8 to about 64.In certain example, more input signal (as up to 256,512,1024 or even more) will be used.

B) type of input signal

Many input signals can be used to nervous tissue.In fact any signal all can so be used) yet, in a preferred embodiment, use signal and will comprise known application signal with neural activity characteristic.Above-mentioned signal include but not limited to α, γ and θ.Preferred input is those the known spatiotemporal modes that can induce synapse viscosity, this spatiotemporal mode includes but are not limited to, have four 100HZ pulses the group " θ-pulse) stimulate " (TBS), described impulse train by 200 milliseconds interval separately so that this impulse train takes place at the 5Hz theta rhythm.In a particularly preferred embodiment, using signal is a θ ripple signal.

1. preferred θ input

θ is a kind of rhythm mode of finding in digitation of hippocamps that is present in the 5-10HZ in multiple behavior and the REM sleep.It is said that (Vertes and Kocsis (1997) Neurosci., 81:893-926) synchronous cell of the middle maximum that can write down is outer movable at normal EEG.By people such as Petsche (1962) Electroenceph.Clin.Neurophysiol., the establishment that 14:202-211 carries out studies confirm that θ is reflected in the neuronic lock-out pulse activity in middle spacer film/bias tape.Studies show that the γ-An Jidingsuan neurokyme and cholinergicly produce one every neuron and have the high-frequency pulse of a pulse period period of wave accordingly afterwards with θ.In addition, this barrier film in the θ frequency stimulates θ (Apostol and Creutzfeldt, (Brain Res., the 15:65-75 that produces normal appearance in digitation of hippocamps; People such as Wetzel (1977) Behav.Biol., 19:534-542; Kramis and Routtenberg, (1977) Brain Res., 125:37-49).These viewpoints combine every outstanding physiological action with digitation of hippocamps is interior, cause such hypothesis, θ causes by the circulation that suppresses to circulate/to disinthibite, and should circulation by every neuronic interpulse/pulse in movable generation the (referring to Vertes and Kocsis (1997) supra. summary)).

Since Green and arduini (1952) J.Neurophysiol., since 17:533-557 confirms that first this rhythm and pace of moving things is an accompaniment of digitation of hippocamps awakening, about θ to the existing considerable supposition of the active effect of digitation of hippocamps.Recently the deep layer relation between θ and the long-term potentiation (LTP) has greatly influenced the viewpoint about this problem, a kind of synapse viscosity that extensively has, and this synapse viscosity is crucial to memory coding.Preliminary study finds to work as pulse at θ frequency (Larson and Lynch (1986) Science, 232:985-988; Staubli and Lynch (1987) Brain Res., when 435:227-234) being repeated to carry out (administer), 30 milliseconds boost pulse of four boost pulses is enough induced current potential.Change outer pulse distance of this θ cycle cause significantly LTP reduce gradually (people (1986) Brain Res. such as Larson, 368:347-350).Secular writing task confirms that in comprising the complex behavior of study digitation of hippocamps neuron demonstrates the θ pulse of the type of inducing LTP ideally (people (1992) Behav.Neural.Biol. such as Eichenbaum, 57:2-36; Okeefe (1993) Curr.Opin.Neurobiol., 3:917-924; Vanderwolf (1969) Electroencephalogr.Clin.Neurophysiol., 26:407-418; Winson (1972) Behav.Biol., 7:479-487; Winson (1974) Electroencephalogr.Clin.Neurophysiol., 36:291-301).

Whereby, the θ pattern causes that the inductive mechanism of LTP is narrated.In brief, the synapse of the pulse activation feedforward inhibition relay cell that imports in the digitation of hippocamps also activates the synapse of target cone neurone.The latter obtains a single synapse and excites input like this, then the prominent axle of very fast acquisition secondary synapse inhibition after-potential (IPSPs).Inhibitory action is by making the inner stimulating current resistance EPSPs originally of this cell hyperpolarization and shunting; Like this, synthetic response is made up of 2 or 3 milliseconds depolarization, is a more persistent hyperpolarization afterwards immediately following this depolarization.One of the derived need of LTP has the depolarization of enough persistent period and sufficient intensity to connect the glutamate receptor of pressure-sensitive NMDA type.Sound to single synapse would not run into these situations.Yet the feedforward inhibition synapse is in a single day intensified, just enters a refractory stage, and this refractory stage reaches the peak behind the 150-200 millisecond, then disappears in next second.The pulse that arrives in the refractory stage peak produces the synthetic response of essentially no IPSPs, then can produce measurable LTP.Pharmacological research point out the autoreceptor of pre-synapse GABAb type be the reason that forms this refractory stage (Mott and Lewis (1991) Science, 252:1718-1720).

2. the signal that changes with the space

This uses signal except changing in time, also can change on applied location.Can only be connected in the special electrodes change signal application location of a particular signal source by change.In other words, signal source is added on the different electrodes, and is switched on or closes, and causes the different spaces of signal to distribute thereby maybe can increase or reduce amplitude.Can change this electrode individually or with collaborative pattern.Can mechanically be switched on or switched off connection by known various electric switching technology in the art, thereby obtain this conversion.

Preferred control pattern signal will include but not limited to, spacing in 10 to 100 milliseconds paired boost pulse, frequency at the repetitive stimulation of 10-100Hz, in the localized mutual stimulation of two or more differences.

C) I/O array and variation

In different preferred embodiments, a plurality of input signals are applied to nervous tissue, and/or detect a plurality of output signals (tissue response signal) and carry out stochastic analysis.

Usually each (different) signal inputs or outputs, and controls and detects by single electrode.A plurality of single electrodes can be applied to this nervous tissue.Yet, simplified analytical specimen by using one or more electrod-arrays.

One " electrod-array " be connected in the common unit, in the tube bank or surperficial a plurality of electrodes (at least two, preferably up to 4,8,16,64,256,512 or even 1024 electrodes or more).The combination of electrode is convenient to be applied to this nervous tissue, and does not need to control respectively (use) each independent electrode.

In a particularly preferred embodiment, the present invention utilizes one or more siliceous multiple electrode arrays, for example the siliceous multiple electrode array (multichannel extracellular recording (MED) system) of Matsushita electronics corporation production.

1) electrode array designs

Preferred multiple electrode array comprises at least eight different electrodes.Yet the number of electrode can (routinely, routinely) change be up to 16,64,128,256,512,1024 or even more regularly.Can under multiple electrode density, (number of the single electrode of per unit area) provide this electrod-array.In a preferred embodiment, " fine and close " or " high density " array preferably have spacing between about 300 μ m or littler electrode, spacing between about 200 μ m or littler electrode is more preferably arranged, most preferably have about 150 μ m or littler or even 100 μ m or littler electrode between spacing.Preferably between about 1mm, change, more preferably between about 750 μ m, change, most preferably between about 450 μ m or 500 μ m, change at about 300 μ m at about 300 μ m at about 300 μ m.

The geometry of many kinds of electrodes is fit to analysis of the present invention.In a preferred embodiment, this electrode is distributed in the uniform basically array (referring to as Fig. 6 A and 6B) regularly.In a further embodiment, in the packing configuration (package configuration) of one substrate and multichip moduleization, provide this electrod-array.This array can comprise a latitude and two-dimensional array of electrodes.Can use based on the flip-chip combined techniques of high density indium bump and realize that the module of multiple-piece is integrated.

Other preferred electrode geometry comprises the conformal mapping of tissue culture, and this conformal mapping is embodied in the main profile relation between input layer and the output layer neuron array.For example, in order to activate a subgroup of digitation of hippocamps synapse by three synapse circuit, with the common subgroup of polymerization in postsynaptic neuron, preferably will import the relative narrow region that (stimulation) passage concentrates on (being limited to) digitation of hippocamps section, promptly one is throwed consistent zone with the topography of its passage.This can realize that this detector has the high-density electrode site and has a spatial distribution that is complementary with the intrinsic nerve circuit by using probe.Similarly, can optimization be used for writing down CA3 and the interior miniature probe size of component that responds the electrical activity of input stimulation of CA1.Like this, in certain embodiments, preferably use a spot of wide element (as changing to about 100 μ m from about 50 μ m), this element can write down the activity of the neuron pool that the movable of adjacent neuron pool rather than space separate.

This electrod-array can be in addition in conjunction with switch (electrode selection) circuit and the preamplifier (preferably insulating) that is used for each active tunnel.Incoming line in the electrod-array can be and instruction system is downloaded to the array that is used for local signal adjustings/amplification and electrode selection gives security.

D) structure of array

Used multiple electrode array can be according to being any assembling in the several different methods known in the art in test of the present invention.For example can use a plurality of one electrodes, this electrode is not connected to an array.On the other hand, a plurality of single people's electrode can connection in groups with a kind of mechanical holder (process as, or in other words make good support piece, this supports piece has a plurality of electrode receptors).This electrode can be combined (as using adhesive, epoxy resin, liquid resin/resin etc.) simply.If the use glass electrode can connect this glass electrode by this glass of heat fused.

In certain embodiments, this electrode is welded into an integrated unit.In this embodiment, particularly go for the place of high concentration and probe concentration (little spatial resolution), the preferred weld method include but not limited to the integrated circuit welding method.To using said method to produce a detailed description of an electrod-array according to Hubbard described (United States Patent (USP) the 5th, 388, No. 577).This Hubbard patents state in one embodiment, a electrode array microchip with CMOS technology welding.This above-mentioned multiple electrode array uses the fabrillation line technology of standard, and metal area is arranged on substrate, has covered one deck glass material on this substrate.Thereby this bell glass cutting is formed electrode to expose metal.Electricity consumption is connected to the leads method of joint or the integrated circuit in detecting plate or the microchip with this electrode.

It should be noted that multiple electrode array also can on market, buy (referring to as, Matsushita electronics corporation, SAGC-5 and SAGC-10 multi-electrode dish (MED)).These electrod-arrays contain 64 microelectrodes lining up 8 * 8 arrays, and these 64 microelectrodes occupy about 1mm at the center of a glass plate ²Area, spacing is 150 μ m (with an interpolardistance between electrodes of 150 μ m) between electrode.Each microelectrode is 50 * 50 μ m, and one 50 kilohms (kilohm) or impedance are still less arranged.

D) location of array in the section

1. the physical positioning of electrod-array

The location (or vice versa) of the nervous tissue on this electrod-array (as the brain section) preferably makes the suitable electrodes number that connects nervous tissue purpose zone in the array reach maximum probability.Fine and close electrod-array provides more accurate localization/arrangement at a reductive area of coverage.In this example, can be by the area that utilizes bigger array or poly array to increase this area of coverage.On the contrary, the low-density array can be used to write down bigger zone.

Fine and close (can have a lot of location as a section on the array of 1mm * 1mm).For example, can place a dense array so that the major part of zone C A3 is contained in this array together with dentate gyrus portion and zone C A1, or so place so as the major part that makes CA1 together with being in the same place with the part of CA3.Wide array is (as 3mm * 3mm) also can be used to best located, so that hippocampal all three main region (DG, CA3, CA1) are contacted with this array, entorhinal cortex also contacts with this array with three all leftover bits and pieces zones, if they are in this section.Usually, will recognize, can change array region, electrode density and electrod-array shape contacting regularly with optimization and specific nervous tissue specimen.

2. the selection of stimulation and/or record position

As implied above, only can select input (stimulation) and/or record position in real time by the electrode of any one or this nervous tissue of more contacts.Can use the microcircuit instruction on " outside switch enclosure " or the electrod-array to obtain the electrode selection.Electrode select or dynamically to reequip by this analysiss installation (as, manually, with programming system or respond specific neural output signal).

It should be noted, a commercially available multi-electrode recording equipment provides a switch enclosure, in experiment once this switch enclosure allow at any time can reselect in real time different electrodes (referring to as, Matsushita electronics corporation, SACC-1MED (multi-electrode dish) adapter, this adapter provide isolate good convertible connection, this connection has per 64 microelectrodes less than 30 ohm contact impedance).

IV. output signal

In analysis of the present invention, the electricity output of this nervous tissue (part of a brain, complete brain circuit etc.) can be arrived in one or more detection and localization.This output signal is amplified at random, is regulated, in time deciphered and/or stored, and is in order to analysis subsequently, as described below.

A) output connects the location/characteristic of (array etc.)

If in one or several (as 2-4) detection and localization output signal, it is quite simple utilizing dispersive electron probe.Yet, in a preferred embodiment, especially when the time in a plurality of detection and localization output signal, this output signal of an available aforesaid electrode (microelectrode) array detection.This electrod-array can be and the identical array of array that is used to control input signal (stimulation), or an independent array.In a preferred embodiment, identical electrode is applied to stimulate and during the response that detects, the different electrodes in the array are used to stimulate and detect.Yet, identical electrode may be used for simultaneously two kinds of activities, especially when between stimulation and signal generation, having one to postpone in advance.This recording electrode can be fixed in this array, or the interrupted or change continuously along with the dynamic recombinant of array.

In a plurality of preferred embodiments, when transmitting input signal, optionally from following this output signal of one or more regional records, this zone comprise granular cell, CA3 pyramidal cell, in smell CA1 deep layer cell any dendron shape zone in pyramidal cell, subiculum cell, presubiculum or side subiculum cell and these zones.In a preferred embodiment, detect output signal in the dendron shape zone of CA3 and/or CA1.

B) amplification of output signal and Noise Suppression

Realize the noise suppressed of preposition amplification, amplification and signal according to known standard method in the electrophysiology field.Usually each signalling channel is attended by preposition amplification period, and makes its as close as possible recording electrode when placing this preamplifier.Like this, in one embodiment, incorporate preamplifier on the electrod-array circuit.Available appropriate filters realizes noise suppressed and/or realizes noise suppressed by algorithm by electronization during analyzing acquired signal.Usually can realize noise suppressed by using a actuator with one or more signals.Signal conditioner is normally computer-controlled, and can use with any lab A/d system is collaborative.Signal conditioner provides multiple filter and noise suppressed facture, includes but are not limited to 4 or 8 bar Bessel low-pass filtering, high-pass filtering, AC/DC coupling, notch filter, variable gain, baseline correction or the like.Suitable preamplifier, amplifier, analogue-to-digital converters, data collecting system, signal conditioner or the like be commercially available (referring to, as, Matsushita electronics corporation, multichannel extracellular recording system, with Axon equipment company, Foster City, preamplifier, amplifier, signal conditioner or the like).

A sketch map of multi-electrode dish (MED), switch enclosure, amplifier, A/D and D/A converter, signal and anacom is shown in Fig. 8 A, 8B and 8C.

V. output signal analysis

A) the simple identification of cognitive function variation

In one embodiment, output signal analysis can be as simple at each output position the record and/or demonstration output signal.The visual analysis of the output signal that so presents is that information is quite abundant, and usually enough satisfies 1) identification changes the chemical compound of cognitive activities; 2) discern the different activity patterns of this chemical compound; 3) provide the characteristic signature of this chemical compound; With 4) make things convenient for this chemical compound comparison (referring to as, the example that provides herein).

In one embodiment, the change of the cognitive function that is caused by a kind of application medicaments can be confirmed by a lot of indication.These changes comprise the change of the signal amplitude, persistent period, frequency or the waveform that respond specific input and cause, the change of different parts output signal mutual relation in the nervous tissue, change of the mutual relation between input signal and the output signal or the like.

Thereby, for example, in a research that detects stable effect to acute digitation of hippocamps section preparation, write down the response (d in as Figure 10, e is shown in the f) in a cellular layer, this response is as the function of the zone of the stratum germinativum epidermidis in CA3 zone internal stimulus.In addition, monitored the activity of another cellular layer (as figure a-c, shown in the g-h) with to the active dependence of initial cell layer.The stimulation location in the stratum germinativum epidermidis zone in CA3 zone cause in this cellular layer (Figure 10, d, e, (Figure 10, a-c g-h) cause more activity by contact more conversely for the f) response at three positions, this response.Field potential is bigger when stable the existence, according to a unexpected result of stabile γ-An Jidingsuan enhancing property effect.Also defer to more advanced analysis as described below by the output signal that analysis of the present invention provides.

B) Xian Jin analytical method

Successive time varying signal, for example the activity pattern that is produced by the section of cultivating normally utilizes hidden Markov model (HMMs) to analyze; In fact above-mentioned analytical method is so ubiquitous, has therefore started the project of being provided with funds by government fully, and the unique purpose of this project is other method of definite and Development Analysis time varying signal.As the achievement of simulation auditory system, an above-mentioned method (people such as Aleksandrovsky, (1996) Proc.Intl.Conf.Pattern Recog.IEEE Comp.Soc.Press, 4:550-554 have been developed; People such as Aleksandrovsky, (1997) Biological and artificialComputation:P104-115:From neuroscience toTechnology, IWANN ' 97Inte ' lConf.on Artificial and Natural NeuralNetworks, Berlin:Springer Verlag; People such as Garzotto (1997) Proc.Intl.Conf.Networks, IEEE Press, 1:564-568).HMMs may be as the statistical model operation of the characteristic sequence of a time series process by structure.For example, for sound, the pronunciation of word (" Bill " or " ball " or " bald ") is divided into the time period, and each time period comprises that a specific dominant frequency set (as is marked with a ₁, a ₂Or the like).The probability that each possible characteristic occurs is calculated in one group of sampling of each pronunciation by each time period, and construct a state transfer model, this state transfer model is consistent to Next transfer probability with the probability density function of forming from feature along with the time (PDF).

HMMs is important for various signal analysis, as sonar with speak, but two shortcomings that well-known cost is high are arranged:, be necessary to obtain an important sampling on statistics of every kind of possible pronunciation or vestige i) in order to construct PDF and the state transfer model that this feature is formed; Ii) the model of Xing Chenging can only use instant preceding time point to calculate the transfer probability of back time point; Can not use any information and activity in the past.This has individual advantage, promptly transmits the combination of avoiding possible on the number in possible prediction and increases sharply, and has still limited the forecasting power of this method, particularly to movable responsive data of past.Construct according to olfactory sensation and auditory system by the described alternative series processing model of people such as Aleksandrovsky (1996 and 1997) supra..

After deliberation model storage and fetch operation, this operation is the series of features that the response to network input phase is occurred.In this embodiment, a series of feature is formed a pattern of the numeral " 3 " of writing.This series comprises two ink dots, follows three ink dots thereafter, follows more ink dot again, arranges according to distance and thickness, if so that be close to, just form the pattern of " 3 ".This network has two different input channels: a topography passage and a non local anatomical passageway.This topography passage optionally activates the topographic region that is used for the particular types characteristic vector of this network by " intermediate layer " of first characteristic vector (points of two arranged verticals) to this network.

By described clustering method (people such as Ambros-ingerson, (1990) science, 247:1344-1348; People such as kilborn (1996) J.Cog.Neurosci. 8:338-353) reads this input on " top layer " of this network.Vertical clustering pattern by discovery thus arrives " deep layer ".This layer is given input structure with its responsive feedback when reading second input.Read next input (points of three arranged verticals) on the top layer and respond cross point between the feedback of first input from deep layer, and in deep layer, read from first clustering to the second cybotactic transmission (by people such as Granger (1994) J.Neurophysiol., the described a kind of sequence of 17:533-557 reads rule).Arrival along with input repeats this process, the successive transmission of storage from a characteristic to next characteristic.In fact total mechanism encode a series of random model along the cognition network of many signs, and each successive modes is from its previous mode producing.

Described network is first network in fractionated organized network family, so the output of this network is transfused to next network, the output of next network is transfused to the network of back again.The cascade of this first network, second network, the 3rd network etc. is extracted feature from input in succession and is summarized, realize that a kind of the superior and the subordinate handle, in these the superior and the subordinate handled, available long sequence information (as a speech) supported shorter sequence with disambiguation (so letter in the speech).

This classification cascade has been used in speech processes and the hand-written letter identification (people (1996 and 1997) supa. such as Aleksandrovsky; People such as Garzotto (1996) supa.).Recently this network has been used to analyze evoked response potential (ERPs) and be used for distinguishing normal, spiritual separate disease and object Alzheimer from their ERPs from human subjects.In this research, during the tone pulses of the example of " audition eccentric disease " shows, from 24 ERP data of separating 28 electrodes of object collection of disease normally with 24 spirit, in this example, when a rare altofrequency tone appears in a succession of low frequency tone, require object to respond.Build this network with 50% data, then with this network of data test of remaining 50%.With a HMM and described model contrast just now.These data are difficult to satisfactory, and this HMM finishes about 70% correct prediction, and auditory model reaches 90% accuracy level.

These initial achievements likely show that this model is useful for the diagnosis of the predictability of EEGs and ERPs.In a section preparation, the remarkable similarity of the synapse response that above-mentioned signal and this bring out shows that also this algorithm is useful for these signals are classified.

VI. medicament is applied to the brain section

A) common medicament kind

In fact all available method of the present invention of any medicament detects and/or characterizes.These medicaments comprise, but be not limited only to influence the drugs of ergasia, (the positively-modulated agent of the gamma-aminobutyric acid receptor such like an elephant benzodiazepines, positively-modulated agent, anticholinergic medicament or the cholinesterase inhibitor of the glutamate receptor resemble the promazine) needs the activity of these drugs is classified, chemical compound such as saccharide, protein, nucleic acid, lipid or little organism storehouse, the environment medicament, the medicament that is used for various manufacture processes, decomposition by-products in the various processing procedures or the like.Medicament also comprises, but be not limited only to, be intended for use in or be used in fact the medicament and/or the lead compound of the treatment usefulness of particular pathologies situation, this pathological condition includes but are not limited to spirit and separates disease, Parkinson's disease, Alzheimer's disease, melancholia, anxiety, various drug dependences or the like.

In a preferred embodiment, this medicament will not comprise known cytotoxin, common buffer agent, salt and the composition that exists usually or the like in tissue culture medium (TCM).

Recently, will concentrate on to utilize the chemical reagent storehouse of combination to help to produce new lead compound.A kind of chemical reagent storehouse of combination is the set of multiple chemical compound, and this chemical compound produces with chemosynthesis or biosynthesis by in conjunction with many chemical building blocks as reagent.For example, as every kind of possible mode, in conjunction with being called as amino acid whose one group of chemistry building block, thereby form the linear combination chemical reagent storehouse as a peptide library according to given chemical compound length (being the amino acid number in a kind of polypeptide compound).Chemical compound up to a million can be synthetic by the combined hybrid of above-mentioned chemical building block.For example, commentator combined hybrid of observing 100 tradable chemical building block systems causes can synthesizing in theory 100,000,000 quaternary part chemical compounds or 1,000,000,000 five element chemical compound (people (1994) 37 (9) such as Gallop: 1233-1250).

The chemicals storehouse of preparation and screening combination has been the technology of knowing in this area.Above-mentioned combinatorial chemistry preparation storehouse include but not limited to, and the peptide storehouse (referring to as United States Patent (USP) the 5th, 010, No. 175, Furka (1991) Int.J.Pept.prot.Res., 37:487-493, people such as Houngton (1991) Nature, 354:84-88).Peptide synthetic unique anything but one that expect and be intended for use method of the present invention.Also can use other chemical substance to produce the number of chemical reagent storage.Above-mentioned chemical substance comprises, but be not limited only to, class peptide (PCT publication number WO91/19735,26Dec.1991), peptide (the PCT publication number WO93/20242 of coding, 14 Oct.1993), irregular biological oligomer (PCT publication number WO92/00091,9 Jan.1992), benzene phenodiazine (United States Patent (USP) the 5th, 288, No. 514), various body as hydantoin, benzene phenodiazine and dipeptides (people (1993) Proc.Nat.Acad.Sci.USA 90:6909-6913 such as Hobbs), alkene class modified polypeptides (people (1992) J.Amer.Chem.Soc.114:6568 such as Hagihara), class peptide compounds (people (1992) J.Amer.Chem.Soc.114:9217-9218 such as Hirschmann) with non-peptide of a β-D-glucose skeleton, the similar organic synthesis of small-sized chemical reagent storage (people (1994) J.Amer.Chem.Soc.116:2661 such as Chen), low polyurethanes people (1993) Science 261:1303 such as () Cho, and/or peptide acyl phosphonate ester (people (1994) J.Org.Chem.59:658 such as Campbell).Usually referring to people such as Gordon (1994) J.Med.Chem.37:1385, nucleic acid library (referring to as Strategene.Corp.), peptide nucleic acid(PNA) storehouse (referring to as United States Patent (USP) the 5th, 539, No. 083) antibody library is (referring to as people such as Vaughn (1996) natural biology technology, 14 (3): 309-314) and PCT/US96/10287, saccharide storehouse (referring to as people such as Liang (1996) Science, 274:1520-1522) with United States Patent (USP) the 5th, 593, No. 853) and small-sized organic molecule library (referring to as the benzene phenodiazine, Bao Mu (1993) C﹠amp; EN, 18,33 pages of Jan, isoprenoid United States Patent (USP) the 5th, 569, No. 588, Thiazolidinone and Metathiazanone United States Patent (USP) the 5th, 549, No. 974, isoprenoid United States Patent (USP) the 5th, 525, No. 735 and the 5th, 519, No. 134, No. the 5th, 506,337, morpholino compounds United States Patent (USP), benzene phenodiazine 5,288,514 or the like).

The equipment of preparation combinatorial libraries on market be commercially available (referring to as 357MPS, 390MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburm, MA, 433A applying biological system, Foster City, CA, 9050Plus, Milipore, Bedford, MA).

Many famous robot systems also have been developed out and have been used for liquid phase chemical.These systems comprise the automatically working station as automatization's synthesis device of being developed by Takeda chemical industry company limited (Osaka, Japan) and much utilize robots arm's robot system (Zymate II, Zymark company, Hopkinton, Mass; Orca, Hewlett-Packard, Palo Alto, Calif), the synthetic operation that this system simulation chemist carries out.Above-mentioned any equipment all is applicable to the present invention.For these equipment are operated as described herein like that, these install improved characteristic and realization (if the words that have) will be conspicuous to persons skilled in the relevant art.In addition, a lot of combinatorial libraries this on market be commercially available (referring to as Comgenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,, MO, Chemstar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, MartekBiosciences, Columbia, MD, or the like).

B) medicament is applied to nervous tissue

According to being any of the known many standard methods of those skilled in the art, this medicament is applied to this nervous tissue, in simple embodiment, when this medicament is solvable, this medicament can be added this culture medium simply especially.When this medicament is soluble, itself and one or more material can be merged, so that itself and this solution (as a kind of emulsion or dispersion) mixes.The method of dissolved compound is the technology of knowing (referring to the Pharmaceutical science as Remington, 15th ed., Mack PublishingCompany, Easton, Pennsylvania (1980)) in this area.

Except that above-mentioned,, this medicament can be directly applied to any exposure of (contact) this nervous tissue as a displaced method.This medicament also can be injected into this tissue, and at the position of wishing long term administration especially, with standard method conduit is inserted this tissue and this medicament is poured into into this tissue.

In another embodiment, this medicament itself can be a kind of tissue (as a kind of nervous tissue), is about to the part that a used mammal brain in analysis of the present invention advances in this tissue transplantation.This be organized on the origin can be from body or allosome, and can from the mammalian tissues of sophisticated, immature, immature (child's) or fetus, obtain.Organizing of this transplanting self can only change cognitive function by cells contacting, or more generally, influences cognitive function (as serotonin, dopamine or the like) by discharging one or more medicaments that influence ergasia.

C) other analytical form (as the format high throughput form)

Any analysis that is used to change the chemical compound of cognitive function as described herein can obtain according to format high throughput screening.High throughput system is utilized the computer control of robotics, information acquisition system, test protocol usually, and the automatization in chemical reagent storehouse is synthetic and screening system between interaction make the maximization of throughput of test agents.

The system of convenient format high throughput screening is available (referring to as Zymark company, Hopkinton, MA on market; Aeronautical technology industry, Mentor, OH; BeckmanInstruments, Inc.Fullerton, CA; Precision Systems Inc., Natick MA, or the like).These systems make usually and comprise that all samplings and the pipetting of reagent, liquid dosage, timing are incubated and are suitable for the whole steps automatization of final reading of the probe of this analysis.These configurable systems both provided the adaptability and the customizations of height, and format high throughput and startup fast also are provided.The manufacturer of said system provides detailed protocol for various format high throughput forms.

VII) signature storehouse

In another embodiment, the invention provides " active signature storehouse ".Active signature storehouse be a plurality of (as 2 or more, more preferably more than 10, more preferably more than 100, most preferably more than 1000,10,000 or even more than 1,000,000) the activity signature collection of different activities signature.The activity of any two kinds of various medicaments signature is diacritic in a preferred storehouse, thereby discerns this medicament uniquely according to other medicament in the storehouse.

Each signature in this storehouse preferably includes the record of sufficient length, so that will (this moment, this test agents produced the difference of an output signal) come in this localized input signal difference in the output signal of a certain position and when this test agents does not exist.Preferred signature comprise about at least one 0.1 second, more preferably about at least one 0.5 second and most preferably about at least 1 second, 10 seconds, with in addition 1 minute or more persistent electrogram, this current potential in mammal brain is cultivated part two or more, preferred four or more, more preferably 8 or more and most preferably 16,64,128,256 or even 512 or more a plurality of different location, this mammal brain is cultivated part and is contacted with the chemical compound that produces signature.In addition when this signature be the chemical compound of a record when not existing, this signature can comprise at random that current potential is at each localized response record.Select the signature of every kind of chemical compound that unique sign signature exists in the storehouse.

This activity signature storehouse provides important resource to be used for classification and/or characterizes medicament.Medicament with similar activity pattern will have similar activity signature.Simultaneously, can according to signature have trickle difference or even the activity pattern of a great difference, disclose the medicament that this organism is had similar or same function.This activity summary can be used to discern treatment instructions about how to take medicine that various medicaments replenish or that change like this.

Characterizing newly when influencing the medicament of ergasia, when estimating possible biohazard, or during the lead compound of search treatment usefulness, this activity Kuku of signing is particularly useful.For example when a kind of medicament just has been confirmed as having the performance that influences ergasia,, only just can in other chemical compound, classify to this chemical compound by activity pattern with the activity signature and the storehouse contrast of active signature of this medicament.In addition, when a kind of characteristic of particular agent is that the unknown (promptly in a kind of composite extract plant) of this reactive compound in the storehouse divides time-like, discern medicament, an indication of the chemical composition of this medicament may be provided, thereby be convenient to purification subsequently with similar activity summary.

If a kind of medicament that influences ergasia is a kind of composition of environment (as natural environment, working environment, waste disposal environment or the like), this medicament classification is helped to estimate by the caused danger of the sort of particular agent by the activity signature.

Allow desired activity signature that influences the medicament of ergasia of prediction at a kind of activity signature that the known compound with physiological effect or side effect that provides in the instructions about how to take medicine is provided.The chemical reagent storehouse has with searching or the approaching medicament that the activity signature of requirement is arranged thereby can screen.The such use of this activity signature is defined as illustrative but not determinate.

When activity signature is easy to be retrieved, sorts, classifies and/or during other systematism, this activity signature storehouse is very useful.So in a preferred embodiment, this signature storehouse of the present invention comprises a data base, most preferably an electronics (as computer control) data base.Like this, this data base ingredient of computer system normally.The computer system that is used to store with operating database is a technology well known in the art, and include but not limited to the distribution node on " personal computer system ", large computer system, the Internet or the Intranet, the data that are stored in (in microchip) in the specific hardware or data base or the like.

VII. workbox

In another embodiment, the present invention also is provided for implementing the workbox of analytical method as herein described.The preferred kit case comprises that is equipped with a following one or more container, comprise an electrod-array, one have the culture apparatus of an electrod-array, a medicament on paper " active signature " storehouse, electronics or optical storage pattern, a cultivation nervous tissue, electroencephalograph device (signal generator, preamplifier, amplifier, data collecting system or the like) buffer, be used for an electrod-array location and be applied to nervous tissue's specimen template, micro-manipulator, be used for VLSI logic element of post processing or analysis or the like.

In addition, this workbox may comprise the guiding material that the explanation (being protocol) of implementing analytical method of the present invention is housed.Though this guiding material generally includes hand-written or materials printed, they are not limited only to these.The present invention has imagined and anyly can store above-mentioned explanation and it is passed to an end user's medium.This medium include but not limited to electronic storage medium (as disk, tape, cassette tape, chip), optical medium (as CD ROM) or the like.This medium may comprise the internet address that above-mentioned guiding material is provided.This workbox optionally comprises material, device or the explanation of any other enforcement the inventive method.

Example

The following invention of example in order to illustrate but to be not limited to be applied for is provided.

Example 1: cultured rat hippocampal toe section

Utilize two different specimen of cultured rat hippocampal toe section, one does not have barrier film input (H specimen), and one has barrier film input (H/S specimen).In these two specimen, input signal is transferred into the granular cell of this dentate gyrus.In both cases, this output signal is recorded in the different layers of this network, that is, and the pyramidal cell of granular cell, CA3 and the pyramidal cell of CA1.

A) specimen of Pei Yanging

((1991) Neurosci.Meth., 37:173-182.) described method prepares the organotypic culture thing of digitation of hippocamps section according to people such as Stoppini.In brief, the Sprague-Dawley Mus brain from 11-22 days under aseptic situation obtains fascia Tarin's, is kept at (Gibco Corp.no.61100-061) in the ice-cold MEM culture medium.This culture medium comprises (unit is mM): HEPES (25), Tris-base (10), D-glucose (10) and MgCl ₂And be placed on one and have on the polytetrafluoroethylene platform that McIllwain organizes chopper (3).Brain section (400 μ m) is cut and transferred on the siliceous multiple electrode array that contains in the tissue culture medium (TCM) (referring to following), this tissue culture medium (TCM) comprises MEM culture medium (GibcoCorp.no.41200-072), and this MEM culture medium comprises glutamine (3mM), HEPES (30mM), NaHCO ₃(5mM), D-glucose (30mM), L-ascorbic acid (0.5mM), CaCl ₂(2mM), MgSO ₄(2.5mM), the insulin of 1 μ g, 20% horse serum and contain penicillin, pH7.2.Then the brain section is stored in 35 ℃ of constant incubators that contain 5% carbon dioxide in the air, and culture medium is changed weekly twice.In initial ten days of incubation, section forms sophisticated characteristic gradually, comprises that myelin forms, forms well (well-developed) dendritic spine and the ability of long-term potentiation.Section is kept in the culture at least 10 days before on-test.

B) secular digitation of hippocamps section produces compound space-time activity pattern.

Be kept at a multi-electrode dish (MED; Matsushita Electronics) the initial experiment in the cultured rat hippocampal toe section in shows that these sections have the of short duration input stimulus (millisecond) of response and produce the strong ability of the activity (second) that prolongs.The digitation of hippocamps section that from 11-13 days the Sprague-Dawley Mus in back that is born, prepares, be placed on the MED device, and be kept on the air and the separating surface between the artificial cerebrospinal fluid (ACSF) of oxygen enrichment, this artificial cerebrospinal fluid comprises: NaCl (124mM), KCl (3mM), KH ₂PO ₄(1.25mM), MgSO ₄(1mM), CaCl ₂(4mM), NaHCO ₃(26mM) and glucose (10mM).These all experiments are all at room temperature carried out.

The activation of response Schaffer-commissural fibers, 4 different position the records from the CA1 zone typically bring out the synapse response.Single response summary with the stimulation that repeats to import into is similar to the response summary that obtains with common recording method.Can be observed the biofeedback of paired pulse at the interpulse interval of 40-80 millisecond.

C) the medelling activity in the culture section is responsive to the excitotoxin of low dosage

In this test, manipulator on two kinds of dissimilar AMPA type glutamate receptors is applied to the digitation of hippocamps section.The digitation of hippocamps section is located so that the major part in one 8 * 8 electrod-array covering CA3 zone.Pyramidal cell activation in the zone C A3a-b is induced asynchronous synapse activity, and writes down this asynchronous synapse activity from the cellular layer of CA3c.Slightly stimulating the regional current potential of (50 μ A) back record in the delivered spaced with 20 seconds is 300 milliseconds.Before medicine adds, behind the cyclothiazide (a kind of benzothiadiazide) of GR120 (a kind of promazine) that pours into 50 μ M or 100 μ M,, calculate the standard deviation of 20 continuous responses of each passage as the intensity and variational a kind of the measuring of neuron activity.GR120 and cyclothiazide all combine with the glutamate receptor of AMPA type, and allosteric ground increases the electric current that is produced by these receptors.

Two kinds of chemical compounds have all increased by 6 neural activity in 8 positioning of electrodes.These differential effect on different electrodes are aroused the attention to this fact, and promptly ampa receptor is positioned in the neuron and the excited neuron of inhibition; Thereby strengthen the neuronic activity that the active drugs of ampa receptor may rely on the local circuit of record to strengthen or suppress to be surveyed.

It should be noted that being seen effect is big unexpectedly for the concentration of used last manipulator.The cyclothiazide that concentration is 100 μ M is the threshold dose of a single synapse response of influence preferably.Effect size seen in the test of report may be because the amplification of many synapses circuit: the influence that each stage in many synapse responses is existed by medicament of surveying, and also obviously combined effect is cumulative at least, and may be non-linear.

This effect was also reported in the acute section with unirecord electrode.Fig. 4 illustrates an experiment, in this experiment, obviously relatively on ampa receptor manipulator in single synapse and response effect in many synapses.The single synapse response of a kind of standard has been brought out in the stimulation of importing the Shaffer-Colaesce of digitation of hippocamps zone C A1 into.To the growth of these single synapses responses of the MGR120 that inculcates 50 μ almost discover less than.In identical experiment, stimulated the porous channel that imports hippocampal gyrus into and measured the interior forward many synapses responses of CA1.Can see that (GR120 of 50 μ M has greatly amplified these responses for people (1996) Neurosci. such as Sirvio, the 74:1025-1035) additional effect on because this medicine is disturbing synapse.Owing to increase the existence of many recording electrodes of the probability that many synapse responses in cutting into slices are detected, by using the MED device to make this experiment that is difficult for finishing become more practicable, and use the section of long-term cultivation to make test agents become possibility, this medicament may only produce destruction for a long time rather than immediately.

In an experiment, measured kainite and stable to the effect in the reflex activity in the digitation of hippocamps section by a single electrode of this multiple electrode array.Galvanism with per 20 seconds 50 μ A should be cut into slices, thereby along with the increase of vertical displacement has shown 20 successive responses.

Kainite has increased the revolving-door in this section significantly, has also increased simultaneously the early stage response amplitude of stimulation and the persistency of consequential reflex activity.Stable stoped this effect, and activity has been suppressed to level when being lower than no kainite in the nutrition base.

Fig. 7 illustrates kainite and the stabile effect with a kind of multichannel array record.Obtained the standard deviation of 20 continuous responses, and, analyzed the time period of 300-400 millisecond persistent period as at the time point shown in the right bottom of panel (panel).Before medicine adds, or behind the kainite of perfusion 10 μ M, or after pouring into the kainite of 10 μ M and 10 μ M stable at the same time, the activity that is illustrated on eight passages of this multiple electrode array is measured.Kainite can activate excitatoty glutamate receptor, and is the same in the experiment of modulator GR120 on having and cyclothiazide, increased the neuron activity that is recorded in great majority record location.Be used for antianxity stabilizing, suppress circuit to increase, greatly suppressed neuron in all localized activities of eight records by acting on gamma-aminobutyric acid receptor.

Example 2: the drug test that replenishes

Digitation of hippocamps zone C A3 has the feature that spreads all over the cortex akrencephalon.Its principal character is the Colaesce feedback system of a densification, and this system allows to occur in one and disperses localized activity to be diffused into other neuron (Fig. 9 left side) rapidly.By several processes that cause a circulation (cycle) of output synchronously can continue to activate and spread.The relay cell that suppresses plays an important role in forming this circulation stimulation, and this circulation stimulation promotes this network cell to a cooperative response.If suppress too many, this network will " downhearted ".If suppress very little, it is excited that this network just becomes.Like this, any variation in feedforward and/or the different groups behavior of feedback γ-An Jidingsuan neurokyme relay cell (Fig. 9 right side) may be influential to the total activity that enlarges with respect to monosynaptic variation.Above-mentioned viewpoint is of value to pharmacological research, because much influence the drugs of ergasia middle neuron is had directly (benzene phenodiazine) or indirect (serotonin reuptake inhibitors) effect.

Therefore, in another experiment, studied stable (valiumTM) effect in the section that an acute digitation of hippocamps half approaches.Figure 10 illustrates when stable existence of 10 μ M (lower tracer) and 8 record in 64 electrodes when not having (lower tracer).Stimulation location in the basal-cell layer zone of zone C A3 causes that (f) response at place, interior three location, this response are passed through to circulate conversely, and (a-c g-h) causes more activity to this cellular layer in contact for d, e.Stable to have the time domain current potential be bigger, and this is a unexpected result according to stabile γ-An Jidingsuan potentiation.The analysis showed that stabile effect be by stimulate and feedback suppression between interaction make response synchronously, thereby less stimuli responsive still causes more consistent bigger regional current potential when existing so that box lunch is stable.

Should be appreciated that example described herein and embodiment only as illustration purpose, in the scope of the application's aim and scope and additional claims, various modifications and variations of the present invention to those skilled in the art all are conspicuous.Therefore, under any circumstance, whole lists of references is included in this all publications, patent, patent application of quoting.

Claims (61)

1. method that is used to screen the medicament that changes the brain function, described method comprises:

The part of the mammal brain that contacts with multiple electrode array is provided; When having the described medicament of two kinds of variable concentrations, use the described mammal brain of time-varying input signal stimulus at least by the one or more electrodes of forming described multiple electrode array;

Two or more electrode detection by forming described multiple electrode array from described mammal brain the time become output signal, wherein said output signal is the function of described input signal;

In the described medicament of at least two kinds of described at least two kinds of variable concentrations, detect the difference of the output signal that produces by given input signal, therefore, the difference of output signal shows under the variable concentrations of described medicament, described medicament works to change the brain function.

2. method according to claim 1, wherein, the described part of mammal brain is the cultivation part of mammal brain.

3. method according to claim 1, wherein, the described part of mammal brain is acute specimen.

4. method according to claim 1, wherein, the characteristic of the influential ergasia of unknown described medicament.

5. method according to claim 1, wherein, known described medicament is to influence ergasia, and in the described part of mammal brain, described medicament exists with a threshold concentration at most.

6. method according to claim 5, wherein, in the described part of mammal brain, described threshold concentration is less than about 10 μ M.

7. method according to claim 1 wherein, also comprises a kind of active signature of determining described medicament and with a described active signature and an active signature storehouse contrast.

8. method according to claim 1, wherein, described time-varying input signal also is a kind of input signal with spatial variations.

9. method according to claim 12, wherein, described input signal has the known spatiotemporal mode that can induce synapse viscosity.

10. method according to claim 1, wherein, described time-varying input signal is the θ pattern.

11. method according to claim 1, wherein, described time-varying input signal is transferred into the group's who is selected from the dentate gyrus composition cellular layer, the cellular layer of CA3, the cellular layer of CA1, the superficial cell layer of entorhinal cortex, the deep cellular layer of entorhinal cortex, the cellular layer of subiculum, the cellular layer of presubiculum, the cellular layer of side subiculum, the dendron shape zone of dentate gyrus, the dendron shape zone of CA3, the dendron shape zone of CA1, the dendron shape zone of entorhinal cortex, the dendron shape zone of subiculum, the dendron shape zone of presubiculum and the dendron shape zone of side subiculum.

12. method according to claim 1, wherein, described at least two kinds of variable concentrations of described medicament comprise the concentration when concentration when described medicament exists and described medicament do not exist.

13. method according to claim 1, wherein, described medicament is a kind of anticholinergic agents.

14. method according to claim 1, wherein, by neural graft's thing contact, described medicament is discharged by described neural graft thing.

15. method according to claim 2, wherein, the described cultivation of mammal brain partly is the mammiferous brain section of Presentation Function network performance.

16. method according to claim 15, wherein, described mammal brain section is the section of digitation of hippocamps brain.

17. method according to claim 15, wherein, described digitation of hippocamps brain section is selected from the group that comprises neopallium section, the section of thalamus cortex, ganglion basal (striatal) and the section of cortex stricture of vagina shape.

18. method according to claim 16, wherein, described digitation of hippocamps section does not have the barrier film input.

19. method according to claim 16, wherein, described digitation of hippocamps section has the barrier film input.

20. method according to claim 16, wherein, described digitation of hippocamps section presents the ability of myelin formation, dendritic spine and long-term potentiation.

21. method according to claim 1, wherein, the described cultivation part of mammal brain is isolating digitation of hippocamps neuron specimen.

22. method according to claim 1, wherein, the described cultivation part of mammal brain is barrier film and hippocampal co-cultivation thing.

23. method according to claim 1, wherein, described output signal is the θ activity pattern.

24. method according to claim 1, wherein, described output signal is by from being selected from following group one or more regional records, the pyramidal cell of described group of pyramidal cell that comprises granular cell, CA3, CA1, the top layer of entorhinal cortex and deep layer cell, subiculum cell, presubiculum cell, side subiculum cell, the dendron shape zone of CA3, the dendron shape zone of CA1, the top layer of entorhinal cortex and the dendron shape zone of deep layer cell, the dendron shape zone of subiculum, the dendron shape zone of presubiculum, the dendron shape zone of side subiculum.

25. method according to claim 1, wherein, described multiple electrode array comprises at least 64 electrodes.

26. method according to claim 1, wherein, the electrode of described multiple electrode array have between the adjacent electrode one less than the extreme electrode of about 300 μ M between spacing.

27. method according to claim 26, wherein, the electrode of described multiple electrode array have between the adjacent electrode one less than the extreme electrode of about 100 μ M between spacing.

28. method according to claim 1, wherein, described multiple electrode array adopts silicon substrate to make.

29. method according to claim 1, wherein, described multiple electrode array comprises silicones, is electroplated layer of metal on the described silicones, and described metal is selected from the group that comprises gold, platinum, copper and silver.

30. method according to claim 1, wherein, described detection comprises uses hidden Markov model (HMM) or the simulation of small echo to concerning between described input signal and the described output signal.

31. the biology-electronics hybrid sensor that is used to screen the medicament that can change the brain function, described pick off comprises:

The part of the mammal brain of In vitro culture, this part contacts with multiple electrode array;

A kind of use is by the device of the described mammal brain of time-varying input signal stimulus of the one or more electrode of the described multiple electrode array of composition;

A kind of one or more electrode detection by forming described multiple electrode array from described mammal brain the time become the device of output signal, wherein, described output signal is the function of described input signal.

32. biosensor according to claim 31, wherein, described device also comprises a storage medium that comprises active signature storehouse.

33. biosensor according to claim 31, wherein, described device provides a kind of time-varying input signal, also can provide a kind of in described electrod-array the input signal with spatial variations.

34. biosensor according to claim 33, wherein, described device provides the time-varying input signal, and the known input signal with spatiotemporal mode that can induce synapse viscosity also is provided.

35. biosensor according to claim 31 wherein, provides the device of time-varying input signal that θ is provided pattern.

36. biosensor according to claim 31, wherein, with described electrod-array location, so that described time-varying input signal is transferred into the cellular layer of dentate gyrus, the cellular layer of CA3, the cellular layer of CA1, the superficial cell layer of entorhinal cortex, the cellular layer of subiculum, the cellular layer of presubiculum, the cellular layer of side subiculum, the dendron shape zone of dentate gyrus, the dendron shape zone of CA3, the dendron shape zone of CA1, the dendron shape zone of entorhinal cortex, the dendron shape zone of subiculum, the dendron shape zone of presubiculum and the dendron shape zone of side subiculum.

37. biosensor according to claim 31, wherein, the described cultivation of mammal brain partly is the mammal brain section of display function network performance.

38. according to the described biosensor of claim 37, wherein, described mammal brain section is the section of digitation of hippocamps brain.

39. according to the described biosensor of claim 38, wherein, described digitation of hippocamps brain section does not have the barrier film input.

40. according to the described biosensor of claim 38, wherein, described digitation of hippocamps brain section has the barrier film input.

41. according to the described biosensor of claim 38, wherein, described digitation of hippocamps brain section presents the ability of myelin formation, dendritic spine and long-term potentiation.

42. biosensor according to claim 31, wherein, the described cultivation part of a mammal brain is isolating digitation of hippocamps neuron specimen.

43. biosensor according to claim 31, wherein, the described cultivation part of mammal brain is barrier film and hippocampal co-cultivation thing.

44. biosensor according to claim 31, wherein, the described device that becomes output signal when being used to detect can be discerned the θ activity pattern.

45. biosensor according to claim 31, wherein, described output signal is selected from following group one or more regional records, the pyramidal cell of described group of pyramidal cell that comprises granular cell, CA3, CA1, the top layer of entorhinal cortex and deep layer cell, subiculum cell, presubiculum cell, side subiculum cell, the dendron shape zone of CA3, the dendron shape zone of CA1, the top layer of entorhinal cortex and the dendron shape zone of deep layer cell, the dendron shape zone of subiculum, the dendron shape zone of presubiculum, the dendron shape zone of side subiculum.

46. biosensor according to claim 31, wherein, described multiple electrode array comprises at least 64 electrodes.

47. biosensor according to claim 31, wherein, the electrode of described multiple electrode array have between the adjacent electrode one less than the extreme electrode of about 100 μ M between spacing.

48. biosensor according to claim 31, wherein, described multiple electrode array is by siliceous manufacturing.

49. biosensor according to claim 31, wherein, described multiple electrode array comprises silicones, electroplates layer of metal on described silicones, and described metal is selected from the group that comprises gold, platinum, copper and silver.

50. biosensor according to claim 31, wherein, the described part of the brain of cultivation be closed and movably.

51. an active signature storehouse, described active signature storehouse comprises storage medium, and described storage medium comprises the activity signature of multiple chemical compound, and wherein, each active signature characterizes and distinguish every kind of chemical compound in the described active signature storehouse uniquely.

52. according to the described signature of claim 51 storehouse, wherein, each described active signature comprises that one of mammal brain portion is cultivated in the part four or at least 0.5 second electrogram of multi-section position more, this is cultivated part and contacts with the chemical compound that produces this signature.

53. according to the described signature of claim 52 storehouse, wherein, when the described chemical compound that produces this signature did not exist, described each active signature comprised in cultivation part of mammal brain portion four or at least 0.5 second electrogram of multi-section position more.

54. according to the described signature of claim 52 storehouse, wherein, the described cultivation of mammal brain partly is the mammal brain section of display function network performance.

55. according to the described signature of claim 54 storehouse, wherein, described mammal brain section is the section of digitation of hippocamps brain.

56. according to the described signature of claim 55 storehouse, wherein, described digitation of hippocamps brain section does not have the barrier film input.

57. according to the described signature of claim 55 storehouse, wherein, described digitation of hippocamps brain section has the barrier film input.

58. according to the described signature of claim 55 storehouse, wherein, described digitation of hippocamps brain section presents the ability of myelin formation, dendritic spine and long-term potentiation.

59. according to the described signature of claim 52 storehouse, wherein, the described cultivation part of mammal brain is isolating digitation of hippocamps neuron specimen.

60. according to the described signature of claim 52 storehouse, wherein, the described cultivation part of mammal brain is barrier film and hippocampal co-cultivation thing.

61. according to the described signature of claim 51 storehouse, wherein, described storage medium is the ingredient of computer system.

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