CN102827768B - System for researching of functions of neural circuits and regulation of animal behaviors and activities - Google Patents
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Abstract
The invention relates to fields like biology, medical science, neural science, etc. and discloses a system for researching of neural circuits and regulation of animal behaviors. According to the invention, visualization of cells and selection of cells of interests in a great perspective are realized; different patterns of rapid photostimulation on sample cells disposed on a fast high-precision two-dimensional electronic-control translation table are dynamically carried out; based on recorded electrophysiological signals or behavioral characteristics and the used stimulus patterns, the system is used for analyzing a selected specific neuronal projection relationship and for researching the processes of operation and integration of neurons, thereby further revealing the functions of a great specific neural circuit and regulating animal behaviors and activities.
Description
Technical field
The present invention relates to the fields such as biology, neuroscience, medical science, relate in particular to light genetics technology Application Areas.
Background technology
Human brain is comprised of nearly hundred billion neurocyte, form network by cynapse between neurone, contact each other, transmission of information, form the systems such as sensation, motion, each system can be divided into some subsystems again, and as vision, sense of smell, the sense of hearing, the sense of taste and body surface sense of touch etc. in sensation, that the enforcement of its function depends on is dissimilar, precisely contact and the neural loop that forms between neural system different sites cell.Neural loop is the bridge between contact molecular cell function and global behavior function, the research of the neural loop of specific function is conducive to understand formation and the modification of neural loop, information coding, processing, with and and behavior between relation, thereby principle of work that can more profound understanding brain.
Neuromodulation is a kind of effective means of the neural loop of research, and interfering effects of drug is a kind of neuromodulation technology of routine, but has the problem that the drug effect time is slow, side effect is large; Electrode stimulating is a kind of neuromodulation technology commonly used, but stimulated zone is large, lack specificity and spatial selectivity, has limited their application in neural loop research.The light genetics technology is a kind of neuromodulation technology of novelty, can can't harm, high-spatial and temporal resolution, both-way operation neurone, is applicable to very much the neural circuit functionality of research, discloses contacting and mechanism of animal behavior activity and neural loop.
The light genetics technology is the product that genetics technology is combined with the light stimulus technology.From genetics technology, developed multiple photaesthesia passage, can conveniently express on culturing cell or in living animal; From the light stimulus technology, there is limitation in current light stimulus method, as adopted the wide field stimulus modality, though can activate or inhibitory neuron, but it can activate whole animal sample or whole neural loop, can't realize that specific cells or the light stimulus of a group cell selective activate; As adopted based on scanning mirror, acousto-optic deflection device, light emitting diode matrix, spatial light modulator, liquid crystal or micro mirror technology, though can realize the stimulation activating cells of high-spatial and temporal resolution, but usually must be in conjunction with being inverted or just putting on fluorescent microscope, the light stimulus that is only applicable to culturing cell, brain section activates, exist the light stimulus scope little, be not suitable for studying the behavioral activity of large neural network and regulation and control living animal.These have limited the application of light genetics technology in neural loop research.
Summary of the invention
Based on this, technical problem to be solved by this invention is to provide a kind of system of studying neural loop and regulation and control living animal behavioral activity, for studying the behavioral activity of large specific function neural network and regulation and control living animal.
For solving the problems of the technologies described above, the present invention proposes a kind of system of studying neural loop and regulation and control living animal behavioral activity, it is characterized in that, comprise the first laser apparatus, shutter, circular adjustable attenuator, coupled lens, single-mode fiber, imaging len, the first dichroscope, prism, cylinder mirror, zooming system, object lens, the two-dimentional electronic control translation stage put successively; And imaging device CCD, computer, interface circuit, shutter controller, electrophysiological recording system and translation stage controller;
The optical axis coincidence of described the first laser apparatus, shutter, coupled lens, single-mode fiber, imaging len, the single-mode fiber incident end face is positioned at coupled lens back focal plane place, and the single-mode fiber output end face is positioned at outside one times of focal length of imaging len; The first dichroscope is placed in angle of 45 degrees with imaging len optical axis, cylinder mirror optical axis respectively, described prism is between cylinder mirror and the first dichroscope, described zooming system is arranged between described cylinder mirror and object lens, and described object lens are installed on described two-dimentional electronic control translation stage top; Described imaging device CCD is installed on the first dichroscope top, and the light path of described imaging device CCD becomes miter angle with the first dichroscope, and imaging device CCD and sample meet the imaging conjugate relation, visual and cells of interest or regional choosing for cell.
Described computer connecting interface circuit, described interface circuit connects respectively described shutter controller, electrophysiological recording system and translation stage controller; Described shutter controller connects shutter, and described translation stage controller connects described two-dimentional electronic control translation stage; Described electrophysiological recording system connection electrode.
Single-mode fiber outgoing end laser facula is positioned at a Jing Qianjiaopingmianchu by real image that imaging len becomes, through cylinder mirror, zooming system, object lens, obtain a laser facula ω ' relevant to the stereoscopic microscope magnification at object lens front focal plane place, its size meets following relation:
The vertical axle ratio of enlargement that wherein α is imaging len, the magnification that M is zooming system, the magnification that β is object lens, ω
0for single-mode fiber outgoing end laser spot diameter.
Further, the present invention also comprises eyepiece, and described eyepiece is positioned at the oblique upper of described prism group, and the position of described prism can translation, the switching for the sample reflected light between eyepiece and imaging device CCD.
Preferably, described circular adjustable attenuator adopts electro-optic crystal or acousto-optic modulator to replace, and described electro-optic crystal or acousto-optic modulator are connected computer by corresponding electro-optic crystal controller with the acousto-optic modulator controller.
Described two-dimentional electronic control translation stage comprises grating scale, servomotor and feedback loop, to improve absolute fix precision and repetitive positioning accuracy.
General technical design based on identical, for providing multi-wavelength's laser to carry out light stimulus, the invention allows for a kind ofly for studying the system of neural loop and regulation and control animal behavior activity, it is characterized in that: comprise the first laser apparatus, acousto-optic tunable filter, coupled lens, single-mode fiber, imaging len, the first dichroscope, cylinder mirror, zooming system, object lens, the two-dimentional electronic control translation stage put successively; And imaging device CCD, computer, interface circuit, acousto-optic tunable filter controller, electrophysiological recording system and translation stage controller;
The optical axis coincidence of described the first laser apparatus, coupled lens, single-mode fiber, imaging len, the single-mode fiber incident end face is positioned at coupled lens back focal plane place, and the single-mode fiber output end face is positioned at outside one times of focal length of imaging len; The first dichroscope is placed in angle of 45 degrees with imaging len optical axis, cylinder mirror optical axis respectively, described prism is between cylinder mirror and the first dichroscope, described zooming system is arranged between described cylinder mirror and object lens, and described object lens are installed on described two-dimentional electronic control translation stage top; Described imaging device CCD is installed on the first dichroscope top, and the light path of described imaging device CCD becomes miter angle with the first dichroscope, and imaging device CCD and sample meet the imaging conjugate relation.
Between described the first laser apparatus and acousto-optic tunable filter, also place successively second laser, the 3rd laser apparatus, N laser apparatus and second dichroscope distinguishing with it correspondence, the 3rd dichroscope, . N dichroscope, described the second dichroscope respectively with the first laser optical axis, the second laser optical axis is placed in angle of 45 degrees, the 3rd dichroscope respectively with the first laser optical axis, the 3rd laser optical axis is placed in angle of 45 degrees, N dichroscope respectively with the first laser optical axis, N laser optical axis becomes 45 angles to place, acousto-optic tunable filter light hole center overlaps with the first laser optical axis.
Described computer connecting interface circuit, described interface circuit connects respectively described acousto-optic tunable filter controller, electrophysiological recording system and translation stage controller; Described acousto-optic tunable filter controller connects described acousto-optic tunable filter, and described translation stage controller connects described two-dimentional electronic control translation stage; Described electrophysiological recording system connection electrode.Described computer is chosen the optical maser wavelength needed by interface circuit, acousto-optic tunable filter controller, and be coupled in single-mode fiber by coupled lens, and then shine on sample by object lens, can and regulate the thorn laser power at several microsecond speed switched laser wavelength.
For reducing system cost, can adopt the spectral filter swiveling wheel to replace acousto-optic tunable filter, realize the optical maser wavelength switching.The spectral filter swiveling wheel be installed different spectral filters, by the circular wheel of electric machine control, under computer control, certain wavelength filter enters the first laser apparatus place optical axis, thus with spectral filter corresponding wavelength laser-transmitting, and other wavelength laser is reflected or absorbed and can't passes through.
The present invention has realized choosing of the visual and cells of interest of the cell under the large visual field or zone, dynamically the animal sample cell be positioned on quick high accuracy two dimension electronic control translation stage is carried out to the fast light stimulation of different mode simultaneously, according to the electricity physiological signal of record or the stimulus modality of behavioural characteristic and use, for analyzing selected specific neurone projection relation, the computing of research neurone, integration process, and then disclose the neural loop function of large specific function, regulate and control the animal behavior activity.
The accompanying drawing explanation
Fig. 1 is the structural representation of the first embodiment of the system for studying neural circuit functionality and the activity of regulation and control animal behavior.
Fig. 2 is the structural representation of the second embodiment of the system for studying neural circuit functionality and the activity of regulation and control animal behavior.
Embodiment
Below by by example, the present invention being described in further detail, but following examples are only illustrative, and protection scope of the present invention is not subject to the restriction of these embodiment.
By genetics technology, by excitability photaesthesia albumen (as ChR2) or inhibition photaesthesia albumen (as NpHR), express on the cell in the animal brain cortex.Under blue light illumination, excitability photosensitive protein (as ChR2) makes the cationic channel on cytolemma open like this, and the positively charged ions such as the outer sodium of born of the same parents, calcium enter cell, and cell produces depolarize and excitement; Under gold-tinted irradiates, inhibition photosensitive protein (as NpHR) is opened chloride channel on film, and the outer chlorion of born of the same parents enters cell and produces hyperpolarization, and cytoactive is suppressed.Different cells can the dissimilar photaesthesia passage of mark, or two kinds of dissimilar photaesthesia passages of same cell expressing, just can activate these cells with the light stimulating method subsequently, and then the neural loop or the whole animal behavior that regulate and control to have specific function.
Activate the cell that these photaesthesia passages are expressed, at first to realize that cell is visual, choose the light source of suitable wavelength with corresponding light stimulus pattern stimuli according to its spectral response curve again, analyze neural circuit functionality in conjunction with electrophysiological technique or study of behaviour register system simultaneously, and then disclose associated with animal behavior of the neural loop of large specific function, for realizing above target, need to set up a set of independently system and corresponding working method, concrete technical scheme is as follows.
As shown in Figure 1, it is the structural representation of the first embodiment of system, it comprises the first laser apparatus 1, shutter 2, circular adjustable attenuator 3, coupled lens 4, single-mode fiber 5, dual-port module 6, prism 12, three order cylinders 7, cylinder mirror 8, zooming system 9, object lens 10 and two-dimentional electronic control translation stage 11, and they are placed according to sequencing.The light path main body of cylinder mirror 8, zooming system 9, object lens 10 constituting body stereomicroscopes, wherein prism 12 and eyepiece 13 are arranged in three order cylinders 7, and prism 12 can pull out, advance, the switching for the sample reflected light between eyepiece and imaging device CCD.
The first laser apparatus 1, shutter 2, coupled lens 4 optical axis centers overlap, the wavelength of the first laser apparatus 1 should be chosen according to the curve of spectrum of expressed photaesthesia passage on cell, shutter 2 is automatically controlled and is opened or closed by circuit, the collimation laser of opening the first laser apparatus 1 output enters circular adjustable attenuator 3, circular adjustable attenuator 3 is coated with broadband dim light film, be operated in visible ray and near-infrared region, the circular adjustable attenuator 3 of axial-rotation can be regulated laser output power continuously.Available electro-optic crystal or acousto-optic modulator replace circular adjustable attenuator, and laser power is regulated can pass through computer controlled automatic.Collimation laser, after circular adjustable attenuator 3, is coupled lens 4 and converges in single-mode fiber 5.Single-mode fiber 5 incident end faces are positioned at the back focus place of coupled lens 4, and single-mode fiber 5 incident end face centers and coupled lens 4 optical axis coincidences.Laser with the single longitudinal mode transmission, shines freeboard at its output end face in single-mode fiber 5.At visible light wave range, the mode field diameter of single-mode fiber 5 is very little, common 3-9 μ m, and establishing the laser spot diameter of single-mode fiber 5 at the output end face place is ω
0.
Dual-port module 6 bottoms are processed with the dovetail-indent of standard, can directly be fixed on three order cylinders 7, and eyepiece 13 is for the eye-observation sample.Be processed with the standard C mouth on dual-port module 6 tops, corresponding imaging device CCD14 is installed; Be processed with interface in dual-port module 6 sides, fixedly single-mode fiber 5 and imaging len 15, imaging len 15 is incorporated into the laser of single-mode fiber output in dual-port module 6; Section is fixed with the first dichroscope 16 within it, the first dichroscope 16 is 45 degree and places with imaging len 15 optical axises, cylinder mirror 8 optical axises respectively, it is reflected in a mirror 8, zooming system 9, object lens 10 place light paths the laser of the first laser apparatus 1 emission, and the light transmission of other wave band.
There is a laser facula in single-mode fiber 5 output end faces, it is positioned at outside 15 1 times of focal lengths of imaging len, according to the lens imaging theory, the laser facula of single-mode fiber 5 output end faces will be in the image space of imaging len 15 real image, as size depend on that laser facula is to the distance of imaging len 15 and the focal length of imaging len.Mobile single-mode fiber 5 outgoing end faces and with the position of imaging len 15, make laser facula just be positioned at the back focal plane place of a mirror 8 by the picture of imaging len 15.According to optical principle, the laser of the first dichroscope reflection is through becoming directional light after cylinder mirror 8, through zooming system 9, be still directional light again, object lens 10 converge to its place, focal plane to the directional light of incident, as are fixed on the pallium place of animal on two-dimentional electronic control translation stage 11.
The vertical axle ratio of enlargement be set as lens 15 is α, and the magnification of zooming system 9 is M, and the magnification of object lens 10 is β, and the laser spot size ω ' of pallium place is
Regulate zooming system 9, or change the object lens 10 of different multiples, can obtain the laser facula of different size, its size and the stereoscopic microscope magnification relation that is inversely proportional to.Can reasonably choose the thorn laser spot according to cell size, the expression amount of photaesthesia albumen on cell like this, the light stimulus that obtains high spatial resolution activates.
System the first embodiment is when work, open the first laser apparatus 1, the animal of expressing the photaesthesia passage is placed on two-dimentional electronic control translation stage 11, observe the animal brain cortex by eyepiece 13, treat that in the visual field, sample is clear, electrode 17 is encapsulated on target cell or the study of behaviour recording unit fixedly with it animal.Then prism 12 is pulled out, eyepiece is unglazed passes through in left side, can only pass through the right side eyepiece.Pallium is the light that sample sends, and after object lens 10, zooming system 9, cylinder mirror 8, the prism 12 of leading up to enters the right side eyepiece, and another road enters in imaging device CCD14 through the first dichroscope 16.CCD14 is positioned at the back focal plane place of a mirror 8, so object lens 10 focal planes and CCD14 photosensitive surface meet conjugate relation, by CCD14, not only can observe sample, but also real time record sample.
The cell electrical signal collected by interface circuit 19 according to computer 18 or the movable information of study of behaviour signal and two-dimentional electronic control translation stage, analyzed, study the function of the neural loop of pallium and the behavioral activity of regulation and control animal, and then the announcement animal behavior relevant to specific neural loop is associated.
Developed at present the photaesthesia albumen of multiple different excitation wavelengths, even system the first embodiment adopts a multiple-wavelength laser, be subject to the restriction of manufacturing process, the laser of a lot of wavelength can't obtain.In addition, the first embodiment adopts shutter 2 to control light stimulus, its mode of operation belongs to mechanical movement, utmost dispatch only can reach several milliseconds, therefore light stimulus speed is slow, can't be competent at the needs that fast light stimulates, and needs to add other long wavelength laser in system for this reason, and adopt other structure control light stimulus, be illustrated in figure 2 the second embodiment structure iron.
In system, increase second laser 22, the 3rd laser apparatus ... N laser apparatus 23, the second dichroscope 24, the 3rd dichroscope ..., a N dichroscope 25, and acousto-optic tunable filter 26, they are placed successively.The second dichroscope 24 is placed in angle of 45 degrees with the first laser apparatus 1 optical axis, second laser 2 optical axises respectively, the 3rd dichroscope is also placed with the first laser apparatus 1 optical axis, the 3rd laser optical axis respectively in angle of 45 degrees .... N dichroscope 25 placed in angle of 45 degrees with the first laser apparatus 1, a N laser apparatus 23 optical axises respectively.Acousto-optic tunable filter 26 light hole centers and the first laser apparatus 1 optical axis coincidence.
During system works, open all laser apparatus, computer 18 is by interface circuit, acousto-optic tunable filter controller 27, apply the electrical signal of certain frequency, certain amplitude to acousto-optic tunable filter 26, the wavelength that acousto-optic tunable filter 26 will be corresponding with signal frequency is chosen out, and with coupled lens 4 optical axis coincidences, converge in single-mode fiber 5 through coupled lens 4, and then realize the light stimulus in cells of interest or zone through object lens.Change the signal amplitude of acousto-optic tunable filter, can regulate diffraction of light efficiency, thereby realize the stimulation of the different luminous powers in sample place.In the second embodiment, the electrical signal be applied on acousto-optic tunable filter 26 is only depended in the switching of optical maser wavelength and the adjusting of power, usually only need several microseconds, so mechanical shutter in relative the first embodiment, light stimulus speed can be brought up to 3 orders of magnitude, and therefore the second embodiment can meet the fast light stimulation activation of multiple photaesthesia protein expression cell.
For reducing system cost, can adopt the spectral filter swiveling wheel to replace acousto-optic tunable filter 26, realize the optical maser wavelength switching.The spectral filter swiveling wheel be installed different spectral filters, by the circular wheel of electric machine control, under computer control, certain wavelength filter enters the first laser apparatus place optical axis, thereby with loaded spectral filter respective wavelength laser-transmitting, go out, and other wavelength laser is reflected or absorbed and can't passes through.This scheme can only be switched for wavelength, and can't regulate laser power.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment, the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (8)
1. study neural circuit functionality and the system that regulates and controls the animal behavior activity for one kind, it is characterized in that, comprise the first laser apparatus, shutter, circular adjustable attenuator, coupled lens, single-mode fiber, imaging len, the first dichroscope, prism, cylinder mirror, zooming system, object lens, the two-dimentional electronic control translation stage put successively; And imaging device CCD, computer, interface circuit, shutter controller, electrophysiological recording system and translation stage controller;
The optical axis coincidence of described the first laser apparatus, shutter, coupled lens, single-mode fiber, imaging len, the single-mode fiber incident end face is positioned at coupled lens back focal plane place, and the single-mode fiber output end face is positioned at outside one times of focal length of imaging len; The first dichroscope is placed in angle of 45 degrees with imaging len optical axis, cylinder mirror optical axis respectively, described prism is between cylinder mirror and the first dichroscope, described zooming system is arranged between described cylinder mirror and object lens, and described object lens are installed on described two-dimentional electronic control translation stage top; Described imaging device CCD is installed on the first dichroscope top, and the light path of described imaging device CCD becomes miter angle with the first dichroscope, and imaging device CCD and sample meet the imaging conjugate relation;
Described computer connecting interface circuit, described interface circuit connects respectively described shutter controller, electrophysiological recording system and translation stage controller; Described shutter controller connects shutter, and described translation stage controller connects described two-dimentional electronic control translation stage, described electrophysiological recording system connection electrode.
2. the system of the neural circuit functionality of research according to claim 1 and regulation and control animal behavior activity, its thorn laser spot meets following relation:
The vertical axle ratio of enlargement that wherein α is imaging len, the magnification that M is zooming system, the magnification that β is object lens, ω
0for single-mode fiber outgoing end face laser spot diameter.
3. the system of the neural circuit functionality of research according to claim 1 and regulation and control animal behavior activity, also comprise eyepiece, described eyepiece is positioned at the oblique upper of described prism, and the position of described prism can translation, the switching for the sample reflected light between eyepiece and imaging device CCD.
4. according to the neural circuit functionality of the described research of one of claim 1-3 and the system that regulates and controls the animal behavior activity, it is characterized in that, described circular adjustable attenuator adopts electro-optic crystal or acousto-optic modulator to replace, and described electro-optic crystal, acousto-optic modulator connect computer by electro-optic crystal controller, acousto-optic modulator controller respectively.
5. the system of the neural circuit functionality of research according to claim 4 and the activity of regulation and control animal behavior, is characterized in that, described two-dimentional electronic control translation stage comprises grating scale, servomotor and feedback loop.
6. a system of studying neural circuit functionality and the activity of regulation and control animal behavior, is characterized in that: comprise the first laser apparatus, acousto-optic tunable filter, coupled lens, single-mode fiber, imaging len, the first dichroscope, cylinder mirror, zooming system, object lens, the two-dimentional electronic control translation stage put successively; And imaging device CCD, computer, interface circuit, acousto-optic tunable filter controller, electrophysiological recording system and translation stage controller;
The optical axis coincidence of described the first laser apparatus, coupled lens, single-mode fiber, imaging len, the single-mode fiber incident end face is positioned at coupled lens back focal plane place, and the single-mode fiber output end face is positioned at outside one times of focal length of imaging len; The first dichroscope is placed in angle of 45 degrees with imaging len optical axis, cylinder mirror optical axis respectively, and prism is between cylinder mirror and the first dichroscope, and described zooming system is arranged between described cylinder mirror and object lens, and described object lens are installed on described two-dimentional electronic control translation stage top; Described imaging device CCD is installed on the first dichroscope top, and the light path of described imaging device CCD becomes miter angle with the first dichroscope, and imaging device CCD and sample meet the imaging conjugate relation;
Described computer connecting interface circuit, described interface circuit connects respectively described acousto-optic tunable filter controller, electrophysiological recording system and translation stage controller; Described acousto-optic tunable filter controller connects described acousto-optic tunable filter, and described translation stage controller connects described two-dimentional electronic control translation stage, described electrophysiological recording system connection electrode;
Between described the first laser apparatus and acousto-optic tunable filter, also place successively second laser, the 3rd laser apparatus, N laser apparatus and second dichroscope distinguishing with it correspondence, the 3rd dichroscope, . N dichroscope, described the second dichroscope respectively with the first laser optical axis, the second laser optical axis is placed in angle of 45 degrees, the 3rd dichroscope respectively with the first laser optical axis, the 3rd laser optical axis is placed in angle of 45 degrees, N dichroscope respectively with the first laser optical axis, N laser optical axis becomes 45 angles to place, acousto-optic tunable filter light hole center overlaps with the first laser optical axis,
Described computer is chosen the optical maser wavelength needed by interface circuit, acousto-optic tunable filter controller, and is coupled in single-mode fiber by coupled lens, and then is coupled in object lens and carries out light stimulus.
7. the system of the neural circuit functionality of research according to claim 6 and regulation and control animal behavior activity, it is characterized in that, adopt the spectral filter swiveling wheel to replace described acousto-optic tunable filter and acousto-optic tunable filter controller, described spectral filter swiveling wheel connects described computer, realizes the optical maser wavelength switching under the control of computer.
8. according to the system of the neural circuit functionality of the described research of claim 6 or 7 and the activity of regulation and control animal behavior, it is characterized in that, described two-dimentional electronic control translation stage comprises grating scale, servomotor and feedback loop.
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| CN104083146B (en) * | 2013-06-25 | 2016-03-16 | 北京大学 | A kind of biological neural loop living imaging system |
| CN106073772A (en) * | 2016-06-07 | 2016-11-09 | 复旦大学附属华山医院 | A kind of animal nerve Function detection platform based on light hereditism's electrophysiological technique |
| CN112382928A (en) * | 2021-01-18 | 2021-02-19 | 北京镭科光电科技有限公司 | Laser based on VCSEL imaging and coaxial visualization design |
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