JP3062457B2 - Analysis method for density distribution of functional glutamate receptor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing the density distribution of a functional glutamate receptor.

[0002]

2. Description of the Related Art Recent medical technology has improved the rate of cure of malignant tumors such as circulatory diseases and cancers, while nervous loss diseases such as senile dementia have been increasing. It has increased. It is known that neurological deficits associated with dementia include Alzheimer-type senile dementia and dementia due to cerebrovascular disorder. In addition, it is known that some are associated with inherited neurological diseases such as Down's syndrome, and some are of a complex type of these multiple diseases, making it difficult to elucidate the mechanism of the onset of the above-mentioned diseases. It is believed that. Furthermore, 2/3 of neurological deficits are considered to be dementia associated with cerebrovascular disease, and therefore, elucidation of the mechanism of elucidation of cerebrovascular disease is desired.

[0003] It is known that the development of cerebrovascular diseases is related to the behavior of glutamate receptors in excitatory synapses of the central nervous system. In particular, in an ion channel type receptor in which the receptor itself is a gate of calcium ions, it is known that calcium ions pass through the receptor due to binding of glutamic acid and flow into cells. Normally, action potential is transmitted by moderate excitation.However, if excessive excitation is transmitted for some reason, more calcium ions will flow into the cell than necessary, and this will cause normal information transmission in the cell. It is thought to inhibit or cause activation of proteolytic enzymes, resulting in neuronal cell death. Conversely, when the excitement of the cells can no longer be transmitted,
It is thought that cell death such as atrophy of cells occurs.

[0004]

Based on the above-mentioned facts known in the prior art, the present inventor has found that moderate activation of the glutamate receptor and consequent regulation of the appropriate intracellular calcium ion concentration are important. It is thought that what is necessary for cells and that such regulation is disrupted will cause cell death. It is known that the glutamate receptor responsible for the above-mentioned regulation has several subtypes based on its pharmacological properties. It is unknown at what location and at what density and at what timing each accepts glutamate and introduces calcium ions into cells, i.e., shares the role. We believe that it is extremely important to develop a method to obtain these findings in order to elucidate the pathogenesis of the disease and to develop therapeutic drugs.

However, in view of the above problems, conventional methods for evaluating drugs have problems. Drugs having a glutamate receptor blocking action are being developed as therapeutics for neurological deficits associated with cerebrovascular neurological deficits, cerebral infarction, cerebral apoplexy, etc. Evaluation methods often use the physicochemical interaction between an isolated and partially purified drug receptor and a drug as an index, and quickly evaluate the drug response in living cells while living cells. How to do this is not yet known. Furthermore, in the conventional method, in addition to the above, an extremely non-physiological method of simply mixing a high concentration of a drug into an extracellular culture solution is used as a drug administration method. Therefore, the method of stimulating neurons, which are highly differentiated cells not only in function but also in morphology, by such a dosage method, cannot accurately analyze various physiological functions inherent in cells. Can not. That is, when neurotransmitter receptors are exposed to high concentrations of transmitters, they become unresponsive to these transmitters (this is called receptor desensitization), and extracellular drug Alternatively, in a conventional drug administration method in which a drug solution mixed with a transmitter is brought close to and administered to a receptor by diffusion, a desensitization phenomenon is caused and the response of the receptor cannot be evaluated. Therefore, there is a strong demand for the development of a technique for administering a drug at a necessary site appropriately and with an appropriate action time while maintaining the function of the original cell.

[0006]

Means for Solving the Problems In order to satisfy the above-mentioned demands, the present inventors have made intensive studies and succeeded in developing a method for analyzing the density distribution of a functional glutamate receptor including the following steps. That is, the present invention provides (1) administering between a nerve cell an agonist in which the active site is inactivated by a protecting group (hereinafter referred to as a caged agonist), and administering a fluorescent dye for detecting calcium ions into the nerve cell; (2) irradiating the micro-region between the cells with light, (3) activating the agonist by the irradiation, administering an agonist to the micro-region, (4) intracellular based on the agonist administration Calcium ions flowing into
Quantifying based on the change in fluorescence intensity of the calcium ion detection fluorescent dye, and (5) analyzing the spatial density distribution of the functional glutamate receptor in the microregion based on the quantification. The present invention relates to a method for analyzing a density distribution of a glutamate receptor.
Furthermore, the present invention relates to the above method, wherein the agonist is glutamic acid. Here, glutamic acid inactivated by the protecting group is referred to as caged glutamic acid.

[0007] The present invention also relates to the present invention, wherein said agonist is α-
It is related to the above method, characterized in that it is amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Here, AMPA inactivated by the protecting group is referred to as caged AMPA.

[0008] The present invention further relates to the present invention, wherein
-Amino-1S3R-cyclopentanedicarboxylic acid (AC
PD) according to the above method. Here, ACPD inactivated by the protecting group is referred to as caged ACPD.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for analyzing the density distribution of a functional glutamate receptor according to the present invention will be described in detail based on embodiments.

Agonists The agonists to which the method according to the present invention can be applied include NMDA which is classically known as an ion channel type.
Type (NMDA, glutamic acid, aspartic acid, quisqualic acid, ibotenic acid), AMPA type (AMPA, glutamic acid, kainic acid, quisqualic acid), kainic acid type (glutamic acid, kainic acid), and metabolic type (glutamic acid,
Quisqualic acid, ibotenic acid, trans-ACPD). In particular, among the glutamate receptors, glutamate which is common to the ion channel type and the metabolic type, AMPA as the ion channel type AMPA type, and trans-ACPD as the metabolic type can be preferably used.

Specifically, when glutamate is used as an agonist, it can act on all subtypes of the glutamate receptor ion channel type and metabolite type. Is possible, and the transformer-
The use of ACPD allows the action of only the metabolic form.

Inactivation by Protecting Group The agonist according to the present invention is a kind of caged reagent whose active site is inactivated by a protecting group that can be dissociated by light irradiation. Therefore, by appropriate light irradiation, the protecting group is dissociated, and the agonist is activated.

In the present invention, the protecting group is not particularly limited, but from the wavelength of irradiation light, stability under physiological conditions, and the like, nitrobenzyl-type photocaged formation based on an ordinary chemical synthesis method is preferable. FIG. 9 shows some cage door gonosts which can be preferably used in the present invention.

1 Glutamate receptors in a drug cell in a nerve cell-proximal microregion are present on the cell membrane surface facing a gap called a synapse where communication between nerve cells is performed. Differences in the density and distribution of activated functional receptors present in these gaps determine the strength of the communication between nerves and the strength of the 'live' of the cells.
Therefore, an agonist whose active site has been inactivated by a protective group in advance is dosed, and the gap between the administered nerve cells is irradiated with a laser beam (approximately 1 μm in diameter) so as to have a sufficiently small irradiation area. This removes the above protecting groups and activates the agonist, thus
It is possible to give the effect of dosing the agonist over a region with a diameter of μm.

By the method described above, it is possible to suppress the desensitization phenomenon of the receptor.

2 Functional glutamate in the microdomain
Analysis of Receptor Density Calcium ions that have flowed into cells by the method according to the present invention described above are subjected to time-resolved and space-resolved changes in the intensity of fluorescence based on the calcium ion-detecting dye previously loaded into the cells. It is quantified by measuring. By the method described above, it is possible to know the density of the functional glutamate receptor in the minute region irradiated with the laser beam.

3 Map of functional glutamate receptor density
Ping By the method described in the above item 1, it becomes possible to administer the drug in the micro area, and therefore, it is possible to suppress the influence on the area other than the administered area.

Therefore, it is possible to continuously or spatially or temporally analyze the glutamate receptor density in a spatially arbitrary or continuous micro region. Furthermore, by analyzing the results of continuous measurement, it becomes possible to know the density distribution of the functional glutamate receptor around the cell surface. As a result, it is possible to analyze the strength of communication between cells and the strength of "live" of cells over time.

As a specific method of performing two-dimensional mapping, a laser beam condensed considerably smaller than this on a cell sample on which an optical microscope stage is arranged is used.
A device for irradiating while scanning in a dimension can be used.
That is, two scanners are placed in the optical path of the laser beam, and the beams are respectively shunted in the X direction and the Y direction.
On the way, the objective lens used for observation is passed through a beam expander for temporarily expanding the beam to converge the beam efficiently and a lens optical system for focusing on the sample surface. To irradiate the cells. The laser uses a pulsed UV light source (in the case of a continuous light source, a combination with a shutter is required). The decomposition of the caged reagent at one point is completed in a short time, and the cell reaction (Ca2 + in the experimental example) is completed.
In the measurement, a change in fluorescence intensity is measured. This is repeated at each point.

The range of scanning in the visual field of the microscope, the distance between irradiation points, the time interval of irradiation, and the irradiation order of each point (whether to irradiate each point in order or to randomly irradiate) are determined by the image processing apparatus. It can be set arbitrarily by an incorporated program. Although there are limitations on mechanical irradiation conditions, the distance between irradiation points is actually about the diameter of the focused beam, and the time interval is about the time when the effects of cell reactions caused by the previous irradiation are eliminated. It is considered to be. According to this device, the response of cells to the active substance released from the caged reagent can be two-dimensionally mapped. This makes it possible to analyze the distribution of molecules (receptors, enzymes, etc.) that cause a chemical reaction with the active substance for living cells, and to correlate them with cell morphology and structure. Is also possible.

4 Glutamate Receptors of Different Subtypes
Analysis of Body Density Distribution Based on the method according to the present invention, it is possible to analyze the properties of receptors for each glutamate receptor subtype. That is, for each subtype-specific agonist, each active site is inactivated by a similar protecting group, and according to the method of the present invention described above, a specific functional glutamate receptor for each subtype is obtained. It becomes possible to know the density distribution.

Specifically, when glutamate is used as an agonist, it can act on all subtypes of the glutamate receptor, such as the ion channel type and the metabolic type, and the density distribution of the functional glutamate receptor can be known. It becomes possible. Furthermore, the use of AMPA makes it possible to know the density distribution of specific functional glutamate receptors by the action of the ion channel type alone. Furthermore, when trans-ACPD is used, it becomes possible to know the density distribution of specific functional glutamate receptors by the action of only metabolites.

Hereinafter, the present invention will be described more specifically with reference to examples.

The three types of caged agonists used in the following examples are represented by the structural formula shown in FIG.

Here, caged AMPA, caged AC
PD is 4,4-dimethoxy-2-nitroacetophenone hydrazon
e is reacted with manganese dioxide to prepare a diazoethane derivative, which is then easily reacted with AMPA or ACPD to obtain a diazoethane derivative. The caged ACPD is obtained by introducing two caged reagents into one molecule of ACPD.

(Example 1) Sample preparation and apparatus 1 Preparation of cells (1) Drosophila egg collection Drosophila eggs 10 days after hatching
elanogaster ₎ and anesthetize it with CO2.
A solution prepared by dissolving 100 mg of dry yeast (Oriental yeast) in 0.7 ml of 00% grape juice (Sunkist) is 60
Transfer to a glass tube containing 3% Agarose coated with μl. Fix in a dark box and leave at room temperature for 5-6 hours to lay eggs.

(2) Treatment of eggs The eggs after laying were treated with 50% perchloric acid for 2 minutes, sterilized with ethanol, washed three times with sterile water, and prepared with a laser puller under a stereoscopic microscope. Aspirate the egg contents with a glass capillary (tip diameter: 20 μm). The contents of the 7-8 eggs are collected and transferred onto poly-L-Lysine-coated cover slips in 100 μl of culture. After 1 hour, add 0.5 ml of culture
Add and leave for 12 hours at room temperature with humidity maintained. During this standing, Fluo3-AM (ester type) is converted into Fluo3 (free type) by the enzyme of the cell itself in the cell, and can bind to calcium ions.

(3) Primary Culture of Eggs Myotube derived from Drosophila eggs was added to Solution I (128 mM NaCl, 2 mM
After washing with KCl, 35 mM Sucrose, 4 mM MgCl2, 5 mM HEPES), the solution is filled with I solution containing 10 μM Fluo3-AM (Dojin Co. Lim.) And 0.2% Plullulan, and left at room temperature for 1 hour.

After washing with the solution I again, the caged reagent-cancelling device observation * (separate sheet) is transferred to the stage.

2. Overview of the device (1) Optical system for release light irradiation Ar ion laser (Cohere
nt, Innova304, wavelength 351 nm, output 100 mW, continuous light) was introduced into an inverted microscope (Nikon, TMD300, an optical system for fluorescence and differential interference observation) 15 through an epi-illumination optical system 16. . In this optical system, the light of the xenon lamp 2 for fluorescence excitation can be introduced at the same time by the optical path vertically coupled to the optical path of the laser light 1 via the dichroic mirror 7.

The laser beam is applied only at the time of releasing the caged reagent. This irradiation time was controlled by a shutter 6 placed on the optical path. Irradiation with the excitation light was performed only during the fluorescence image acquisition time in order to avoid fading of the dye and damage to the sample, and the shutter was able to block the excitation light at other times. Excitation wavelength is bandpass filter 5
Can be selected according to the fluorescent dye used by inserting it into the optical path. In the case of this experiment, the one of 470-490 nm was used according to the characteristics of fluo3.

Both the laser light and the excitation light are reflected upward by the same dichroic mirror (510 nm) set in the microscope, and the objective lens (Nikon, Fluor 100x, oil)
The sample 9 is irradiated through 8. The resulting fluorescence passed through an objective lens 8 and a dichroic mirror 7 and was photographed by a TV camera 10 connected after its wavelength was selected by a long pass filter (> 520 nm) 5 below it.

(2) Intracellular calcium ion (Ca2 +) concentration measuring device Image analyzer for microscope (Hamamatsu Photonics, ARGUS-50) 17
It was used. With this apparatus, an arbitrary number of images can be continuously acquired at arbitrary intervals according to image acquisition conditions set in advance by an experimenter. The configuration consists of an ICCD camera that captures a microscope image, an image processing device that records and calculates images, a data analysis device (PC) that drives a program that controls image acquisition and analyzes the obtained image data, and an ICCD camera. Consists of a monitor that displays the raw image that is being taken or the image that has already been recorded.

The intracellular calcium ion concentration can be estimated based on the fluorescence intensity of the calcium ion detecting fluorescent dye introduced into the cell. The fluo3 used here is a dye that does not cause the excitation or shift of the fluorescence wavelength as the intracellular calcium ion concentration increases, but has the property of increasing the fluorescence intensity. Therefore, when a TV camera is used as a detector as in this device, the change in intracellular calcium ion concentration can be temporally and two-dimensionally determined based on the change in the fluorescence intensity of the fluorescence image obtained at a constant excitation and fluorescence wavelength. It can be monitored spatially.

3. Measurement procedure (1) Confirmation of irradiation position of release light The irradiation range of the used laser light is considerably smaller than the visual field of the microscope. About 95x90μm when using 100x objective lens
Is irradiated to about 1 μmφ with respect to the field of view. Because the irradiation position is slightly shifted depending on how the laser light is introduced into the microscope,
Before the experiment, where the laser beam is irradiated in the field of view is confirmed in advance by observing the reflected light on the metal or glass surface with a television camera. Since the cells used have a size of about 20 × 5 μm in plan view, the laser light is also applied to a small part of the cells.

(2) Determination of target cells The sample is subjected to transmission illumination using a differential interference optical system, and a cell morphological image is observed on a monitor to determine the cells to be tested and the position at which the caged reagent is to be released. . After the determination, the stage of the microscope is operated to match the position of the cell with the irradiation position of the laser beam. At this time, the morphology of the cell and the irradiation position of the light are recorded in association with each other.

(3) Acquisition of Fluorescence Image and Irradiation of Release Light The observation system of the microscope is switched to fluorescence, and a fluorescence image of the cells by fluo3 is recorded as needed by an image analyzer. Parameters for image acquisition, such as the number of images to be acquired, acquisition intervals, and integration time, are input to the program in advance. In this experiment, seven images were acquired at 10-sec intervals (total experiment time was 60 sec), and the integration time was set to 8 frames (about 0.26 sec). In addition, since this apparatus has a function of graphing and monitoring the time change of the fluorescence intensity of an arbitrary part while acquiring an image, the measurement part is set near the position where the release light is irradiated.

After the start of the image acquisition, the change in the fluorescence intensity up to the third sheet (20 seconds after the start) is monitored, and if it is confirmed that the value serving as the baseline is stable, the image before the next image acquisition is obtained. At 25 seconds after the start, the release light was introduced into the microscope only for 1/16 sec, and the cells were irradiated. At this time, in order to prevent excessive light from being incident on the TV camera and damaging the photocathode, the incident on the camera was temporarily blocked by the shutter, but these operations were performed during the image acquisition interval (10 sec). To complete, data acquisition was not affected. The obtained series of images were stored in an analyzer and used for analysis. Hereinafter, the same operation was repeated for the same cell while changing the irradiation position of the release light to obtain an image. In the present invention, as shown in FIGS. 2 and 3, by irradiating light to a specific position, the amount of calcium ions flowing into the cell can be measured at various positions on the cell (see above). 2 and 3 show the results at six arbitrary positions), so that the fluorescence measurement can be performed.

The image analysis procedure is as follows, for example.

[1] Starting the image analyzer [2] Setting image acquisition parameters (interval, number of sheets,
[3] Determination of target cell and release light irradiation position (differential interference observation, recording of image and irradiation position) [4] Switching to fluorescence observation system [5] Setting of fluorescence intensity measurement window for monitor [ 6] Acquisition of a dark image [7] Acquisition of a fluorescent image (according to the setting in [2], irradiation of release light during acquisition) [8] Saving of the image [9] Image operation (Ratio calculation R _a tio = (in −D) / (i1 ₋ D ₎ =
I _n / I1 in is the fluorescence intensity of the n-th image, i1 is the fluorescence intensity of the first image, D is the value of the dark image. [10] Saving the image after calculation [11] Data analysis (4) Data Analysis Since the change in intracellular Ca2 + concentration is reflected by the change in fluorescence intensity of fluo3, the change in fluorescence intensity at any site in the cell was analyzed using a series of image data. At this time, in order to make the amount of change in the fluorescence intensity easy to understand, the ratio of the fluorescence intensity of the second and subsequent images to the fluorescence intensity of the first image (Rati
o = In / I1) was calculated for each pixel in the image. By this method, unevenness in fluorescence intensity due to differences in cell thickness, distribution of fluo3 in the cell, and distribution of excitation light intensity can be canceled.

Example 2 Outline of Experimental Protocol and Apparatus 1. Primary culture of rat hippocampus (1). From rat brain on day 20 of embryo (plug confirmation day 20),
The brain is removed and the tunic is carefully peeled off. The left and right brains are separated from the upper brain along the midline, and the hippocampus located between the cerebral cortex and the hypothalamus is separated from the midline section using tweezers along a structure called hippocampus fimbriae. The separated hippocampus is temporarily stocked in serum-free ice-cold D-MEM (minimus essential medium, Dulbecco's modified Eagle's medium) and ice-cooled. When the required amount of tissue has been collected, gently centrifuge, remove the supernatant, add 5 ml of trypsin solution to the precipitate, and digest at 30 ° C for 15 minutes. Thereafter, a DNase I solution for degrading DNA leaking from the tissue and DNase I solution are added, and the mixture is digested at 30 ° C., and 1 ml of fetal bovine serum is added to stop the reaction. The operation of sucking up the solution with a pipette and returning the solution to the original container is repeated 5-10 times to crush the tissue. Thereafter, the mixture is filtered through a nylon mesh having a diameter of 100 μm, and the filtrate is centrifuged to remove the supernatant. The cells are washed by adding a fresh culture solution, and the supernatant is discarded after centrifugation. Next, D-MEM containing 10% fetal bovine serum is added, the cell density is adjusted to 2.4 × 10 6 cells / ml, and 300 μl is transferred onto a poly-L-Lysine-coated cover glass. One hour later, after the cells have adhered, 2 ml of the culture solution was added, and the mixture was allowed to stand at room temperature for 12 hours or more while keeping the humidity. After the standing, the culture medium is exchanged with D-MEM containing B27 additive (manufactured by Gibco) which is 1/100 amount of rat brain extract, and the culture is further continued.

2. Synthesis and Purification of Caged AMPA (1) Synthesis of diazoethane derivative 25 mg, 0.1 mmol of 1- (4,5-Dimethoxy-2-Nitrophenyl) Diaz
Oethane (DMNPE) (manufactured by Molecular Probe) is dissolved in 1 ml of DMF, 100 mg of manganese dioxide is added, and the mixture is stirred for 2 hours at room temperature. The reaction solution is filtered with a syringe equipped with a filter.

(2) Coupling reaction of diazoethane derivative with AMPA (synthesis of Caged-AMPA) In another tube, 1 m of anhydrous DMF and 16 mg (0.085 mmol) of α were added.
-Amino-3-hydroxy-5methylisoxazol-4-propionic acid
(Research Biochemicals LP; One Strathmore Road, N
atick, MA 01760-2418 USA; APMA), and the diazoethane derivative synthesized in the previous reaction was added little by little.

(3) Purification of caged AMPA The above reaction solution was applied to a Silica Gel column (2 × 20 cm) / CHCl 3, developed with CHCl 3 -MeOH (7: 1), and analyzed at UV 254 nm and Ninhydr
Spots that reacted with the rin reagent were collected. Further, fractions having a purity of 95% or more were collected by HPLC, concentrated to dryness, and concentrated to dryness.
6 (21 mg) was obtained.

HPLC conditions Column: YMC pack ODS-A (6.0 mm ID × 150 mm), A312, S-5
μ120A eluent; methanol-water (58:42) flow rate; 0.6 ml / min. detection; UV254 nm temperature; 40 ° C. Further, degradation of the obtained caged AMPA by light irradiation was examined by HPLC. As a sample, 1 μl of a 5 μM aqueous solution of caged AMPA was added onto a cover glass, and laser irradiation (conditions: argon laser 50 mW, 10 seconds, manufactured by Nikon Corporation)
Ultra-Fluorx100), add orthophthalaldehyde, and perform HPLC analysis (HPLC conditions: column; TSKGELOctade)
cyl-4p (manufactured by Tosoh Corporation), eluent: 30% methanol, 50 mM
Phosphate Buffer (pH 6.5), flow rate: 1 ml / min., Detection: fluorescence (excitation wavelength: 350 nm, observation wavelength: 445 nm), temperature: 40
° C). As a result, the peak (3.8 minutes) of only caged AMPA before irradiation disappeared, and AMPA (9.5 minutes) was newly added.
Min) and diazoethane derivative (7.4 min) peaks.

TOFMS (MALDI-4, Shimadzu Corporation)
Mass spectrometry results by dissolving in 50% DMF and using no matrix): (M + Na) + (418), ⁽ M + K) + (4
34).

1H-NMR (DMSO-d6, TMS, δ ppm) analysis result: 7.56 (2nd or 5th position of s, 1H, nitrophenyl), 7.16
And 7.12 (2nd or 5th position of s, 1H, Nitrophenyl), 6.24-6.
19 (m, 1H, benzyl position), 3.84 (methoxy group at 3 or 4 position of s, 3H, Nitrophenyl), 3.86 (3, s, 3H, Nitrophenyl 3)
Position or 4-position methoxy group), 3.90 to 3.64 (m, 1H, propio
nicacid 2nd), 2.73 ~ 2.50 (m, 2H, propionic acid 3
Methylene), 2.17 and 2.07 (methyl group at 5-position of s, 3H, Isoxazolyl), 1.55 (methyl group at Benzyl position of d, 3H, Nitrophenyl).

3. Synthesis and purification of caged trans ACPD The same procedure as in synthesis of caged AMPA of 3 was performed. However, the reagent used was α-Amino-1S3R-cyclopentane dicarboxy
lic acid (Research Biochemicals LP; One Strathmor
e Road, Natick, MA 01760-2418 USA; ACPD) 9mg (0.05mmo
l) and 1- (4,5-Dimethoxy-2-Nitrophenyl) Diazoethane
(DMNPE) (Molecular Probe) 25 mg (0.1 mmol) and manganese dioxide 100 mg.

4. General Procedure of Measurement (1) Microscope of Cell and Determination of Stimulation Position Fixing on stage, 100 × objective lens (Nikon; Fluor)
Oil immersion oil is dropped, and the power of the fluorescent observation mercury lamp is turned on, and the lamp light is projected on the sample on the stage. The Nomarski prism of the microscope is inserted into the optical path, and the sample is inspected using a differential interference image. When the observation field of view is determined, turn on the power of the calcium measuring device, and turn on the computer and the observation display. Switch the optical path to the CCD camera side and project the observation field of view on the display.
In this state, the stimulation position of the cell is determined. After determining the stimulation position of the cells, the microscope stage is operated and moved so that the stimulation position matches the irradiation position of the laser light recorded on the display. At this time, the morphology of the cell and the stimulation position are recorded in association with each other.

(2). Laser irradiation and image capture Once the recording position is determined, the calcium measurement program (Fl
uo-3; Hamamatsu Photonics) is activated, and a Nomarski image is captured with the Nomarski prism inserted. Here, the light of the mercury lamp is removed from the optical path. Next, the Nomarski prism is removed from the optical path. Image capture time
Set the sampling time interval (image capture interval) to 60-90 seconds, and adjust the sensitivity of the ARGUS-50 image processor to an appropriate value. When the preparation is completed, an imaging command is activated to start capturing an image. At the end of the second capture from the start of capture, confirm that the baseline is constant.If the base is stable, quickly change the optical path from the CCD camera to the laser during the interval until the next image capture. Switch. Here, an argon ion laser with an output of 100 mW and an irradiation time of 1/16 is applied.
Immediately after irradiation, the optical path is switched back to the camera side, and changes after irradiation are imaged. By changing the recording position and irradiating the laser in the same manner, the response of the cell is recorded.

(3) Analysis of Data Next, the imaged data is analyzed at a wavelength ratio of 360 nm / 490 nm by a command of a calcium measurement program ratio.

Measurement Example 1 (Effect of caged glutamate on primary cultured hippocampal cells) FIG. 4 shows the time course of intracellular calcium concentration at arbitrary points 1 to 4 on the cells. At the time of the arrow, light was irradiated to make glutamic acid appear. It was shown that the density of receptors at the irradiation position was different.

Measurement Example 2 (Effects of caged APMA and caged ACPD on primary hippocampal cells) Changes in intracellular calcium concentration over time at arbitrary points 1 to 4 on cells are shown. At the time of the arrow, irradiate the light, AMPA
(FIG. 5) and the change when ACPD (FIG. 6) appears. It was shown that the density of receptors at the irradiation position was different.

Measurement Example 3 (MBP-
Loss of action of 105 and MBP-106 by their respective specific inhibitors) Action when a specific competitive inhibitor for AMPA type (FIG. 7) and trans ACPD sensitive metabolic type (FIG. 8) receptors was added Was examined. The upper panel shows the coexistence of DNQX (6,7-dinitroquinoxaline-2,3-dione), an inhibitor of AMPA-type receptors, and the lower panel shows the coexistence of MCPG (α-methyl-4-carboxyphenylglycine) on ACPD, resulting in loss of action. It was investigated. The suppression of each response indicated that the synthetic product acted specifically on AMPA and metabotropic glutamate receptors.

[0055]

According to the method of the present invention, an agonist whose active site has been inactivated by a protective group in advance is administered to a gap called a synapse where a glutamate receptor is exposed in a cell, and a sufficiently small irradiation area can be obtained. By irradiating with squeezed light, it becomes possible to remove the above-mentioned protecting group, activate the agonist, and thus to give an effect of administering the agonist to a region having a diameter of 1 μm. The phenomenon can be suppressed.

The calcium ions that have flowed into the cells as a result can be measured in a time-resolved and spatially-resolved manner based on the change in the intensity of fluorescence based on the calcium ion detection dye previously loaded into the cells. It can be quantified. Therefore, the density of the functional glutamate receptor can be arbitrarily known spatially and temporally in the minute region irradiated with the light, and further, mapping can be performed.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a method for analyzing the density distribution of a functional glutamate receptor (indicating a laser light source type) according to the present invention.

FIG. 2 is a diagram showing a temporal change in the concentration of intracellular calcium of calcium ions when light is irradiated at one position, and is a diagram measured at six arbitrary points in the cell.

FIG. 3 is a diagram showing a temporal change in the concentration of intracellular calcium of calcium ions when irradiated with light at another position different from that in FIG. 2, and a diagram measured at six arbitrary points in the cell. is there.

FIG. 4 is a diagram showing a temporal change in intracellular calcium concentration in a release region when caged glutamate is released in an arbitrary local region of rat hippocampal neurons.

FIG. 5 is a diagram showing a temporal change in intracellular calcium concentration in a release region when caged AMPA is released in an arbitrary local region of rat hippocampal neurons.

FIG. 6 is a diagram showing a temporal change in intracellular calcium concentration in a release region when caged ACPD is released in an arbitrary local region of rat hippocampal neurons.

FIG. 7. Caged AMPA is applied to any local area of rat hippocampal neurons in an inhibitor of AMPA-type receptor (DN
It is a figure which shows the time-dependent change of the intracellular calcium density | concentration of the release area | region when release | release is carried out in presence or absence of QX).

FIG. 8. Caged ACPD was applied to metabolic receptor inhibitors (MCP) in any localized area of rat hippocampal neurons.
It is a figure which shows the time-dependent change of the intracellular calcium density | concentration of the release area | region when release | release is carried out in presence or absence of G).

FIG. 9 shows caged agonists, caged glutamate, caged AMPA, and caged agonists used in embodiments of the invention.
It is a figure which shows the chemical structure of caged trans ACPD.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Laser, 2 ... Fluorescence excitation light source, 3 ... Transmission light source, 4 ... Condensing lens, 5 ... Bandpass filter, 6 ...
Shutter, 7 dichroic mirror, 8 objective lens, 9 sample, 10 television camera, 11 image processing device, 12 monitor, 13 data analysis device, 14
... mirror, 15 ... microscope, 16 ... optical system for irradiating release light,
17 ... Image analysis device

──────────────────────────────────────────────────の Continued on the front page (72) Inventor Takayuki Suga 5000 Hiraguchi, Hamakita City, Shizuoka Prefecture Inside the Biophotonics Research Laboratory Co., Ltd. (72) Inventor Minoru Saito 388-201 Nakaizumi, Gunma-cho, Gunma-gun, Gunma-gun (72) ) Inventor Masahiko Hirano 5000 Hiraguchi, Hamakita-shi, Shizuoka Pref. Inside the Biophotonics Research Laboratory Co., Ltd. (72) Inventor Akihiko Watanabe 5000 Hiraguchi, Hamaguchi-shi, Shizuoka Pref. Inside the Biophotonics Research Laboratories Co., Ltd. 4-2-104 Aramaki-machi, Maebashi-shi, Gunma (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/48 G01N 33/483 G01N 33/58 G01N 33/84

Claims (4)

(57) [Claims] 1. A method for analyzing the density distribution of a functional glutamate receptor comprising the following steps: (1) administering an agonist having an active site inactivated by a protective group between nerve cells, and applying a fluorescent dye for detecting calcium ions; Administering to nerve cells, (2) irradiating the microregion between the cells with light, and (3) irradiating the agonist, the active site of which is inactivated by a protecting group, Administering an agonist to the region, (4) quantifying calcium ions flowing into cells based on the agonist administration, based on the change in fluorescence intensity of the calcium ion detection fluorescent dye, (5) the quantification Analyzing the spatial density distribution of functional glutamate receptors in the microregion based on the transformation. 2. The method according to claim 1, wherein the agonist is glutamic acid. 3. The method according to claim 1, wherein the agonist is α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 4. The method according to claim 1, wherein the agonist is 1-amino-1S3R.
2. The method according to claim 1, which is -cyclopentanedicarboxylic acid (ACPD).