JPWO2014115571A1 - D-cell visualization method - Google Patents

Abstract

A method for visualizing D-cells contained in brain tissue obtained after autopsy after human death. A method of fixing brain tissue obtained after autopsy after human death, slicing the fixed brain tissue, and inactivating peroxidase contained in the sliced brain tissue; Reacting a primary antibody obtained by reacting an animal with an antigen to brain tissue inactivated with the peroxidase; A method for visualizing D-cells, comprising: reacting a secondary antibody; and developing a color of brain tissue reacted with the secondary antibody. [Selection] Figure 1

Description

  The present invention relates to a method for visualizing D-cells, and more particularly to a method for visualizing D-cells, which are considered to be one of cells that produce a neuroactive substance that acts on a trace amine-related receptor.

The molecular mechanism of mesolimbic dopamine overactivity in schizophrenia has been a mystery.
In recent years, research on receptors for trace amines such as tyramine and β-phenylethylamine, which regulate the action of dopamine, has progressed, and trace amine-related receptor type 1 (trace amine associated with ventral tegmental dopamine neurons) -Associated receptor, type1: TAAR1) has been shown to increase the firing frequency of ventral tegmental dopamine neurons in the midbrain.
The trace amine-related receptor type 1 is positioned as a target receptor for developing a novel antipsychotic drug that improves the pathology of neuropsychiatric disorders such as schizophrenia and sleep disorders.
Compounds such as 2-azetidinemethanamine and 2-pyrrolidinemethanamine that actually act on the trace amine-related receptor type 1 are depression, anxiety disorder, bipolar disorder, attention deficit hyperactivity disorder (ADHD), Stress-related disorders, schizophrenia, neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, epilepsy, migraine, hypertension, substance abuse and metabolic disorders such as eating disorders, diabetes, diabetic complications, obesity, lipid metabolism It has been reported that it can be used to improve symptoms of abnormalities, energy consumption and assimilation, body temperature homeostasis and dysfunction, sleep and circadian rhythm disorders and cardiovascular disorders (Patent Document 1).

  D-cells, which are one of the cells that produce neuroactive substances that act on the trace amine-related receptors, were identified by Jaeger et al. In 1983 as "aromatic L-amino acid decarboxylase: AADC". = dopa decarboxylase (DDC), but do not contain monoamines such as dopamine (DA) and serotonin ”and is reported to Science as a trace amine-synthesizing cell in the rat central nervous system. Patent Document 1).

On the other hand, “D-cell hypothesis” has been proposed by Ikemoto in relation to the molecular basis of mesolimbic dopamine system overactivity in the pathology of schizophrenia (Non-patent Document 2).
FIG. 1 is a block diagram for explaining the “D-cell hypothesis”.
The outline of the “D-cell hypothesis” will be described with reference to the block diagram of FIG.
Trace amine present in the vicinity of the nucleus accumbens D-neuron due to a decrease in the number of nucleus accumbens D-neurons in the human brain due to a decrease in the function of neural stem cells in the subventricular zone of the lateral ventricle in the human brain The concentration of. This decrease in trace amine attenuates stimulation of the trace amine-related receptor type 1 on the ventral tegmental dopamine nerve ending, and increases the firing frequency of ventral tegmental dopamine neurons in the brain. And the amount of dopamine released in the nucleus accumbens increases in the brain, and the activity of the mesencephalic marginal dopamine system in the brain becomes active. Excessive activity of the mesobrain dopamine system in the brain is thought to increase D2 receptor stimulation on neural stem cells by dopamine and suppress proliferation of neural stem cells.
Conventional antipsychotic drugs that have been developed so far have been developed with the intention of blocking step 100 in FIG. 1, for example.

  However, although the presence of the D-cell is known, the D-cell may increase or decrease depending on in which part of the human brain the D-cell exists and the progression of the pathology of schizophrenia. Until now, it has been difficult to accurately grasp the basic information.

A technique for visualizing dopamine neurons in the ventral tegmental area has also been studied (Non-patent Document 3).
However, this prior art does not have a detailed reference to a method for visualizing D-cells.

JP 2010-534701 A

28. Jaeger, CB, Teitelman, G., Joh, TH, Albert, VR, Park, DH and Reis, DJ (1983) Some neurons of the rat central nervous system contain aromatic-L-amino-acid decarboxylase but not monoamines. Science, 219: 1233-1235. Keiko Ikemoto: Why D-neuron? Importance in schizophrenia research, Open Journal of Psychiatry, 2012, 2, 393-398 OJPsych doi: 10.4236 / ojpsych.2012.224055 Published Online November 2012 (http://www.SciRP.org/journal/ ojpsych /) Keiko Ikemoto et al .: Case Reports in Neurological Medicine Volume 2011, Article ID 381059

  An object of the present invention is to provide a method for visualizing D-cells contained in brain tissue obtained after autopsy after human death.

As a result of intensive studies by the inventor to solve the above problems, after the death of a human, a step of fixing brain tissue obtained after autopsy,
Slicing the fixed brain tissue;
Inactivating the peroxidase contained in the sliced brain tissue;
Reacting a primary antibody obtained by reacting an animal with an antigen against brain tissue inactivated by the peroxidase;
Reacting a secondary antibody with the primary antibody as an antigen to brain tissue reacted with the primary antibody;
Coloring the brain tissue reacted with the secondary antibody;
Having
The inventors have found that the method for visualizing D-cells meets the purpose of the present invention, and have completed the present invention.

That is, the present invention
[1] (1) fixing a brain tissue obtained after autopsy after human death;
(2) slicing the fixed brain tissue;
(3) inactivating peroxidase contained in the sliced brain tissue;
(4) reacting a primary antibody obtained by reacting an animal with an antigen against brain tissue inactivated by the peroxidase;
(5) reacting a brain antibody reacted with the primary antibody with a secondary antibody having the primary antibody as an antigen;
(6) coloring the brain tissue reacted with the secondary antibody;
including,
A method for visualizing D-cells is provided.

One of the present invention is
[2] The step (1) of slicing the fixed brain tissue is a step of slicing the fixed brain tissue to 30 to 100 μm,
The step (4) of reacting the primary antibody obtained by reacting an antigen with an animal to the brain tissue inactivated with the peroxidase is an antigen with respect to the brain tissue inactivated with the peroxidase. A step of reacting anti-animal serum, which is a primary antibody obtained by reacting aromatic L-amino acid decarboxylase protein,
The method for visualizing D-cells according to [1] above is provided.

One of the present invention is
[3] In the step (5) of reacting the primary antibody with the secondary antibody using the primary antibody as an antigen to the brain tissue reacted with the primary antibody, the brain tissue reacted with the primary antibody is labeled with biotin. Reacting with a secondary antibody anti-animal serum;
Reacting brain tissue reacted with the biotin-labeled anti-animal serum with an avidin-biotin complex in which many avidins and biotinylated peroxidases are bound;
The method for visualizing D-cells according to [2] above, comprising:

One of the present invention is
[4] The step (6) of developing the brain tissue reacted with the secondary antibody is a step of developing the brain tissue reacted with the avidin-biotin complex.
The method for visualizing D-cells according to [3] above is provided.

One of the present invention is
[5] The step (1) of fixing the brain tissue includes:
Slicing brain tissue obtained after autopsy to a thickness of 5 mm to 3 cm and immersing in a phosphate buffer solution containing paraformaldehyde for 1 to 3 days (1-1);
Dipping in a phosphate buffer solution containing sucrose and sodium azide in a range of 3 days or more and less than 20 years (1-2);
The method for visualizing D-cells according to [1] above, comprising:

One of the present invention is
[6] The step (3) of inactivating the peroxidase comprises:
The method for visualizing D-cells according to the above [1], comprising a step (3-1) of immersing the sliced brain tissue in a phosphate buffer solution containing methanol and hydrogen peroxide solution for 5 to 120 minutes. It is to provide.

One of the present invention is
[7] The step (4) of reacting the primary antibody comprises:
Anti-animal serum, which is a primary antibody obtained by reacting an animal with an aromatic L-amino acid decarboxylase protein as an antigen to brain tissue inactivated with the peroxidase, is in the range of 1-10 ° C., 7 Including a step (4-1) of reacting for 10 days,
The method for visualizing D-cells according to [2] above is provided.

One of the present invention is
[8] The group consisting of rabbit, mouse, rat, chicken, goat, sheep and guinea pig, wherein the anti-animal serum is a primary antibody obtained by reacting the animal with an aromatic L-amino acid decarboxylase protein as an antigen. The method for visualizing D-cells according to [7] above, which is derived from at least one selected from the above.

One of the present invention is
[9] The step (5) of reacting the brain tissue reacted with the primary antibody with a secondary antibody using the primary antibody as an antigen labels the brain tissue reacted with the primary antibody with the biotin. The step (5-1) of reacting the obtained anti-animal serum in the range of 1 to 10 ° C for 12 to 48 hours
The method for visualizing D-cells according to [3] above is provided.

One of the present invention is
[10] The anti-animal serum labeled with biotin is anti-rabbit serum labeled with biotin, anti-mouse serum labeled with biotin, anti-rat serum labeled with biotin, anti-chicken serum labeled with biotin, anti-biotin labeled with biotin. The method for visualizing D-cells according to [9] above, which is at least one selected from the group consisting of goat serum and anti-sheep serum labeled with biotin.

One of the present invention is
[11] The step of reacting an avidin-biotin complex in which a large number of the avidin and biotinylated peroxidase are bound,
Step of reacting a brain tissue reacted with the biotin-labeled anti-animal serum with an avidin-biotin complex in which a large number of avidin and biotinylated peroxidase are bound (0.5-2) )including,
The method for visualizing D-cells according to [3] above is provided.

One of the present invention is
[12] The step of coloring the brain tissue reacted with the avidin-biotin complex,
Performing in a solution containing tris (hydroxymethyl) aminomethane, diaminobenzidine tetrahydrochloride and aqueous hydrogen peroxide (6-1),
The method for visualizing D-cells according to the above [4] is provided.

According to the D-cell visualization method of the present invention, D-cells contained in brain tissue obtained from a human autopsy brain can be visualized easily and clearly.
From the definition described in Non-Patent Document 1, D-cells are “aromatic L-amino acid decarboxylase immunity-positive cells that do not contain DA or serotonin”. DA and serotonin biosynthetic enzymes, ie tyrosine hydroxylase and tryptophan hydroxylase, are not expressed.
Therefore, immunostaining was performed by the above method using a primary antibody against tyrosine hydroxylase and tryptophan hydroxylase, and the cells were positive for aromatic L-amino acid decarboxylase immunity. If it can be shown that the enzyme cannot be visualized, it can be proved to be a D-cell by definition.
This step is performed by applying immunodouble staining with the same and serial sections. Since DA and serotonin are rapidly oxidatively decomposed in the human postmortem brain, it is difficult to visualize DA and serotonin itself, and it is necessary to label and visualize biosynthetic enzymes that are more stable biological materials. This is because the.
Using this method, localization identification, morphological observation, and quantification of D-cells contained in the brain tissue can be performed.
By the method for visualizing D-cells of the present invention, the relationship between the pathology of a neuropsychiatric disorder such as schizophrenia and D-cells is elucidated, and the relationship between the pathology and the increase or decrease in D-cells before and after administration of a novel antipsychotic drug Can be compared.
Based on the knowledge obtained by the D-cell visualization method of the present invention, the “D-cell hypothesis” shown in FIG. 1 proposed by the present inventor can be specifically verified.
Using the D-cell visualization method of the present invention, the molecular state of schizophrenia is further elucidated in detail, the signal transduction of D-cells is analyzed, and the molecular state of other neuropsychiatric diseases is elucidated. Can be dramatically accelerated.

FIG. 1 is a block diagram for explaining the D-cell hypothesis.

  The present invention will be described in detail below based on examples. In addition, this invention is not limited at all by the following examples.

[Step of fixing brain tissue obtained after autopsy after human death (1)]
After human death, brain tissue obtained after necropsy is removed, sliced to a thickness of 1 cm, and fixed with 0.1 M phosphate buffer solution (pH 7.4) containing 4% paraformaldehyde for 1 to 3 days.
During this fixation, the 0.1M phosphate buffer solution containing 4% paraformaldehyde is exchanged once or twice with a fresh 0.1M phosphate buffer solution containing 4% paraformaldehyde.

  The fixed brain tissue is then transferred to a 0.1 M phosphate buffer solution (pH 7.4) containing 15% sucrose and 0.1% sodium azide. 0.1M phosphate buffer solution containing 15% sucrose, 0.1% sodium azide and 0.1M phosphate buffer solution containing 15% sucrose, 0.1% sodium azide once or twice Change and store in 0.1 M phosphate buffer solution containing 15% sucrose, 0.1% sodium azide for a period of not less than 3 days and less than 20 years.

[Step (2) of slicing fixed brain tissue to 30 to 100 μm]
Next, a block including a region predicted to contain D-cells is prepared, and sliced into a thickness of 50 μm with a cryostat or a microtome to prepare a sliced section. The obtained sliced sections are stored in a storage solution.

<Immunohistochemistry: (Free floating method)>
[Step (3) for inactivating peroxidase contained in sliced brain tissue]
In order to suppress endogenous peroxidase contained in the brain tissue of the sliced slices obtained, treatment with 0.1 M phosphate buffer solution containing 40% methanol and 1% hydrogen peroxide at room temperature for 20 minutes, Rinse 10 minutes × 3 times with 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl.

[Step (4) of reacting a primary antibody obtained by reacting an aromatic L-amino acid decarboxylase protein with an animal on brain tissue inactivated with peroxidase]
Although there is no limitation in particular in the animal used for this invention, if an example is given, 1 type or 2 types or more, such as a rabbit, a mouse | mouth, a rat, a chicken, a goat, a sheep, a guinea pig, can be mentioned. In this example, a rabbit was used as the animal. The same applies to the secondary antibody described later.
A primary antibody (self-specific antibody) obtained by reacting an aromatic L-amino acid decarboxylase protein with a rabbit is treated with a 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl. Dilute 1 to 70,000 times, put in a container, immerse the section in it, and allow to react for 1 week to 10 days using a shaker with gentle shaking at 4 ° C.
Here, shaking slowly refers to shaking the shaker preferably at a rotational speed of 0.2 to 2 rpm. The same applies to the following.

[Step (5) of reacting anti-animal serum labeled with biotin to the brain tissue reacted with the primary antibody]
After rinsing with a 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl for 10 minutes × 3 times, anti-rabbit serum labeled with biotin as a secondary antibody was treated with 0.3% Triton. Dilute 1000-fold with 0.01 phosphate buffer solution containing X-100 and 0.9% NaCl, in which the reaction is similarly carried out overnight at 4 ° C. with gentle shaking.

[Step of reacting avidin-biotin complex in which avidin and biotinylated peroxidase are bound to brain tissue reacted with anti-animal serum labeled with biotin]
After rinsing with a 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl for 10 minutes × 3 times, the brain tissue reacted with the biotin-labeled anti-animal serum, The mixture is reacted with a 0.01 M phosphate buffer solution containing 0.3% Triton-X and 0.9% NaCl containing an avidin-biotin complex in which a large number of avidin and biotinylated peroxidase are bound, with gentle shaking for 2 hours.

[Step (6) of developing color of brain tissue reacted with avidin-biotin complex]
After rinsing with 0.01 M phosphate buffer solution containing 0.3% Triton X-100 for 10 minutes × 3 times, the brain tissue is developed with the following reaction solution.
Reaction solution: In 50 ml of 0.05 M Tris-hydrochloric acid buffer (tris (hydroxymethyl) aminomethane and hydrochloric acid-containing buffer, pH 7.6), 0.5 g of ammonium nickel sulfate was dissolved while stirring with a stirrer. After dissolution, 5 mg of diaminobenzidine sodium tetrahydrochloride (DAB) is completely dissolved.
This solution is put into a 10 ml petri dish, and the above section is placed. Then, 3 μl of 1% hydrogen peroxide solution is dropped, and the color reaction is observed with occasional shaking under a stereomicroscope.
When the color has developed sufficiently, transfer to 0.05 M Tris-HCl buffer (pH 7.6) to stop the reaction. If the color reaction is insufficient, add 1% hydrogen peroxide to the reaction solution. If the reaction is insufficient even after observing for more than 15 minutes, transfer to a new reaction solution and try to develop color in the same way.
Thereafter, it is mounted on a slide glass coated with gelatin, dried, dehydrated and transparent, and encapsulated using enteran and cover glass.
After drying, the visualized D-cells can be observed by observing under an optical microscope.

[Comparative Example 1]
In the case of Example 1, when a similar experiment was performed using tyrosine hydroxylase protein instead of the aromatic L-amino acid decarboxylase protein, no tyrosine hydroxylase protein was present in D-cells. Therefore, D-cells cannot be visualized.

[Comparative Example 2]
In the case of Example 1, when the same experiment was conducted using tryptophan hydroxylase protein instead of the aromatic L-amino acid decarboxylase protein, no tryptophan hydroxylase protein was present in D-cells. Therefore, D-cells cannot be visualized.

According to the present invention, it is possible to easily visualize a D-cell that is considered as one of cells that produce a neuroactive substance that acts on a trace amine-related receptor.
Another possible method for visualizing D-cells is in situ hybridization, which visualizes a labeling substance as particles. However, in order to obtain information about the localization that mRNA identified as particles is present in the cell body, even if the mRNA generated when synthesizing the protein from the aromatic L-amino acid decarboxylase gene is visualized. The procedure becomes complicated. Therefore, at present, no attempt has been made to visualize D-cells.
Trace amine is an unstable biological material contained in trace amounts in the human brain and cannot be quantified in the autopsy brain after human death. Therefore, attention was paid to aromatic L-amino acid decarboxylase, which is one of the synthases and a relatively stable biological substance in the postmortem brain. At present, this is the only way to visualize trace amine neurons. In the future, if other pathways related to biosynthesis and metabolism of trace amines in humans are elucidated, it will be necessary to label biological substances other than aromatic L-amino acid decarboxylase and try the same visualization method.
By applying the present invention, it is possible to elucidate the mechanism of action at the molecular level of mesobrain dopamine system overactivity in the pathogenesis of schizophrenia and the role of D-cells in the pathogenesis of other neuropsychiatric disorders. It can provide information essential for developing new antipsychotic drugs.
In addition, the molecular level pathogenesis of schizophrenia is further elucidated using the D-cell visualization method of the present invention, the signal transduction of D-cells is analyzed, and the pathological level of other neuropsychiatric disorders is analyzed. Elucidation can also be accelerated dramatically.

  100 steps

Claims (12)

(1) fixing a brain tissue obtained after autopsy after human death;
(2) slicing the fixed brain tissue;
(3) inactivating peroxidase contained in the sliced brain tissue;
(4) reacting a primary antibody obtained by reacting an animal with an antigen against brain tissue inactivated by the peroxidase;
(5) reacting a brain antibody reacted with the primary antibody with a secondary antibody having the primary antibody as an antigen;
(6) coloring the brain tissue reacted with the secondary antibody;
including,
D-Cell visualization method. Slicing the fixed brain tissue (1) is a step of slicing the fixed brain tissue to 30 to 100 μm,
The step (4) of reacting the primary antibody obtained by reacting an antigen with an animal to the brain tissue inactivated with the peroxidase is an antigen with respect to the brain tissue inactivated with the peroxidase. The method for visualizing D-cells according to claim 1, which is a step of reacting anti-animal serum, which is a primary antibody obtained by reacting aromatic L-amino acid decarboxylase protein. The step (5) of reacting the primary antibody to the brain tissue reacted with the primary antibody comprises reacting the primary antibody with the secondary antibody labeled with biotin to the brain tissue reacted with the primary antibody. Reacting with an anti-animal serum that is
Reacting brain tissue reacted with the biotin-labeled anti-animal serum with an avidin-biotin complex in which many avidins and biotinylated peroxidases are bound;
The method for visualizing D-cells according to claim 2, comprising:   The method for visualizing D-cells according to claim 3, wherein the step (6) of developing the brain tissue reacted with the secondary antibody is a step of developing the brain tissue reacted with the avidin-biotin complex. . The step (1) of fixing the brain tissue comprises:
Slicing brain tissue obtained after autopsy to a thickness of 5 mm to 3 cm and immersing in a phosphate buffer solution containing paraformaldehyde for 1 to 3 days (1-1);
Dipping in a phosphate buffer solution containing sucrose and sodium azide in a range of 3 days or more and less than 20 years (1-2);
The method for visualizing D-cells according to claim 1, comprising: The step (3) of inactivating the peroxidase comprises:
Immersing the sliced brain tissue in a phosphate buffer solution containing methanol and aqueous hydrogen peroxide for 5 to 120 minutes (3-1),
The method for visualizing D-cells according to claim 1. Reacting the primary antibody (4) comprises:
Anti-animal serum, which is a primary antibody obtained by reacting an animal with an aromatic L-amino acid decarboxylase protein as an antigen to brain tissue inactivated with the peroxidase, is in the range of 1-10 ° C., 7 The method for visualizing D-cells according to claim 2, comprising a step (4-1) of reacting for 10 days.   The anti-animal serum, which is a primary antibody obtained by reacting the animal with an aromatic L-amino acid decarboxylase protein that is an antigen, is selected from the group consisting of rabbits, mice, rats, chickens, goats, sheep, and guinea pigs. The method for visualizing D-cells according to claim 7, which is derived from at least one.   The anti-animal labeled with biotin on the brain tissue reacted with the primary antibody in the step (5) of reacting the primary antibody with the brain antibody reacted with the primary antibody. The method for visualizing D-cells according to claim 3, comprising a step (5-1) of reacting serum in the range of 1 to 10 ° C for 12 to 48 hours.   The anti-animal serum labeled with biotin is biotin-labeled anti-rabbit serum, biotin-labeled anti-mouse serum, biotin-labeled anti-rat serum, biotin-labeled anti-chicken serum, biotin-labeled anti-goat serum and The method for visualizing D-cells according to claim 9, wherein the method is at least one selected from the group consisting of anti-sheep serum labeled with biotin. Reacting the avidin-biotin complex in which a large number of the avidin and biotinylated peroxidase are bound,
Step of reacting a brain tissue reacted with the biotin-labeled anti-animal serum with an avidin-biotin complex in which a large number of avidin and biotinylated peroxidase are bound (0.5-2) The method for visualizing D-cells according to claim 3. Coloring the brain tissue reacted with the avidin-biotin complex,
The method for visualizing D-cells according to claim 4, comprising a step (6-1) performed in a solution containing tris (hydroxymethyl) aminomethane, diaminobenzidine tetrahydrochloride and aqueous hydrogen peroxide.

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