CN104846015B - The composition of GABA serotonergic neurons in special heat nucleus accumbens septi and its application in schizophrenia difference behavior is improved - Google Patents

Abstract

The present invention provides a composition for specifically exciting GABAergic neurons in the nucleus accumbens and its application in improving abnormal behaviors of schizophrenia. A viral vector carrying a light-sensing gene for neurons; the vector includes a promoter, a light-sensing gene, and a green fluorescent marker gene, wherein the promoter is a Syn promoter; an illumination device that can produce blue light is used for treating post-infection The nucleus accumbens undergoes photoregulation to excite it. The composition of the invention can precisely and specifically excite the GABAergic neurons in the nucleus accumbens, and can effectively improve the abnormal behavior of schizophrenia.

Description

Composition of specifically excitatory GABAergic neurons in nucleus accumbens and its role in improving mental Application in Abnormal Behavior of Schizophrenia

technical field

The invention relates to a composition for specifically exciting GABAergic neurons in the nucleus accumbens and its application in improving abnormal behaviors of schizophrenia.

Background technique

The epidemiological survey of mental illness in 12 regions in China shows that there are currently about 100 million people in the country suffering from mental illness, and about 16 million people with severe mental illness, and about 250,000 people die by suicide every year. Among the 340 million children and adolescents under the age of 17 in China, about 30 million suffer from emotional disorders and psychological and behavioral problems. Neuropsychiatric diseases currently rank first in the total burden of disease in my country, accounting for one-fifth of all diseases and trauma-induced disabilities and labor losses. According to experts' prediction, the 21st century will be the century of the prevalence of mental illnesses. With the acceleration of my country's economic construction and social development, the pace of life and competition are increasing, and mental illnesses will continue to increase. It is estimated that by 2020, mental illnesses will account for the total 25% of the burden will be accounted for. The World Health Organization has designated the first year of the new century as the Year of Mental Health, so whether we like it or not, we are inexorably entering the "age of mental illness".

Schizophrenia (schizophrenia) is one of the most serious mental diseases of human beings, characterized by basic personality changes, splits in thinking, perception, emotion, and behavior, and incoordination between mental activities and the environment. Epidemiological survey data show that the patients suffering from schizophrenia and neurological disorders account for about 2% of the total population in the world. Schizophrenia has the characteristics of high disability rate, high recurrence rate, poor prognosis, and heavy social burden, which seriously affects the population health and economic development of our country.

At present, the mainstream believes that the symptoms of schizophrenia patients are somehow related to the hyperactivity of dopamine in the brain. Clinically, D2-type dopamine receptor D2 (Dopamine Receptor D2, DRD2) antagonists have shown obvious therapeutic effects as drugs for treating psychosis. Conversely, repeated use of amphetamines, neuroactive substances that increase dopamine release, can induce psychosis in healthy individuals and exacerbate psychotic symptoms in schizophrenia. Similarly, administration of levodopa, the precursor of dopamine, to Parkinson's disease induces psychotic symptoms (often hallucinations and delusions) in many patients.

Dopamine drugs for schizophrenia have been used for more than 30 years now. There are both positive and negative evidences, but there is no breakthrough. Moreover, there is also a lack of direct evidence of abnormal dopamine levels in patients with schizophrenia: compared with healthy individuals, whether neurons in patients with schizophrenia release more dopamine to synapses under stimulation is still inconclusive; some studies have also shown that , patients with schizophrenia may have abnormal regulation of dopamine transport and function. Therefore, the current control of the onset of mental illness mainly relies on drug treatment and family and social support. It is impossible to specifically regulate the dopamine system in the abnormal neural circuit and cannot achieve the purpose of real treatment. It is only a "palliative".

In recent years, the role of γ-aminobutyric acid (GABA) network in schizophrenia, depression, autism, anxiety and other diseases is gradually being recognized. The latest international research evidence shows that the abnormality of GABA neurons plays a very important role in schizophrenia. The nucleus accumbens is located at the junction of the basal nucleus and the limbic system, the outer lower part of the septum, the inner lower part of the caudate putamen, the front is connected with the anterior olfactory nucleus, the subsequent bed nucleus of the stria terminalis, and the ventral globus pallidus and olfactory tubercle are located on the ventral side. Is a larger nucleus of the basal forebrain. The basic cell type of this nucleus is the medium spiny neuron. The neurotransmitter produced by these neurons is gamma-aminobutyric acid (GABA). However, whether the nucleus accumbens can be used as a target for regulating abnormal behaviors in schizophrenia has not been studied, and the prior art has not yet provided a method for specifically intervening in the nucleus accumbens to improve abnormal behaviors in schizophrenia.

Contents of the invention

The main purpose of the present invention is to provide a technology for specifically regulating targets and effectively improving abnormal behaviors of schizophrenia.

The present invention uses optogenetics regulation technology to specifically regulate the cells carrying photosensitive channel protein in the nucleus accumbens of animals with schizophrenia, and simultaneously monitors the changes in behavior. Abnormal behaviors of fear attenuation and/or anxiety, so that the nucleus accumbens is likely to be a new target for intervention of abnormal behaviors in schizophrenia.

Thus, on the one hand, the present invention provides a composition for specifically exciting GABAergic neurons in the nucleus accumbens, the composition comprising:

A viral vector carrying a light-sensing gene for infecting GABAergic neurons in the nucleus accumbens; the vector includes a promoter, a light-sensing gene, and a green fluorescent marker gene, wherein the promoter is a Syn promoter;

A lighting device capable of producing blue light is used to light-regulate the infected nucleus accumbens to excite it.

In the present invention, the lentiviral vector carrying the photosensitive gene is injected into the nucleus accumbens region, which will infect the GABAergic neurons in the nucleus accumbens region, and express the corresponding photosensitive receptors in the GABAergic neurons. GABAergic neurons expressing photosensitive receptors are stimulated by light to excite them, and after being excited by light, GABAergic neurons can improve the abnormal behavior of schizophrenia. The present invention proves through the elevated maze experiment that after stimulating the nucleus accumbens with the composition of the present invention, abnormal behaviors of schizophrenia can be significantly improved, such as congenital weakening of fear and/or anxiety, and the animal model of schizophrenia can be effectively improved. The misjudgment of factors can improve its ability to avoid related adverse environments. Therefore, the present invention provides a new therapeutic target aimed at the characteristic abnormal behaviors of schizophrenic patients who lack safety awareness, and improves their cognitive ability to avoid danger; meanwhile, it provides a new strategy for the development of new drugs.

According to a specific embodiment of the present invention, the composition of the specific excitatory GABAergic neurons in the nucleus accumbens described in the present invention can also be referred to as a "set" or "kit", which includes the composition of the present invention Reagents, materials, instruments and other components used to specifically excite GABAergic neurons in the nucleus accumbens, as a product in the form of a kit, each component can be placed separately in a large package according to a predetermined position or Individually packaged and sold as a set.

According to a specific embodiment of the present invention, the composition for specifically exciting GABAergic neurons in the nucleus accumbens described in the present invention further includes: making the virus vector carrying the light-sensing gene infect the GABAergic neurons in the nucleus accumbens. Reagent materials for neurons. Specifically, for example, it may include: a stereotaxic instrument and related sample injection system, which can accurately inject the virus vector carrying the light-sensitive gene into the nucleus accumbens region, so that it can infect the GABAergic neuron cells in the nucleus accumbens region.

In a specific embodiment of the present invention, a viral vector carrying a light-sensing gene for infecting GABAergic neuron cells or their precursor cells in the nucleus accumbens is constructed; the promoter in the vector is the Syn promoter; The sensing gene is ChR2; the green fluorescent marker gene is eYFP; the virus is a lentivirus; the specific construction process can be carried out with reference to the prior art in the field.

According to a specific embodiment of the present invention, the viral vector carrying the photosensitive gene is a lentiviral vector carrying a Syn plasmid. After the GABAergic neuron cells in the nucleus accumbens are infected with the lentiviral vector containing the Syn plasmid, the promoter Syn starts to carry the photosensitive gene to start transcription, and the corresponding photosensitive receptors are expressed in the GABAergic neuron cells in the nucleus accumbens region. After being stimulated by light, the nucleus accumbens brain area is excited, which plays a role in improving the abnormal behavior of schizophrenia. According to a specific embodiment of the present invention, the composition for improving abnormal behaviors of schizophrenia described in the present invention, wherein the lentiviral vector carrying the light-sensitive gene is a lentiviral vector containing a Syn-ChR2-eYFP plasmid , whose structure is shown in Figure 1A. In a specific embodiment of the present invention, the Syn-ChR2-eYFP can be constructed by the following method, the plasmid containing the photosensitive gene, the green fluorescent marker gene and the promoter Synapsin (Syn) for specific labeling of neurons Together with the mixed plasmids pCMVΔR8.74 and pMD2.G related to lentiviral packaging, they were transfected into 293FT cells using liposomes, and the membrane was ruptured to obtain lentiviral vectors containing Syn-ChR2-eYFP plasmids.

According to a specific embodiment of the present invention, in the composition for specifically exciting GABAergic neuron cells in nucleus accumbens according to the present invention, the blue light is blue light with a wavelength of 460-480 nm, preferably blue light with a wavelength of 470 nm.

On the other hand, the present invention provides the application of the composition that specifically excites GABAergic neurons in the nucleus accumbens in preparing a system for improving the abnormal behavior of schizophrenia. For example, the composition that specifically excites the GABAergic neurons in the nucleus accumbens is the composition of the present invention, and the application is that the composition of the present invention is used to improve the weakening of innate fear caused by schizophrenia and/or or application of anxiety systems.

According to a specific embodiment of the present invention, in the application of the present invention, the system for improving the abnormal behavior of schizophrenia is to use the virus vector carrying the light-sensing gene in the composition to infect the nucleus accumbens of the brain The GABAergic neurons of the schizophrenia were stimulated by blue light light to improve the abnormal behavior of schizophrenia.

According to a specific embodiment of the present invention, in the application of the present invention, the system further includes an optical fiber for transmitting blue light to the infected nucleus accumbens. In a specific embodiment of the present invention, a 200 μm optical fiber is used to introduce the light generated by the illuminating device into the nucleus accumbens of the brain, so as to light-regulate the infected nucleus accumbens.

According to a specific embodiment of the present invention, in the application of the present invention, the blue light illumination conditions are: the blue light wavelength is 470-480nm, the output power of the optical fiber is about 12-20mW, and the light with an illumination frequency of 40-80Hz; 1 : The duty cycle of 1 lasts for more than 5 minutes.

On the other hand, the present invention also provides a system for improving abnormal behaviors of schizophrenia, which includes the composition described in the present invention. For example, the schizophrenic abnormal behavior is attenuation of innate fear and/or anxiety.

Beneficial technical effects of the present invention: the technology of the present invention can accurately and specifically excite the GABAergic neurons in the nucleus accumbens, and can effectively improve the abnormal behavior of schizophrenia. Compared with the traditional dopamine antagonist drug treatment, the specificity is truly achieved , is of great significance.

Description of drawings

Figure 1A is a schematic diagram of the Syn-ChR2-eYFP plasmid;

Figures 1B-1C are schematic diagrams of successful expression of Syn-ChR2-eYFP by GABAergic neurons in the nucleus accumbens of mice, in which Acbc is the nucleus accumbens, and Acbsh is the shell of the nucleus accumbens;

Fig. 2 is a schematic diagram of lighting the nucleus accumbens region of a mouse by light, in which Acbc is the nucleus accumbens, and Acbsh is the shell of the nucleus accumbens;

Fig. 3 is the experimental result figure that has improved the excitability of the nucleus accumbens brain region under light conditions in embodiment 1;

4 is a schematic diagram of the results of the elevated plus maze experiment of wild-type, DISC1::ChR2 and DISC1::eYFP mice in Example 1;

Fig. 5 is a graph showing statistical results of the elevated plus maze experiment results of wild-type, DISC1::ChR2 and DISC1::eYFP mice in Example 1.

Detailed ways

In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solutions of the present invention are now described in detail in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, generally follow the conventional conditions in the field or the conditions suggested by the manufacturer.

In the following examples, the raw reagents and materials used are all commercially available, or can be prepared according to the description of the prior art.

Example 1

(1) Preparation of a lentiviral vector carrying the Syn-ChR2-eYFP plasmid.

Prepare a lentiviral vector for infecting the nucleus accumbens in living animals, which carries a Syn-ChR2-eYFP plasmid: the plasmid (0.5 μg) (the fusion plasmid structure is shown in Figure 1A), together with the mixed plasmids pCMVΔR8.74 and pMD2.G. (1.5 μg) related to lentiviral packaging, they were transfected into 293FT cells (ATCC company product, the cells need to be replaced with a new batch of cell lines after 25 passages) (6 μL liposomes per 200,000 cells). After 24 hours, the culture medium for culturing 293FT cells was replaced with serum-free DMEM containing 5 mM sodium pyruvate and continued incubation. After 16 hours, the ultracentrifuge was used to rupture the membrane at a speed of 50,000 g, the suspension was passed through a 20% sucrose filter column, and the supernatant was collected. The virus titer can reach more than 3×10 ⁸ TU/mL, and the produced virus particles are resuspended in sterilized phosphate buffer solution at a volume ratio of 1:1000. The obtained virus suspension was mixed into serum-free DMEM at a volume ratio of 1:400, and used to infect the target brain region of the animal model: nucleus accumbens.

(2) Inject the lentiviral vector carrying the Syn-ChR2-eYFP plasmid into the regulatory region to infect the cells.

The lentivirus carrying the light-sensitive gene prepared above was injected into the nucleus accumbens of the mouse using conventional stereotaxic technology in the field of neuroscience and a micro-injection system. The injection volume was generally 0.4 μL. The brain tissues of ~3 mice were identified by immunohistofluorescence staining, and the identification results showed green fluorescence, indicating that Syn-ChR2-eYFP was successfully expressed in situ (see Figure 1B and Figure 1C), and it was ready to carry out the light-regulated Behavioral detection of mice suffering from schizophrenia, wherein the mice suffering from schizophrenia are transgenic mice induced by tamoxifen (tamoxifen) to express the DISC1 mutant protein.

(3) Behavioral detection of mice suffering from schizophrenia under light regulation.

As shown in Figure 2, a commercially available optical fiber with a diameter of 200 μm was vertically inserted into the nucleus accumbens brain region of the above-mentioned mouse in which Syn-ChR2-eYFP was successfully expressed in situ, and a 470 nm blue light was stimulated with a light intensity of 1.1 mW and a light frequency of 60Hz, every 50ms irradiated for 50ms, the duty ratio was 1:1, and lasted for 5 minutes, the discharge characteristics of neurons in the nucleus accumbens brain area were extracted and analyzed by the method of in-body light-electric integrated stimulation and recording, and the results are shown in the figure As shown in Figure 3, the results in Figure 3 show that after light stimulation, the excitability of the nucleus accumbens brain region of the brain is improved, and then the elevated plus maze experiment is performed on mice (numbered as DISC1::ChR2), and wild-type mice and DISC1 Type and schizophrenia mice transfected only with the eYFP gene (coded as DISC1::eYFP) for comparative experiments, the results are shown in Figure 4, and Figure 5 shows the statistical results. In Figure 4 and Figure 5, DISC1::ChR2 represents the DISC1 transgenic mice transfected with ChR2 and expressed channelrhodopsin in the GABAergic neuron cells in the nucleus accumbens suffering from schizophrenia, and DISC1::eYFP represents the psychiatric The GABAergic neuron cells in the nucleus accumbens of schizophrenia were only infected with eYFP and expressed DISC1 transgenic mice. In the figure, ON means that light stimulation is turned on, and the way of light stimulation shown is as above, and OFF means that it is turned off Photostimulation.

The elevated plus maze (High plus maze) is to investigate the anxiety state of animals by using the animal's exploration characteristics of novel environments and the fear of high hanging open arms to form conflicting behaviors. The elevated plus maze has a pair of open arms and a pair of closed arms. Rodents tend to move in the closed arms due to their dark phobia, but they will move in the open arms out of curiosity and exploration. When stimulated, animals have the impulse to explore and fear at the same time, which causes the conflict behavior of exploration and avoidance, resulting in anxiety. However, anti-anxiety drugs can significantly increase the number and time of entering the open arm, and the cross maze is higher than the ground, which is equivalent to a person standing on a cliff, causing fear and anxiety in the experimental subjects. The maze of the present embodiment is 60 cm high from the ground, the open arm is 25×5 cm (length×width), and the closed arm is 25×5×14.3 cm (length×width×height).

It can be seen from Figure 4 and Figure 5 that the elevated plus maze experiment showed that in the OFF state before regulation, DISC1 schizophrenia mice (DISC1::ChR2 and DISC1::eYFP) had lower innate fear of open arms than For wild-type mice, the probability of entering the open arm is greater than that for wild-type mice. However, in the ON state, after optogenetics technology specifically regulates the NAc brain region (DISC1::ChR2) of DISC1 mice (injection containing optogene ChR2 in the NAc brain region, and directly stimulates the brain region with light), the comparison with wild type In the control group, there was no significant difference in the probability of entering the open arm, suggesting that the regulation took effect; in addition, compared with DISC1 mice injected with empty shell virus (DISC1::eYFP), it significantly reduced the probability of entering the open arm.

Claims (7)

1. Application of a composition that specifically excites GABAergic neurons in the nucleus accumbens in the preparation of a system for improving abnormal behaviors in schizophrenia; The composition includes: A viral vector carrying a light-sensing gene for infecting GABAergic neurons in the nucleus accumbens; the vector includes a promoter, a light-sensing gene, and a green fluorescent marker gene, wherein the promoter is a Syn promoter; The photosensitive gene is ChR2, and the green fluorescent marker gene is eGFP; A lighting device capable of producing blue light is used to light-regulate the infected nucleus accumbens to excite it. 2. The application according to claim 1, wherein the composition further comprises: a reagent material for infecting the GABAergic neurons in the nucleus accumbens with the virus vector carrying the light-sensing gene. 3. The application according to claim 1, wherein the blue light is blue light with a wavelength of 460-480nm. 4. The application according to claim 3, wherein the blue light is blue light with a wavelength of 470nm. 5. The application according to claim 1, wherein the system for improving the abnormal behavior of schizophrenia is to utilize the virus vector carrying the light-sensing gene in the composition to infect the GABAergic neurons in the nucleus accumbens, and Stimulating the infected nucleus accumbens with blue light through a lighting device to improve abnormal behaviors in schizophrenia. 6. The application according to claim 5, wherein the system for improving abnormal behaviors in schizophrenia further comprises an optical fiber for transmitting blue light to the infected nucleus accumbens. 7. The application according to claim 6, wherein the blue light illumination conditions are: the blue light wavelength is 470-480nm, the output power of the optical fiber is 12-20 mW, and the light with an illumination frequency of 40-80Hz; a duty ratio of 1:1 , for more than 5 minutes.