KR20140125659A - Method for treatment of depressive disorder with optogenetic stimulation at left medial prefrontal cortex - Google Patents

Abstract

The present invention relates to a method for treating depression by optogenetic stimulation at left medial prefrontal cortex. More specifically, it is verified that a social interaction due to stress is closely associated with left medial prefrontal cortex, and that the social interaction can be controlled by optogenetic stimulation at the left medial prefrontal cortex based on the above results. Therefore, the method for expressing channel rhodopsin at the left medial prefrontal cortex can be effectively used to treat depression using an optogenetic stimulation means.

Description

[0001] The present invention relates to a method for treating depression by mediastinal stimulation of the medial prefrontal cortex of the left brain,

The present invention relates to the treatment of photogenetic depression in the left brain, and more particularly to a method of treating depression through optogenetic stimulation of the left medial prefrontal cortex.

The mental impairment that occurs in modern societies is not caused by a great psychological impact or stimulation but rather by the weak but continuous and repetitive stress that occurs in everyday life. These stresses are easily overlooked by the hospital even though the patients themselves are not aware of it. The weak stimuli accumulate and cause depression in the individual.

Depression is an emotional pathology that occurs irrespective of the objective situation, and all of the patients' lives are covered with depressed mood, decreased in interest, become anhedonia, deteriorated in mental exercise, It is a disease that leads to suicidal ideation by feeling suicidal, and shows various physical symptoms such as decreased appetite, insomnia, constipation, loss of libido, and increased sensitivity to diseases caused by immune function deterioration.

To date, depression has been largely dependent on drug therapy. However, about 60% of patients respond to a drug, and even if the drug is used, the probability of the patient not being able to recover from the depression reaches 10%. In addition, the medication treatment is delayed because the treatment effect is delayed by starting treatment and waiting until 1 month. In such a case, the physical and psychological distress of the patient is great, . In addition, 10% of the patients who did not respond to medication were treated with classical electrical shock therapy or light cranial magnetic stimulation. Deep head stimulation (DBS) is emerging as a new treatment for these patients. Although it is limited to invasive methods in which electrodes are inserted into the patient's brain, the effectiveness of the procedure is considered to be excellent in most cases.

Optical genetics (optogenetics) is a field of biotechnology that has recently come to the fore, which means manipulating genes to control something with light. Early studies of photogenetics have been applied to regulate the activity of nerve cells using light, which means that nerve cells are activated when the light is applied to the nerve cells, and the activity of the nerve cells is inhibited when the light is not applied. By regulating the activity of neurons using light, it is possible to control the activity of neurons more precisely than controlling neurons using conventional electrical stimulation. Because electrical stimulation irritates many nerve cells indefinitely, photogenetics technology can precisely stimulate specific cells of interest.

The channel of channel rhodopsin means something passes, and the word rhodopsin is related to light. This is because when the light is received, the channels of the channel rhodopsin are opened, and something enters the cell, and when there is no light, the channel does not open. In this regard, it has been confirmed that the activity of specific cells can be regulated by regulating light to open and close channels of rhodopsin, and applied to neurons. Therefore, channel rhodopsin protein is injected into neurons to regulate the opening and closing of channels in neuronal cell membranes. When neuronal channels are opened, cations such as potassium (K) enter cells, This causes a potential difference between the inside and outside of the cell to activate the neuron.

In this regard, it has been established that certain neurons of the amygdala control the anxiety of rats by existing studies. The mice instinctively tended not to walk well in the open space due to the fear of predators and the like. When light was applied to the rat's amygdala by applying photogenetics technology, the mice showed a result of striding the space without showing an uneasy appearance . This indicates that photogenetics technology can be a new tool for the development of therapeutic agents for mental disorders such as anxiety disorders. In addition, by applying photogenetics technology to mice modeled on Parkinson 's disease, we found that the disease of these rats can be improved almost in the same way as normal rats. In addition, there is a research result of applying optical genetics technology to restore visual acuity of blind mice to basal level. This is because the retinal cells are excited by photogenetics and the signal is transferred to other cells to activate the optic nerve network. Indicating that photogenetics can also be used to activate the retinal nerve.

Accordingly, the inventors of the present invention have attempted to develop a method for treating depression that can minimize damage to the brain. As a result, the social interaction according to the stress is closely related to the left medial prefrontal cortex, , We confirmed that it is possible to control the social interaction through the optical stimulation of the left frontal lobe of the brain. We have shown that it can be useful for the treatment of depression by using the means of expressing channel rhodopsin in the left frontal lobe and using optical stimulation Thus completing the present invention.

It is an object of the present invention to provide a method for treating depression through optogenetic stimulation of the left medial prefrontal cortex, a neural cell processing method for providing control information of a left-sided brain specific social interaction using the same, The present invention also provides a method of manufacturing a mouse model having a controlled degree of accuracy.

In order to achieve the above object, the present invention provides a depression treatment kit including a vector including a channel rhodopsin gene and a laser device.

The present invention also provides a method of treating depression comprising the steps of:

1) transforming a channeled rhodopsin protein to be expressed in the left medial prefrontal cortex; And

2) irradiating the transformed left brain inner prefrontal cortex of step 1) with a laser.

The present invention also provides a neural cell processing method for providing control information of left-brain specific social interaction including the following steps:

1) transforming a channel rhodopsin protein to be expressed in the neurons derived from the left frontal lobe; And

2) irradiating the transformed neuron of step 1) with a laser.

In addition, the present invention provides a method of preparing a depression-level modulated mouse model comprising the steps of:

1) transforming a vector containing channelrodoxine or halorhodopsin into the left frontal lobe of the left ventricle of a mammal other than a human, and expressing said channelrodoxine or halorodoxine; And

2) irradiating the left prefrontal cortex of the left ventricle expressing channel rhodopsin or halorodoxine in step 1).

The method for treating depression including a vector comprising a channel rhodopsin gene and a laser device of the present invention has advantages in that the electrode size is reduced and the intensity of the electrode used is reduced as compared with the method used in conventional electrode treatment In addition, it is possible to reduce the brain damage caused by the electrode treatment and to reduce the brain damage that may occur during the treatment by irradiating the laser to only one brain as compared with the method of treating the patient by irradiating both brains with the laser Can be useful in the treatment of depression.

Figure 1 is a diagram illustrating other effects in the hemispherses of the brain in terms of social stress.
a: After the obstruction of the left or right brain, confirmation of the time change of the interaction with the presence of the social target,
b: After confirming the change in the interaction rate in the unstressed (black circle), resilient (blue circle), and sensitive (red circle)
c: Time change of interaction by electrical stimulation of 100 ㎐.
FIG. 2 is a diagram showing excited neural patterns of the left and right frontal lobe of the brain between the group of the sensitive mouse and the group of the restorative mouse.
a: In the control, sensitive, and resilient arm, the utterance unit of excited neurons in the left and right brains,
b: averaged comparison of the firing rate of excited neurons between hemispheres, and
c: Confirmation of the relationship between the left brain or right brain medial prefrontal neuron activity and social interaction.
FIG. 3 is a diagram showing the recovery of sociability in a group of sensitive mice caused by the light-induced stimulation of the left frontal lobe.
a: vector structure and immunohistochemical staining,
b: multiunit spikes responsive to 473 nm laser stimulus, and
c and d: Identification of the social behavioral effects of left-brain or right-sided prefrontal cortex stimulation in stressed mice.
Fig. 4 is a graph showing inhibition of excitatory neuronal activation of the medial prefrontal lobe in resilient mice.
a: vector structure and immunohistochemical staining,
b: inhibition of neuronal activity by halorhodopsin, NpHR (3.0)
c and d: Behavioral responses to social targets in stressed and restored mice, and
e: A proposed model for the lateralized role of the medial prefrontal cortex in the regulation of sociality.

Hereinafter, the present invention will be described in detail.

The present invention provides a depression treatment kit comprising a vector comprising a channel rhodopsin gene and a laser device.

The channel rhodopsin is most preferably the channel rhodopsin-2 described in SEQ ID NO: 1, but it can be used if it is a protein capable of opening and closing the ion channel of the cell membrane in response to exposure to laser.

The vector is preferably selected from the group consisting of a linear DNA vector, a plasmid DNA vector, and a recombinant viral vector, but is not limited thereto. In addition, the recombinant viral vector is preferably selected from the group consisting of retrovirus, adenovirus and adeno-associated virus. However, according to a preferred embodiment of the present invention, it is more preferable that the recombinant viral vector is adeno-associated virus.

According to a preferred embodiment of the present invention, the vector is preferably composed of CaMKIIa promoter, channel rhodopsin, and mCherry. In the channel rhodopsin, the 134th histidine from the N-terminal is more preferably substituted with arginine But is not limited thereto.

In addition, the depression treatment kit may further include an optical fiber, but the present invention is not limited thereto. According to a preferred embodiment of the present invention, the optical fiber is a ferrule having a length of 0.02 mm But is not limited thereto.

In the use of the depression treatment kit, the vector having channel rhodopsin is preferably administered to the medial prefrontal region, and is preferably administered intravenously at a dose of from about 0.02 to about 0.04 cm lateral, from 0.2 to 0.5 cm from the breeding, More preferably 0.2 cm, and most preferably about 0.03 cm, 0.23 cm, and 0.15 cm, respectively, but not always limited thereto. The intensity of light required for stimulation is preferably 1 to 2 mW, but is not limited thereto.

In addition, the depression treatment kit preferably includes but is not limited to instructions for use.

In a preferred embodiment of the present invention, the inventors of the present invention found that a group of mice having social avoidance and a group of mice having no significant social avoidance to confirm the relationship of social interactions according to stress to the left brain As a result of performing a behavioral test using the mouse, it was confirmed that the stress-induced social avoidance in the mice having the left frontal intra-prefrontal lesion decreased the interaction time between the mice and became more sensitive thereto (see FIGS. 1A and 1B).

In addition, in order to confirm the relevance of the social interaction according to the electrical stimulation with the left brain, the present inventors investigated the change of social interaction after stimulating the medial prefrontal lobe of each of the left brain and the right brain with electrodes. As a result, (FIG. 1C), while the mice receiving the electrical stimulation of the left intraventricular prefrontal cortex did not have social avoidance.

In addition, the inventors conducted electrophysiological recording to confirm the role of the medial prefrontal hemispheres in stress sensitivity. As a result, in the case of obtaining stress due to the medial prefrontal cortex of the right brain, a sensitive mouse and a resilient mouse No differences could be found (see Figs. 2A and 2B).

In addition, the present inventors used optogenetic method to increase the activity of stagnation in the left intraventricular prefrontal cortex in order to determine whether the stimulation of the left interphalangeal prefrontal cortex can regulate social interactions. As a result, Sensitive mice showed a general increase in sociability, but did not show a tendency to recover from the sensitive rats that increased stunted neuronal activity in the right frontal lobe (see Figures 3B and 3D).

In addition, immunohistochemical staining of the excitatory neurons of the left frontotemporal prefrontal cortex by optical stimulation confirmed the overexpression of channelrodoxine (see FIG. 3A), whereas when using halorodoxine, It was confirmed that stimulation could suppress the neural activity of the medial prefrontal cortex of the left brain (see FIG. 4).

Thus, it was confirmed that the vector containing the channel rhodopsin gene and the laser device of the present invention can be effectively recovered from stress-induced depression by significantly activating neuronal activity in the left frontal lobe.

The present invention provides a method of treating depression comprising the steps of:

1) transforming a channeled rhodopsin protein to be expressed in the left medial prefrontal cortex; And

2) irradiating the transformed left brain inner prefrontal cortex of step 1) with a laser.

In addition, the present invention provides a neural cell processing method for providing control information of left brain-specific social interaction including the following steps:

1) transforming a channeled rhodopsin protein to a neural cell derived from the left medial prefrontal cortex; And

2) irradiating the transformed neuron of step 1) with a laser.

Preferably, the modulation of the social interaction is performed on a target having an extremely low social interaction ratio of less than 0.15, but the present invention is not limited thereto. It is preferable to use the optogenetic method, Not limited.

The channel rhodopsin is most preferably a channel rhodopsin-2 represented by SEQ. ID. NO. 1, but it can be used as a protein capable of opening and closing ion channels of a cell membrane in response to exposure to a laser.

The transformation may be carried out by any method selected from the group consisting of transformation, transfection, electrophoresis, transduction, microinjection and balliatic introduction. And it is most preferable to use transduction according to the preferred embodiment of the present invention, but the present invention is not limited thereto.

In addition, the laser irradiation is preferably 1 to 2 mW, but is not limited thereto.

In a preferred embodiment of the present invention, the present inventors confirmed that when irradiated with a laser after injecting channel rhodopsin or halorodopsin, the effect of increasing or inhibiting specific brain activity of the left brain was demonstrated, And to provide neural cell processing method to provide control information of left brain specific social interaction.

The present invention also provides a method of preparing a depressed level modulated mouse model comprising the steps of:

1) transforming a vector containing channelrodoxine or halorhodopsin into the left frontal lobe of the left ventricle of a mammal other than a human, and expressing said channelrodoxine or halorodoxine; And

2) irradiating the left prefrontal cortex of the left ventricle expressing channel rhodopsin or halorodoxine in step 1).

The above-mentioned depressive level is preferably used to activate neurons using channel rhodopsin, and to inhibit neurons using halorodopsin, but the present invention is not limited thereto, and the neuron processing method uses an optogenetic method But is not limited thereto. In addition, the channel rhodopsin is preferably channel rhodopsin-2 described in SEQ ID NO: 1, and halodorocycline is preferably halodiposide described in SEQ ID NO: 2, but is not limited thereto. According to a preferred embodiment of the present invention, it is preferable that the optical fiber has a length of 0.02 mm in the center of 200 탆 in the form of a ferrule, But is not limited thereto.

The transformation may be carried out by any method selected from the group consisting of transformation, transfection, electrophoresis, transduction, microinjection and balliatic introduction. And it is most preferable to use transduction according to the preferred embodiment of the present invention, but the present invention is not limited thereto.

In the laser irradiation, the vector having channelrodoxine or halorodoxine is preferably administered to the medial prefrontal region, and it is preferable to administer the medial prefrontal region in an amount of from 0.02 to 0.04 cm in lateral, 0.2 to 0.5 cm in brema, More preferably 0.2 cm, and most preferably about 0.03 cm, 0.23 cm, and 0.15 cm, respectively, but not always limited thereto. The intensity of light required for stimulation is most preferably 1 to 2 mW in the case of channeled rhodopsin, or 1.5 to 2.5 mW in the case of halolodopsin, but is not limited thereto. In addition, in the case of including rhodopsin in the above step 1), it is preferable to irradiate a blue laser beam of 473 nm in step 2), but not limited thereto, when halodiodine is contained in step 1) It is preferable, but not limited, to irradiate a green laser of 532 nm.

In a preferred embodiment of the present invention, the present inventors confirmed that when irradiated with laser after injecting channel rhodopsin or halorodoxine, the effect of increasing or inhibiting specific brain activity in the left brain was demonstrated, It is possible to provide a manufacturing method of a model.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> Social Interaction with Left Brain and Stress social interaction )

<1-1> Preparation of experimental animals

In the present invention, male C57B / 6J mice (13-15 weeks old) were used in all examples. The breeding and manipulation of experimental animals was carried out in accordance with the instructions of the Animal Care and Use Committee of KINS. The mice were free to consume food and water, and were maintained in an environment where day and night were repeated for 12 hours.

<1-2> Effects of chronic social defeat stress on social interactions in mice

Behavioral tests were performed using mouse group with social avoidance and group without significant social avoidance to confirm that chronic social defeat stress induces a decrease in social interaction in mice.

Specifically, to prepare mice with chronic social defeat stress, mice anesthetized with avertin (Sigma, USA) were fixed in a stereotaxic device (KOPF instrument) and an electrolytic lesion (Bregma +0.23, ML ± 0.003, depth of 0.25 cm from the smoke film) at the junction of sagittal and coronal sutures. A monopolar stainless electrode (A-M system 564410) was used and 1 ㎃ was poured into a stimulus isolator (WPI, A365) for 15 seconds. One week after recovery, the mice were given chronic social defeat stress for 10 days, and at the end of the stress schedules, the stressed mice underwent a social interaction test. The social interaction test was carried out in a dark room box (42 x 42 x 42 cm) with wire netting (6 x 6 x 10 cm). The social interaction test results were recorded in a digital file and the time in the interaction zone was analyzed by Ethovision 3.1 (Noldus). The social preferences index was obtained by the time ration consumed in the interaction area (when there is no target / no target). In addition, to visualize electrolyte disturbances, the brains of the mice were obtained and fixed in 4% formaldehyde. The cooled portion was prepared to a thickness of 30 [mu] m and cresyl-violet staining was performed. Comparing damaged areas with mouse brain atlas (The Mouse Brain 2nd edition, Academic pression 2001), if prematurity area (M2), ventral orbital cortex (VO) or corpus callosum are affected by electrolyte disturbances If received, the mice were excluded from the experimental results.

As a result, stress-induced social avoidance in mice with left-sided prefrontal cortex lesions as shown in Figs. 1A and 1B resulted in a decrease in the social interaction time of the mice, thereby increasing sensitivity to social interactions, Stress-induced social avoidance in the mice having an increased social interaction time of the mice showed resilience (Figs. 1A and 1B). In addition, mice with impaired right ventricular medullary prefrontal lobe may be deficient in stress hormone response as long as the medial prefrontal lobe mediates the secretion of stress hormones when stressed.

< Example  2> Confirming the relevance of social interactions due to left brain and electrical stimulation

In addition to chronic social defeat stress, changes in social interactions were observed when stimulated with the inner prefrontal cortex of the left or right brain, respectively, with an anode electrode.

Specifically, the electrode stimulus was applied to the target location at an angle of 20 [deg.] To minimize damage: Bregma +0.23 mm, side to side 0.1, depth from the smoke 0.18 cm. The electrode for the stimulation was a custom-made SNEX-100 (Rhode Medical) and adjusted to the brain of the mouse (0.1 mm inside exposure, 0.1 mm external exposure and 0.2 mm cross-exposure interval). The electrodes were fixed to prevent movement and dropping to three sites of the skull (two temporal and one cerebellum) and were also fixed with dental cement. The experimental mice were well adapted to wire attachment for 5 minutes after having a wire. During the first 3 days, it was well adapted to the social interaction test box for 15 minutes. After 3 minutes adaptation from 4th to 14th day, a new CD1 mouse was placed in the iron glass of the box. After the experimental mouse was placed in the box, electrical stimulation was applied. A bipolar square pulse (20 ms wide, 100 Hz, 20 uA) was applied by a stimulus insulator (WPI, RBMA-200C) and NII (National instrument, Noldus) 1 second. On the last day, at least 6 hours after the last stimulus, the probe test is performed in a hexagonal probe black box (28 × 28 × 28 × 28 × 28 × 28 cm) with black and white lines on the background wall . Wire netting (6 × 10 × 10 cm) was placed on one corner of the box, and a weak Raven flavor was added before the experiment.

As a result, as shown in FIG. 1C, experimental mice experiencing electrical stimulation in the right frontal medial prefrontal lobe in a social environment showed a decrease in social interaction and behavioral effects similar to those of social defeat. On the other hand, experimental mice that experienced electrical stimulation in the left frontal lobe of the brain did not have social avoidance (Fig. 1C).

< Example  3> In stress sensitivity, medial prefrontal hemisphere ( hemispheres Check the role of

Chronic social defeat shows the result of behavioral spectrum expression in stressed mice despite similar stress hormone levels between stressed and untreated mice. This means that there is an independent metabolic process for the recovery of stress sensitivity or stress awareness. Therefore, the role of medial prefrontal hemispheres in stress sensitivity was confirmed by electrophysiological recording.

Specifically, a probe test was carried out, and mice were fixed by urethane anesthesia (1.5 g / kg) in a stereotaxic apparatus from day 5 to day 10, (Homothermic blanket system, Harvard Apparatus). Quartz-coated tetrodes (0.5 to 2 MΩ, THOMAS Recording) coated with quartz were placed on the left and right medial prefrontal lobe (distance from the braggam (mm), +1.7, ± 0.3, 1.3 to 2.5: Anterior-posterior (AP), medio-lateral (ML), and dorsal-ventral (DV) axes (aexs). To determine the recording position, the tip of the quadrupolar tube was slightly immersed in a fluorescence dye solution (Dil, 50 ㎎ / ㎖, Sigma) before passage through the tissue, and electrode tracing was performed in the brain slice And visualized with a confocal microscope using a rhodamine filter. The signal was amplified using an AC amplifier (PGMA, THOMAS Recording) and sampled at 30 kHz (DT3010, Neuralynx). To measure the multi-unit activity, 300 to 6000 And filtered at 1.52 to 50 Hz to measure the localization potential (㎐) or local-field potential. The resulting spike sorting was performed with SpikeSort3D (Neuralynx) and the units were classified as excitatory or inhibitory by waveform analysis.

As a result, as shown in Figs. 2A and 2B, the firing rate of the excitatory neurons in the left hemisphere of the sensitive mouse was significantly decreased when a sensitive mouse, a restorative mouse, and a negative control group were compared Respectively. On the other hand, the right hypothalamic prefrontal activity of stressed mice (both sensitive and resilient mice) was higher than negative control, but no difference between sensitive and resilient mice (FIGS. 1A and 1B). In addition, the firing rate of the left intraventricular neurons in the left brain was proportional to their sociability in each mouse, but the firing rate of the right inner brain prefrontal neurons was not related to sociability (FIG. 2C). These results indicate that the stress experience was similar in the sensitive or resilient mice in the acquisition of the right ventral medial prefrontal mediator, but not in the behavioral differences of the two mouse groups.

< Example  4> Left anterior medial frontal Optic stimulation  Confirming the moderating effect of social interaction through

<4-1> Optical stimulus  Medial prefrontal cortex through the left Stagnant  Increased neuronal activity

To observe whether the observed activity stagnation of the left intraventricular prefrontal cortex was functionally related to the sensitive mouse, we observed the social behavior after increasing the stunted neuronal activity in the left frontal lobe of the brain through the optogenetic method.

More specifically, 4.09 × 10 ¹³ particles / ㎖ of AAV2 station having a horizontal CaMKIIa.hChR2 (H134R) -mCherry vector (vector core Penn, USA) / 9 virus and 4 × 10 ¹² particles / ㎖ the inverse horizontal rAAV2 / 5 / AAV2 / 9 and halorhodopsin virus with the CaMKIIa-eNpHR3.0-eYFP vector (Penn vector core, USA) were used with slight manipulation. Socially defeated mice to be used in the experiment were selected as the time to stay in the interaction area when social objects were present, and were classified into the sensitive group when staying less than 40 seconds and the resilient group when staying more than 90 seconds. One of the AAV2 / 9-CaMKIIa.hChR2 (H134R) or rAAV2 / 5-CaMKIIa-eNpHR was injected into each of the medial prefrontal regions [lateral ± 0.03 cm, braguma 0.23 cm, ventral 0.15 cm] . Optic fiber (200 μm center, 0.2 mm length) was inserted at the site of administration in the form of a ferrule (D280-2350, Doric, Canada) and fixed to the surface of the skull with screws and dental cement . The procedure was performed within 48 hours after the end of chronic defeat stress, and social interaction tests were performed 10 days after surgery. The next day, the experimental mouse was placed in a 10 minute orientation to familiarize with the fiber optics to which the FC / PC connector was connected. Optic stimulation (10 ㎐, 5 ms) and stimulating insulator (S48, Grass) were then used with a 473 nm blue laser (DSSPL, Shanghai laser) for ChR2 detection and NpHR detection It was used to stimulate a 532 nm green laser (DSSPL, Shanghai laser). The intensity of the light was used up to 1 ㎽ for the ChR2 experiment and 1.3 ㎽ for the NpHR (3.0) experiment. Prior to all light stimulus behavior test, light intensity was measured with an optical power meter (Thorlabs, Germany). In addition, the virus administration and light stimulation positions were confirmed by post-mortem examination, and the mice used in the experiment had seizures or abnormal motor behaviors (e.g., muscle switch, head nodding, Or jumping], it is considered that the optical fiber is inserted at the wrong position. In the analysis of the experimental data, the result of the mouse was excluded. As a control group, mice that underwent the same surgery except laser stimulation were used.

As a result, a sensitive mouse that received a high frequency stimulus (10 ㎐) (Fig. 3B) on the left frontal lobe without spike adaptation increased the time consumed in the interaction zone and the general social level (or interaction rate) Respectively. On the other hand, the sensitized mice that received the light-mediated stimulation on the medial prefrontal cortex did not show a tendency to recover (Figs. 3C and 3D). These results confirmed that the left frontotemporal neurons are important for stress - mediated changes in sociability.

<4-2> Optical stimulus  Confirming the increase in the firing rate of excitatory neurons in the medial prefrontal lobe of the left brain

Immunohistochemical staining was used as a positive control to determine whether the ignition points of excitatory neurons increase in the left and right frontal lobes of sensitive mice with extremely low social interaction rates (less than 0.15).

Specifically, the brain was cut into 50 쨉 m thickness with a vibratome (Leica, VT1000S) after 12 to 16 hours with ice cold heparin sprinkled and fixed in 4% formaldehyde. Immunohistochemical staining was performed using an anti-cfos antibody (Santa Cruz, USA) and a cy5 or cy3 labeled anti-rabbit secondary antibody (Jackson Immunoresearch, USA), and the signal of the brain slice was analyzed using a confocal microscope LSM 710).

As a result, as shown in Fig. 3A, the structure of the vector was shown on the upper side, and immunohistochemical staining was performed on the lower part. In the left brain of a sensitive mouse having an extremely low social interaction ratio of less than 0.15 It was confirmed that CaMKII promoter-driven channel rhodopsin (CaMKII promoter-driven channel rhodopsin) was overexpressed to increase the firing rate of excitatory neurons in the right frontal lobe of the prefrontal cortex (FIG. 3A).

<4-3> Optical stimulus  Suppression of neuronal activity of the medial prefrontal cortex through left ventricle

The light-dependent chloride channel CaMKII promoter-driven halorhodopsin (CaMKII promoter-driven halorhodopsin, NpHR3 (SEQ ID NO: 2)) in resilience mice was prepared using the methods of Examples 3 and 4 above. .0) suppressed neuronal activity in the left and right brains, and then social behavior was observed.

As a result, as shown in FIG. 4A, the structure of the vector was shown on the upper side, and immunohistochemical staining results were shown on the lower side (FIG. 4A). In addition, the restorative mouse inhibited the activity of the left frontal nerve prefrontal neurons, leading to severe social avoidance (Fig. 4B), whereas inhibition of the right inner nerve prefrontal neurons did not result in significant social behavior changes (Figs. 4C and 4D). These results indicate that, in resilient mice, social avoidance, such as a consequence of chronic social defeat, can be suppressed by increased neuronal activity of the left prefrontal cortex.

Thus, FIG. 4E shows the role of the medial prefrontal cortex in functional responses, stress adaptation, and stress sensitivity in the action of the stress response confirmed by the inventors of the present invention (FIG. 4E).

<110> Korea Advanced Institute of Science and Technology <120> Method for treatment of depressive disorder with optogenetic          stimulation at left medial prefrontal cortex <130> 13P-03-03 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 684 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > channelrhodopsin-2 (H134R) <400> 1 tcgttgctca ggccggtgag gttgctcagg cggataagga tgacaggaca agtgagcagc 60 cactctgcat agcgcagcca ctgcacccgg tgtcctgtgg caaggtagag catagaggga 120 ttcttaaact caaaaaagaa ctcgagaatc accttaacca tttcaatggc gcacacatag 180 atctcctccc agccgcatgt agatttccag gtttggtagg catagaacat cagcagcaaa 240 atgctgaatc ctgctgcaag ccactgcagg acatttgacg cggtctgagc gccgttcgtg 300 ccgcgagatt caatccatcc ggcacagtaa cattgatcct cagggaccag gacggacccg 360 ttcaccacca caggattagt aacgaacaaa agttcgcgtc cgacggcaga caaagcgccg 420 ccatagtcca tggtggccta ccggtacccg gggatccggt accgctctag agctgccccc 480 agaactaggg gccactcgcc tgcccgtgct cctgagtgca aacggagaac cggcttgact 540 gacgagcttg gggcttctga gcagggcact gtggctgctc acagcctcgc gagccttcgt 600 cagcatccag gtccccatgg caaccacctc cgaaacgccc tgttcccgtt gccccggtaa 660 ctgcctcccc aacacctgcc tgcc 684 <210> 2 <211> 725 <212> DNA <213> Artificial Sequence <220> <223> Halorhodopsin <400> 2 gcacaaggat ggttgacaca tcgggtcata aaaacgaaca gcagtatact cagtcctgca 60 agtgcgatgt tgatatagag actgcttgca agcaaagggt cgttcagcac gaactcgaac 120 aactcccttt gggtaacctc ggcttgaagg gccacggcac tctcggtcac gggaggcagg 180 gtctctgtca tggtggccta ccggtacccg gggatccggt accgctctag agctgccccc 240 agaactaggg gccactcgcc tgcccgtgct cctgagtgca aacggagaac cggcttgact 300 gacgagcttg gggcttctga gcagggcact gtggctgctc acagcctcgc gagccttcgt 360 cagcatccag gtccccatgg caaccacctc cgaaacgccc tgttcccgtt gccccggtaa 420 ctgcctcccc aacacctgcc tgcctttcac tttaaaacct gctcctcttt gccctggcct 480 tatatacaca atactgcagg actgtgtggg cccaggagca agtggaccct gttcccccag 540 ggtgatgtac tgagggggtt ggagaagggg atgcgaacaa acttagtcca caagtgatca 600 tcgctcttcc tacctccccc acctccaact atagggccct tttaacagac gggatgggat 660 taagtggggc aaagaggcag ttgctatggt aacggctagt gggtgccaca ttctgggatg 720 gtcct 725

Claims (14)

A depression treatment kit comprising a vector comprising a channel rhodopsin gene and a laser device.
The therapeutic kit for depression according to claim 1, wherein the channelrodoxine is channelrodoxine-2 described in SEQ ID NO: 1.
2. The kit for depression according to claim 1, wherein the vector is selected from the group consisting of a linear DNA vector, a plasmid DNA vector and a recombinant viral vector.
4. The kit of claim 3, wherein the recombinant viral vector is selected from the group consisting of retrovirus, adenovirus, and adeno-associated virus.
2. The depression treatment kit of claim 1, wherein the depression treatment kit further comprises an optical fiber.
3. The depression treatment kit of claim 1, wherein the depression treatment kit further comprises instructions for use.
1) transforming a channeled rhodopsin protein to a neural cell derived from the left medial prefrontal cortex; And
2) irradiating the transformed neuron of step 1) with a laser beam to provide information on control of the left-brain specific social interaction.
The method according to claim 7, wherein the channel rhodopsin of step 1) is channel rhodopsin-2 described in SEQ ID NO: 1.
8. The method according to claim 7, wherein the transformation of step 2) is performed by transformation, transfection, electrophoresis, transduction, microinjection, and balliatic introduction ). &Lt; / RTI &gt;
8. The method of claim 7, wherein the laser irradiation in step 2) is at most 1 to 2 mW.
1) transforming a vector containing channelrodoxine or halorhodopsin into the left frontal lobe of the left ventricle of a mammal other than a human, and expressing said channelrodoxine or halorodoxine; And
2) irradiating the left prefrontal cortex of the left frontal lobe with rhodopsin or halodipodin expressed in step 1) to a laser with a controlled degree of depression.
12. The method of claim 11, wherein the laser irradiation in step 2) is performed by inserting an optical fiber into the left frontal lobe of the brain.
12. The method of claim 11, wherein when the step includes the channel rhodopsin in step 1), a blue laser beam of 473 nm is irradiated in step 2).
12. The method of claim 11, wherein when step (1) comprises haloaldoxine, a green laser of 532 nm is irradiated in step (2).

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