KR102150417B1 - Sleep promoting animal model by optogenetic stimulation of astrocytes in the ventrolateral preoptic area and method for screening sleep control agents using the same - Google Patents

Abstract

The present invention relates to a sleep-inducing animal model through optogenetic stimulation of astrocytes in a ventrolateral preoptic nucleus (VLPO) and a method for screening a sleep control agent using the same. In order to regulate cells, an adenovirus containing a light-responsive gene regulated by a cell-specific (GFAP) promoter was injected into the brain of a rat to express the photoreactive gene in astrocytes. Astrocytes were stimulated by implanting an optical fiber tube and irradiating blue light through the installed optical fiber tube in order to transmit light stimulation into the brain. Through the present invention, it was confirmed that light stimulation of astrocytes in the ventral lateral vision area can induce an increase in sleep, and it has been confirmed that it can induce the activity of glia cells and neurons. Therefore, the technology developed through the present invention can be usefully used to study the regulatory action of astrocytes in sleep induction, as well as the possibility of developing a stellate cell-centered treatment strategy for controlling sleep and treating sleep-related diseases. present.

Description

Sleep promoting animal model by optogenetic stimulation of astrocytes in the ventrolateral preoptic area and method for screening sleep control agents (Sleep promoting animal model by optogenetic stimulation of astrocytes in the ventrolateral preoptic area and method for screening sleep control agents) using the same}

The present invention relates to a sleep-inducing animal model through optogenetic stimulation of astrocytes in a ventrolateral preoptic nucleus (VLPO) and a method for screening a sleep control agent using the same.

Maintaining nerve activity is an energy consuming process, and sleep is essential to recovering energy. In addition, sleep is associated with a number of brain functions, including memory consolidation and synaptic plasticity. Sleep is regulated by two processes. One is circadian rhythm and the other is sleep propensity, also known as sleep pressure, and is dependent on pre-brain activity during waking. In this regard, sleep stimulation based on sleep desire requires both "sleep-on" and "wake-off" signals at the same time, thereby promoting mutual inhibition between sleep and arousal centers. Plays a pivotal role in promoting sleep. In particular, the ventral lateral vision area (VLPO) regulates several arousal centers of the brain through GABA inhibitory projection, which is one of the major nuclei that promote sleep under sleep pressure. Indeed, lesions in VLPO cause insomnia and impairment of NREM sleep. In a group of neurons in VLPO, expression of the c-Fos protein is particularly increased during sleep, but not during waking.

There is growing evidence that astrocytes interact with adjacent neurons in response to appropriate stimuli, such as neuronal activity, through the release of neuroactive substances (called gliotransmitters), including glutamate, ATP, D-serine and GABA. As a result, astrocytes can regulate several physiological brain functions such as synaptic transmission, respiration, perception, learning, and memory. In addition, astrocytes play an important role in sleep homeostasis and cognitive decline associated with sleep disorders. Changes in sleep homeostasis can be mediated by the release of ATP from astrocytes and hydrolysis of ATP to adenosine and is dependent on the adenosine A ₁ receptor. Indeed, adenosine is believed to be an intrinsic sleep-promoting substance, and extracellular adenosine concentrations in several brain regions increase with life propensity.

Adenosine accumulates during waking, and the somnogenic effect of adenosine applied externally in several brain regions, including VLPO, has been reported. However, the mechanism for the sleep-promoting effect of endogenous adenosine in VLPO has not been identified.

Korean Patent Publication 10-2018-0022150 (published on March 6, 2018)

An object of the present invention is a method for manufacturing a sleep-induced animal model comprising the step of optogeneically stimulating astrocytes in a ventrolateral preoptic nucleus (VLPO) of the brain, a sleep-induced animal model prepared by the method, and It is to provide a method for screening a sleep control drug used.

The present invention provides a method of manufacturing a sleep-induced animal model comprising the step of optogeneically stimulating astrocytes in a ventrolateral preoptic nucleus (VLPO) of an animal other than humans.

In addition, the present invention provides a sleep-inducing animal model other than humans produced by optogenetic stimulation of astrocytes in the ventrolateral preoptic nucleus (VLPO) of the brain.

In addition, the present invention comprises the steps of administering a sleep control drug candidate to a sleep induction animal model; And it provides a method for screening a sleep control drug comprising the step of selecting a drug candidate exhibiting a sleep control effect compared to the control substance administered animal group.

The present invention relates to a sleep-inducing animal model through optogenetic stimulation of astrocytes in a ventrolateral preoptic nucleus (VLPO) and a method for screening a sleep control agent using the same. In order to regulate cells, an adenovirus containing a light-responsive gene regulated by a cell-specific (GFAP) promoter was injected into the brain of a rat to express the photoreactive gene in astrocytes. Astrocytes were stimulated by implanting an optical fiber tube and irradiating blue light through the installed optical fiber tube in order to transmit light stimulation into the brain. Through the present invention, it was confirmed that light stimulation of astrocytes in the ventral lateral vision area can induce an increase in sleep, and it has been confirmed that it can induce the activity of glia cells and neurons. Therefore, the technology developed through the present invention can be usefully used to study the regulatory action of astrocytes in sleep induction, as well as the possibility of developing a stellate cell-centered treatment strategy for controlling sleep and treating sleep-related diseases. present.

1 shows the results that when the rat is in a waking state, optogenetic stimulation of VLPO astrocytes induces sleep. A, The location of the cannula used to stimulate VLPO astrocytes was visually shown, and the results of quantifying the ChR2-Kat1.3 expression profile at the VLPO site are shown. B, a fluorescence image of the VLPO site is shown. This is the result of quantification of ChR2-Kat1.3 and GFAP-positive cells around C and VLPO sites. D, shows the photostimulation effect during 12:30-14:30 (La). E, shows the photostimulation effect during 15:30-17:30 (Lb). F, frontal and parietal EEG (FC EEG and PC EEG, respectively) before and during photostimulation (Lb), motor activity-related vibrations and hypnograms are shown.
2 shows the results that astrocytes release ATP during optogenetic stimulation. A, shows a schematic diagram of microdialysis of extracellular fluid during photostimulation at the VLPO site. B, ATP concentration of dialysate (extracellular fluid, ECF) before and after photostimulation (120 minutes) is shown. Photostimulation (120 min)-induced changes in the levels of D-serine, glutamate and glycine in microdialysates obtained at C and VLPO sites. D, shows the extracellular ATP concentration measured in astrocyte-conditioned medium (ACM), which was collected after photostimulation of the cultured astrocytes for 120 min. E, on the release of ATP from cultured astrocytes, it shows the effect of Brilliant Blue G (BBG, a P2X7 receptor antagonist, 100 nM) or carbenoxolone (CBX, a hemichannel blocker, 3 μM).
3 shows the results that optogenetic stimulation of astrocyte ChR2 reduces the excitability of NE-DP VLPO neurons. A, Records of voltages measured before, during and after 100 μM NE application in the same neuron (top panel), and voltage records before, during, and after photostimulation of astrocyte ChR2 (473 nm, middle panel) Represents. The bottom panel shows the voltage response records before and during photostimulation on an enlarged time scale. B, shows NE-induced changes in AP frequency (left) and membrane potential (right). C, shows photostimulation-induced changes in AP frequency (left) and membrane potential (right). D, change of membrane potential by ATP (100 μM, n = 8) or adenosine (Ade, 100 μM, n = 17) and DPCPX (1 μM, selective adenosine A ₁ receptor antagonist, n = 16) or TNAP- Shows the photostimulation-induced change in membrane potential in the presence of I (10 μM, TNAP inhibitor, n = 6). AP frequency changes induced by E, ATP (100 μM, n = 6) or adenosine (Ade, 100 μM, n = 15) and DPCPX (1 μM, n = 16) or TNAP-I (10 μM, n = 7) Shows the photostimulation-induced AP frequency change in the presence.
4 shows the result that optogenetic stimulation of astrocyte ChR2 increases the excitability of NE-HP VLPO neurons. A, Records of voltages measured before, during and after 100 μM NE application in the same neuron (top panel), and voltage records before, during, and after photostimulation of astrocyte ChR2 (473 nm, middle panel) Represents. The bottom panel shows the voltage response records before and during photostimulation on an enlarged time scale. B, shows NE-induced changes in AP frequency (left) and membrane potential (right). C, shows photostimulation-induced changes in AP frequency (left) and membrane potential (right). D, change in membrane potential by ATP (100 μM, n = 7) or adenosine (Ade, 100 μM, n = 10) and DPCPX (1 μM, selective adenosine A ₁ receptor antagonist, n = 12) or TNAP- Shows the photostimulation-induced change in membrane potential in the presence of I (10 μM, TNAP inhibitor, n = 7). AP frequency changes induced by E, ATP (100 μM, n = 6) or adenosine (Ade, 100 μM, n = 8) and DPCPX (1 μM, n = 12) or TNAP-I (10 μM, n = 7) Shows the photostimulation-induced AP frequency change in the presence.
5 shows the results that optogenetic stimulation of VLPO astrocytes decreases GABAergic transmission to NE-HP VLPO neurons. A, Current recordings of astrocyte ChR2 in NE-HP neurons before photostimulation (473 nm), during photostimulation, and after photostimulation are shown. B, shows the cumulative probability distribution for the internal-event interval (left, p <0.01, KS test) and current amplitude (right, p = 0.17, KS test) of GABAergic sIPSCs. C, Current recordings of astrocyte ChR2 in the presence of 1 μM DPCPX before photostimulation (473 nm), during photostimulation, and after photostimulation are shown. D, Photostimulation-induced change of GABAergic sIPSC frequency according to the presence or absence of DPCPX. E, the results of immunohistochemical analysis for GAD67 (red), TNAP (green) and galannine (GAL, white) at the VLPO site are shown. The results of quantification of immune response cells to GAD67, TNAP, and galatin at F and VLPO sites are shown.
6 shows the results that optogenetic stimulation of VLPO astrocytes reduces the excitability of TMN histaminergic neurons. A, Astrocyte ChR2 before photostimulation (473 nm), during photostimulation, after photostimulation, and current analysis results recorded in histaminergic neurons are shown. B, shows the cumulative probability distribution for the internal-event interval (left, p <0.01, KS test) and current amplitude (right, p = 0.78, KS test) of GABAergic sIPSCs. C, Current recordings measured from histaminergic neurons before, during, and after photostimulation of astrocyte ChR2 in the presence of 1 μM DPCPX (473 nm) are shown. D, Photostimulation-induced change of GABAergic sIPSC frequency according to the presence or absence of DPCPX. E, voltage records measured before and during photostimulation (473 nm) of astrocyte ChR2 in histaminergic neurons are shown. F, it shows the photostimulation-induced change in AP frequency (a) and film potential (b).
Figure 7 shows the astrocyte-mediated sleep induction mechanism in the VLPO site proposed in the present invention.
8 shows a schematic diagram of an expression vector including a channelrhodopsin-2 (ChR2) gene, which is a light-activated ion channel used in the present invention. A, a schematic diagram of Ad-sGFAP-ChR2 (H134R)-Katushka1.3 (Ad-ChR2), an adenovirus vector containing a ChR2 structure, is shown. Expression of ChR2 is regulated by GfaABC1D, a shortened version of the GFAP promoter. The actuation of mCMV in the antisense direction regulates the chimeric transcriptional activator Gal4p56, and the specificity for expression in both directions is measured by GfaABC1D. A schematic diagram of AAV-GFAP-hChR2(H134R)-eYFP(AAV-ChR2), which is an adeno-associated viral vector containing B, ChR2 construct is shown. Expression of ChR2 is regulated by GfaABC1D, a shortened version of the GFAP promoter.

The present invention provides a method of manufacturing a sleep-induced animal model comprising the step of optogeneically stimulating astrocytes in a ventrolateral preoptic nucleus (VLPO) of an animal other than humans. Preferably, the animal may be a rat, but is not limited thereto.

Preferably, the step of optogeneically stimulating astrocytes in VLPO comprises: 1) transforming astrocytes in VLPO with an expression vector containing a light-activated ion channel gene, and the photoactivation Expressing an ion channel; And 2) photostimulating astrocytes in the VLPO, but is not limited thereto.

In addition, the present invention provides a sleep-inducing animal model other than humans produced by optogenetic stimulation of astrocytes in the ventrolateral preoptic nucleus (VLPO) of the brain. Preferably, the animal may be a rat, but is not limited thereto.

Preferably, the optogenetic stimulation transforms astrocytes in VLPO with an expression vector containing a photoactivated ion channel gene, thereby expressing the photoactivated ion channel, and photostimulating astrocytes in the VLPO. , But is not limited thereto.

In the present invention, optogenetics is one of the biotechnology fields that have recently been in the spotlight, and it means controlling something with light by manipulating genes. Early research in optogenetics was applied to control the activity of nerve cells using light, which refers to a technology that activates nerve cells when light is irradiated to them. In this way, by using light to control the activity of nerve cells, you can control the activity of nerve cells more precisely than to control nerve cells using conventional electrical stimulation. This is because, while electrical stimulation stimulates a large number of unspecified nerve cells, optogenetic technology can accurately stimulate specific cells of interest. Accordingly, an object of the present invention is to transform a photoactivated ion channel capable of activating astrocytes by the photostimulation under the control of an astrocyte-specific promoter.

In the present invention, the light-activated ion channel is a group of membrane-permeable proteins that open and close pores by light stimulation. In the present invention, the photo-activated ion channel can be used without limitation as long as it is a protein capable of opening an ion channel of a cell membrane in response to exposure to a light stimulus, and channelrhodopsin, halohodopsin, or alkyrodopsin Proteins such as (archaerhodopsin) can be used. Preferably, in one embodiment of the present invention, channelrhodopsin-2 (ChR2) was used. On the other hand, channelrhodopsin-2 used in the present invention is a variant in which the 401th base of genbank number EF47017 (ChR2) is point mutated to guanine, and is also indicated as ChR2 (H134R).

The channel of the channel rhodopsin means that something passes, and the word rhodopsin is related to light. This has the characteristic that when it receives light, the channel of channel rhodopsin opens and something enters the cell, and the channel does not open without light. Therefore, the channel rhodopsin-2 protein was injected into astrocytes to control the opening of the channel in the astrocyte cell membrane with light, and when the channel of the astrocyte is opened, cations such as potassium (K) enter the cell into the cell. , As a result, a potential difference between the inside and outside of the cell occurs, and the astrocyte is activated.

In the present invention, the method of transforming astrocytes with a photoactivated ion channel may use a transformation method known in the art without limitation, and non-limiting examples thereof include transformation, transfection, and It can be selected from the group consisting of electrophoresis, transduction, microinjection, and balliatic introduction.

In the present invention, the expression vector may be selected from the group consisting of a linear DNA vector, a plasmid DNA vector, and a recombinant viral vector, but is not limited thereto, and all conventional vectors used for transformation in the art are the methods of the present invention. Can be used for

Preferably, the expression vector used in one embodiment of the present invention may be an adenovirus vector or an adeno-associated viral vector, and more preferably, the expression vector is a ChR2 structure Ad-sGFAP-ChR2 (H134R)-Katushka1.3 (Ad-ChR2), an adenovirus vector comprising, or AAV-GFAP-hChR2 (H134R)-eYFP (AAV-ChR2), an adeno-associated viral vector comprising a ChR2 structure ), but is not limited thereto. A schematic diagram of the Ad-ChR2 and AAV-ChR2 is shown in FIG. 8.

In the present invention, the optical stimulation is preferably irradiated with blue light having a wavelength of 450 to 480 nm, and the irradiation time for the optical stimulation is 30 to 120 minutes, and it is preferable to perform optical stimulation once to three times. On the other hand, the method of irradiating the light stimulation is not particularly limited, and the animal may be immobilized and the light may be irradiated from the outside, and the VLPO may be directly irradiated with light stimulation by implanting an optical fiber into the VLPO. The wavelength of the light may vary depending on the type of photoactivated ion channel to be transformed, and the optimal light wavelength may be easily selected by a person skilled in the art through repeated tests.

In addition, the present invention comprises the steps of administering a sleep control drug candidate material to the sleep-inducing animal model; And it provides a method for screening a sleep control drug comprising the step of selecting a drug candidate exhibiting a sleep control effect compared to the control substance administered animal group. Preferably, the sleep control drug may be a narcolepsy therapeutic agent, a stimulant agent, or a sleeping agent, but is not limited thereto.

In the present invention, the type of the sleep control drug candidate is not particularly limited, and preferably natural products, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, metabolites and bioactive molecules of bacteria or fungi It may be selected from the group consisting of.

The method of administering the sleep control drug candidate to an animal can be used without limitation in the art of administering the drug to an animal, for example, intraperitoneal administration, oral administration, intravenous administration, negative administration, intrathecal administration, etc. Can be used. The timing of administration of the candidate substance may be immediately after irradiation with light stimulation to within 1 day, but is not limited thereto, and depending on the type of the candidate substance, the sleep control effect may be evaluated by administering a drug in advance before irradiation with light stimulation . The timing of administration, route of administration, administration interval, and dosage of the drug can be applied by a person skilled in the art by easily selecting and applying optimal conditions through preliminary experiments according to the kind, nature, and mechanism of action of the candidate substance.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each of the examples according to the present invention.

1. Experimental animals

All experiments were conducted according to the recommendations of the Kyungpook National University Ethics Committee, and every effort was made to minimize the pain of animals used in the experiments. All animals were reared in Plexiglas cages (28 × 42 × 18 cm) at 21-24° C. and 12:12 h in a controlled environment. Animals were fed standard commercial diet, and water was freely available except on the day of recording. Except where specifically indicated, adult male Sprague Dawley (SD) rats (275-300 g, 8-12 weeks old) were used for all behavioral studies and microdialysis experiments. There was no difference in the number of animals between the start and end of the invention. All relevant data shown in the present invention were obtained from the inventors.

2. Electrophysiological experiment

(1) tissue section culture

Newborn SD rats (3-5 days old, regardless of gender) were sacrificed under ketamine (100 mg/kg, ip) anesthesia. The brain was cut, and cold artificial cerebrospinal fluid (ACSF; in mM: 120 NaCl, 2 KCl, 1 KH ₂ PO ₄ , 26 NaHCO ₃ , 2 CaCl ₂ , 1 MgCl ₂ ) using a microslicer (VT1000S; Leica, Nussloch, Germany) , and 10 glucose, saturated with 95% O ₂ and 5% CO ₂ ) in cross section with a thickness of 400 μm. Sections containing the VLPO site were placed on a membrane insert (diameter, 30 mm; Millicell-CM, Millipore, Billerica, MA, USA) and transferred to a 6-well culture plate containing 1 ml culture medium per well. Then, adenovirus vector [Ad-sGFAP-ChR2(H134R)-Katushka1.3, Ad-ChR2, 1 μl; titer 1.6 × 10 ¹¹ PFU/ml] was carefully injected into the VLPO site using a micropipette under a binocular microscope. The culture medium consisted of 50% minimum essential medium, 25% Hank's balanced salt solution, 25% heat-inactivated horse serum (all from Sigma, St. Lois, MO, USA) and 1 mM glutamine. To bring the final concentration to 10 mM, glucose was added to the medium. Before the experiment was performed, section culture was maintained at 37° C., 5% CO ₂ and 100% humidity for 2-3 days. Thereafter, the section was transferred to the recording chamber, and the VLPO site was confirmed under an upright microscope (E600FN; Nikon, Tokyo, Japan) equipped with a submerged objective lens (×40). As a subset of the experiment, brain slice sagittal planes containing VLPO and TMN were cultured in the same medium.

(2) Preparation of acute section

Young adult male SD rats (3-4 weeks old) were peritoneal anesthesia using a cocktail (2 ml/kg) of ketamine hydrochloride (72.6 mg/kg) and xylazine hydrochloride (5.1 mg/kg), and stereotactic fixation. Placed on the device (stereotaxic frame). Adeno-associated viral vector [AAV5-GFAP-ChR2(H134R)-eYFP, AAV-ChR2, 1 μl; in the lower region of VLPO; titer 1.6 Х 10 ¹² PFU/ml] was microinjected. 7-10 days after microinjection, rats were sacrificed under anesthesia with ketamine (100 mg/kg, ip). The brain was cut and sectioned transversely or sagittally at 350 μm thick in cold artificial cerebrospinal fluid using a microslicer (VT1000S). For at least 1 hour prior to electrophysiological recording, the brain section cross-sections or fibroblasts were stored in ACSF and maintained at room temperature (22-25° C.) with 95% O ₂ and 5% CO ₂ . Thereafter, the section containing both VLPO or VLPO and TMN was transferred to a recording chamber, and VLPO neurons or histaminergic neurons were identified under an upright microscope (E600FN) equipped with a submerged objective lens (×40). Images were acquired using a digital microscope camera (ProgRes® MF; Jenoptik, Yena, Germany or DS-Ri1; Nikon, Tokyo, Japan).

(3) Whole cell patch clamp recording

All electrophysiological measurements were performed using a computer-controlled patch-clamp amplifier (Axopatch 200B; Molecular Devices; Union City, CA, USA). For whole cell recording, borosilicate capillary glass (1.5 mm outer diameter, 0.9 mm inner diameter; G-1.5; Narishige, Tokyo, Japan) using a pipette puller (P-97; Sutter Instrument Co., Novato, CA, USA). ) To make a patch pipette. The resistance of the recording pipette filled with the internal solution (in mM; 140 K-gluconate, 10 KCl, 2 EGTA, 2 Mg-ATP, and 10 HEPES, pH 7.2 with Tris-base) was 2-5 MΩ. The liquid-to-liquid contact potential was corrected. In a subset experiment, Alexa 594 (Alexa Fluor dye 594 hydrazide sodium salt; Thermo Fisher Scientific Korea Ltd. Seoul, Korea) was added to the pipette solution to a final concentration of 50 μM. Pipette capacitance and series resistance were compensated by 50-70%. For current-clamp recording, the amplifier was switched to current-clamp (I-CLAMP) mode with series resistance compensation. Through this, the same effect as the'bridge balance' of other amplifiers could be obtained. Membrane voltage or current was low-pass filtered at 2-5 kHz and acquired at 10-20 kHz (Digidata 1440; Molecular Devices). All experiments were conducted in a temperature range of 22-24°C. The water bath was perfused with ACSF at 2 ml/min using a peristaltic pump (MP-1000; EYELA, Tokyo, Japan).

(4) Photostimulation of ChR2

The optical fiber tube (200 μm inner diameter, 0.37 NA) connected to the laser light source (blue laser [473 nm]; SDL-473-100MFL; Shanghai Dream Lasers Technology Co., Shanghai, China) is used with a micromanipulator. Placed on the surface of the VLPO site. Photostimulation (500 ms duration) was stimulated at a frequency of 1 Hz (500 ms on/off) using a stimulator (SEN-7203; Nihon Kohden, Tokyo, Japan), and applied for 3-5 minutes. The maximum light intensity at the end of the fiber optic tube was 1.7 mW (PM204; Thorlabs Co., Newton, NJ, USA).

(5) data analysis

APs and GABAergic sIPSCs were counted and analyzed using pClamp 10.3 (Molecular Devices) or MiniAnalysis program (Synaptosoft, Inc., Decatur, GA, USA). The frequency of sIPSCs or APs during the control period (5-10 min) and photostimulation period (3-5 min) was calculated for each condition. The effect of photostimulation was quantified through the percentage change in the frequency of APs or sIPSCs, and the membrane potential was compared with the control value. Using standardized control values, numerical results were expressed as mean±SEM, except for those indicated separately.

(6) drugs

The drugs used in the present invention are ATP, Bz-ATP, adenosine, DPCPX, suramin, PPADS, ARL67156, BBG, CBX (Sigma Aldrich, St. Louis, MO, USA), SR95531, 2MeCCPA, CGS 21680, BAY 60-6583 , HEMADO (Tocris, Bristol, UK) and 2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (TNAP inhibitor, TNAP-I, from Merck Millipore, Darmstadt, Germany). All drugs were administered via bath application.

(7) single cell RT-PCR

After whole cell patch clamp recording, the recorded neuronal constructs containing the mRNA were carefully aspirated through a recording pipette. Then, the material harvested in the patch pipette was transferred to a PCR tube. Reverse transcription and the first PCR reaction were performed in the same tube using a one-step RT-PCR kit (Qiagen, Hilden, Germany). Primers used for RT-PCR are as follows. galanin (1st) 5'-tggctcctgttggttgcaacc-3'/5'-gtggtctcaggactgctctag-3', (2nd) 5'-aacagcgctggctaccttctg-3'/5'-tcttctgaggaggtggccaag-3' (product size 251 bp), GAD67 (1st) 5 '-gtgtgctgctccagtgttctg-3'/5'-gaagttggccttgtccccttg-3', (2nd) 5'-tgcaaccagatgtgtgcaggc-3'/5'-ttgatcttgggagccaccctg-3' (product size 350 bp), histidine decarboxylase (HDC) (1st) 5'-atctgccagtacctgagcacc-3'/5'-ggcaacaagacgagcgttcag-3', (2nd) 5'-atgtgaagcctgggtacctgc-3'/5'-ggactcatcagcattgggctc-3' (product size 440 bp) and GAPDH (1st) 5'-catcttccaggagcg'/5'-cagtgagcttcccgttcagct-3', (2nd) 5'-tggagtctactggcgtcttcac-3'/5'-gatgcagggatgatgttctggg-3' (product size 350 bp). Secondary nested PCR was performed using a GoTaq ^ⓡ DNA polymerase (Promega), it was used for each of the first round PCR product as a template (2 μl). The amplified PCR product was electrophoresed on a 2% agarose gel to which RedSafe ^TM Nucleic Acid Staining Solution was added, and the gel was then photographed.

3. Molecular science , Cytology and histological studies

(1) in vivo (in vivo) virus gene transfer

Adult rats were anesthetized with 2-4% isoflurane (Baxter, Deerfield, IL, USA) and placed on a stereotaxic frame. An adenovirus vector (Ad-ChR2; generously provided by Dr. Kasparov at University of Bristol, Bristol, UK) was microinjected into the lower region of VLPO in one or both directions as follows. Antero-posterior (AP) -0.4 mm from the bregma, lateral (L) ±1.1 mm and ventral (V) -8.5 mm from the dura. Meanwhile, an adeno-associated virus vector (AAV-ChR2; University of North Carolina Gene Therapy Program Vector Core) was similarly injected into the same VLPO site. After microinjection, the wound was closed. Postoperative treatment was performed immediately, and animals were recovered for 7 days before the experiment to confirm the high expression level of the transgene. In all behavioral experiments using viral vectors (Ad-ChR2, Ad-eGFP, AAV-ChR2, and AAV-eYFP), expression of related proteins in the VLPO site was confirmed through fluorescence signals after each experiment. The results of behavioral experiments in which no fluorescence signal was detected in the VLPO site were excluded.

(2) Confocal image analysis of Ca ^{2 +} reaction in cultured astrocytes

After infection with Ad-ChR2 or AAV-ChR2, a calcium marker Fluo-4 or rhodamine 2 was used for primary cultured astrocytes. Images were obtained using a confocal microscope equipped with a submerged objective lens (x40). To activate ChR2, a 488 nm Argon laser was used and measured using a bandpass filter (505-550 nm). The laser intensity and intensity were kept to a minimum (at 0.5-0.7% of laser output).

(3) Microdialysis

Adult rats were anesthetized using isoflurane and fixed to a stereotaxic frame. Of the two guide cannulaes, one was for photostimulation and the other was for microdialysis, which was implanted at the VLPO site. The tip of the guide cannula was placed 1.0 mm below the target. AP -0.4 mm from the bregma, L ±1.1 mm and -8.5 mm from the dura. They were fixed to the skull with 3 anchor screws using dental adhesive. The cannula for photostimulation was inserted at an angle (20° angle), and the microdialysis probe cannula was inserted vertically into the VLPO. After cannula implantation, it was recovered for 7 days in the breeding room. 24 hours before the start of microdialysis, a microdialysis probe (CMA Microdialysis AB, Stockholm, Sweden) was inserted into the VLPO through a guide cannula. The probe was connected to a polyethylene tubed microperfusion pump and perfused with artificial cerebrospinal fluid at a flow rate of 0.5 μl/min. Extracellular fluid (ECF) from the end of the tube outlet was collected in a plastic vial kept on ice. Samples were collected for 120 minutes at 60 minute intervals. The collected samples were immediately frozen at -80°C and maintained until analysis of ATP, adenosine and other neurotransmitters. For some experiments, ECF was collected after administration of an astrocyte metabolism inhibitor (L-α-AA, 10 nM; Sigma Aldrich, St. Louis, MO, USA).

(4) Immunohistochemistry

Animals were anesthetized with diethyl ether, first perfused through the heart with saline, and then perfused with 4% paraformaldehyde diluted in 100 mM PBS. Brains were fixed in 4% paraformaldehyde for 1 day, and after dehydration using 30% sucrose solution, they were cryopreserved in a cryogenic freezer. The fixed brain was embedded with an OCT compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan), and sliced to a thickness of 20-25 μm. For double or triple immunofluorescence analysis, tissue sections were prepared with primary antibody mouse anti-GFAP antibody (1:500 dilution; BD Biosciences, San Diego, CA, catalog number: 556330), goat anti-TNAP antibody (1:500 dilution; Santa Cruz Biotech, Dallas, TX, catalog number: sc-23430, mouse anti-GAD67 (1:500 dilution; Millipore, Billerica, MA, catalog number: MAB5406), rabbit anti-galanin (1:500 dilution; Peninsula Laboratories; San Carlos, CA, catalog number: T-4334), mouse anti-c-Fos (1:500 dilution; Santa Cruz Biotech, Dallas, TX, catalog number: sc-8047), rabbit anti-GFAP (1:1000 dilution ; Dako, Glostrup, Denmark, catalog number: Z0334), rabbit anti-Iba-1 (1:1000 dilution; Wako, Osaka, Japan, catalog number: 019-19741) and mouse anti-NeuN (1:500 dilution; Millipore , catalog number: MAB377) After that, secondary antibodies Cy3-, Cy5- and FITC-conjugated anti-mouse, anti-rabbit or anti-goat IgG antibody (The Jackson Laboratory, Bar Harbor, ME; Cy3-mouse , catalog number: 715-165-151; Cy3-rabbit, catalog number: 711-165-152; Cy3-goat, c atalogue number: 705-165-147; Cy5-rabbit, catalog number: 711-175-152; FITC-mouse, catalog number: 715-095-151; FITC-rabbit, catalog number: 711-096-152; FITC-goat, catalog number: 705-095-147) and confirmed under a fluorescence or confocal microscope. Fluorescence intensity was quantified using ImageJ software version 1.44 (National Institutes of Health, Bethesda, MD, USA). Images were obtained from randomly selected non-overlapping areas in VLPO for quantitative analysis.

(5) Bioluminescence assay

ATP bioluminescence assay kit (Sigma) and Synergy 4 Multi-Detection Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA) were used to measure the extracellular ATP content of the sample. For ATP analysis, primary cultured astrocyte-derived culture medium (100 μl) or microdialysis brain tissue fluid (25 μl) was pipetted into a 96-well microplate. ATP assay mixed solution (100 μl) was added to each well, and the luminescence signal was measured dynamically within 1 minute.

(6) Analysis of neuroactive substances (gliotransmitters) using an automatic amino acid analyzer

A sample of 50 μl dialysate hydrolyzed with 6 N HCl was injected into an automatic amino acid analyzer (L-8900, Hitachi, Japan). With an ion exchange column (2622 SC PF, Hitachi), free amino acids were separated at 50° C., and a mobile phase buffer set (PF-1, PF-2, PF-3, PF-4, PF-6, PF-RG, It was eluted in R-3 and C-1). The eluted free amino acids were reacted after column at 135° C. with a ninhydrin solution (Wako, Japan). The detection of ninhydrin-labeled free amino acids, measured at 570 and 440 nm wavelengths, was quantified. A mixture of type ANII and type B (Wako) was used as an internal standard for quantification of free amino acids.

(7) Primary culture of astrocytes

New astrocyte cultures were prepared from mixed glial cell cultures. Briefly, the entire brain of SD rats (2-3 days old, regardless of gender) was cut and mechanically broken using a nylon mesh. The obtained cells were inoculated into culture flasks, and Dulbecco's modified Eagle's medium (DMEM) to which 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin (Gibco-BRL, Rockville, MD, USA) were added. ) At 37° C., 5% CO ₂ under atmospheric conditions. After 5 days, the culture medium was changed for the first time, after which it was changed every 3 days. Cells were used after incubation for 14-21 days. For mixed glial cells, astrocytes and microglia were separated by applying 250 rpm for 12 hours in a stirrer. The culture medium was removed, and astrocytes were separated using trypsin-EDTA (Gibco-BRL), and collected by centrifugation at 1,200 rpm for 10 minutes. Primary cultured astrocytes were cultured in a DMEM medium supplemented with 10% FBS and penicillin-streptomycin, and incubated at 37° C. and 5% CO ₂ .

4. In vivo ( In vivo ) Sleep record

(1) surgery and microinjection of viral vectors

Adult rats were subjected to peritoneal anesthesia using a cocktail (2 ml/kg) of ketamine hydrochloride (72.6 mg/kg) and xylazine hydrochloride (5.1 mg/kg). If maintenance of anesthesia was required, the cocktail (1 ml/kg) was additionally repeatedly injected. In order to implant the EEG electrode in the flat skull area, the rat was placed in a stereotactic fixation device (David-Kopf Instrument Co., Tujunga, CA, USA). Two 22-gauge stainless steel guide cannulaes were implanted on both sides, the ends 1.0-3.0 mm below the VLPO (-0.6 mm from bregma, ±1.4 from midline, -8.8 mm from dura) or 1 mm below the hippocampus (- 4.3 mm from bregma, ±4.0 from midline, -3.0 mm from dura) to prevent damage to the target area. All electrodes and guide cannula were fixed with dental adhesive. A 27-gauge injection needle was injected on each side so that the tip reached the VLPO. Adenovirus vector (Ad-ChR2 or Ad-eGFP) or adeno-associated virus vector (AAV-ChR2 or AAV-eYFP) was injected for 10 minutes at a rate of 0.1 μl/min. The needle was removed 10 minutes after injection. Then, an optical fiber cannula (200 μm diameter, 0.37 NA) was inserted into each guide cannula, the tip was placed 0.5 mm on VLPO and fixed with dental adhesive.

(2) photostimulation

After recovery from surgery, for photostimulation, an optical cable connected to a laser light source (473 nm; SDL-473-100MFL, Shanghai Dream Lasers Co., Shanghai, China) was connected to the optical cannula. Photostimulation was applied in both directions for 30 to 120 minutes at a frequency of 1 Hz (500 ms on/off) through a stimulator (Model S-88, Grass Inc., USA). The light intensity at the end of the fiber optic cable was set to 1.3-1.7 mW (PM204, Thorlabs Co.). In general, the light intensity at the end of the optical cannula was 80-85% of the optical cable. First of all, during most sleep (12:30-14:30; sleep period in the light phase) and waking state (15:30-17:30; wake period in the light phase), 2 photostimulation for 120 minutes each. It was carried out and tested. If the photostimulation promotes sleep, the cage was tilted (3 times by lifting a part of the cage and making the angle 45 degrees from the floor) and immediately 30 minutes after waking the animal, stimulation was performed 3 times (11:00-11. :30, 14:00-14:30 and 17:00-17:30), the sleep latency was measured. In order to confirm reproducibility with other viral vectors, additional light injecting AAV-ChR2-eYFP and AAV-eYFP into rats in the light state (12:00-14:00) and dark state (19:00-20:00) A stimulation experiment was performed. The hippocampus was selected as a control brain region to confirm that it was a specific effect on VLPO astrocytes.

(3) microinjection of drugs

After 3 consecutive stable recording sessions, the experimental microinjection was started. The probe was removed from the guide cannula, and a 27-gauge injection needle was inserted so that the tip was extended to VLPO (-0.6 mm from bregma, ±1.4 from midline, -8.8 mm from dura). 2-chloroadenosine (adenosine A ₁ receptor agonist, 10 nmol per side) or control (100 mM PBS solution, 1 μl per side) using a syringe pump (Model 22; Harvard Apparatus, Inc., Holliston, MA, USA) Thus, perfusion was performed through an injection needle connected to a 50-μl Hamilton syringe through polyethylene tubing (PE-25) for 10 minutes at a rate of 0.1 μl/min. TNAP-I or control (3 μl per side, 0.1 μl/min for 30 minutes) were perfused in the same manner.

(4) Recording and scoring sleep-wake status

Recording was started at least 7 days after surgery. Once the animals have adapted to the recording setup (e.g., chamber and connecting cable), recordings are performed daily from 09:30 to 18:30 for 3 days. The EEG signal from the cortex and the vibration signal from the vibrating plate were amplified by ×10,000 and ×10, respectively, and filtered at 0.5-100 Hz (Model 3500; A-M Systems, Inc., WA, USA). The signal was digitized by a commercially made LabView program (National Instruments Inc.) at 200 Hz (DAQ PAD6015; National Instrument Inc., San Francisco, CA, USA) speed. Sleep-wake state was assessed by two experienced scorers, according to EEG and vibrational signals recording motor activity. Each 10-s period was recorded as one of the following three states. Wake (W), slow-wave sleep (S or SWS) and paradoxical sleep (P or PS). All procedures related to behavioral experiments, such as surgery and viral injection and scoring of sleep-wake states, were performed with a random double-blind method to establish fairness and avoid errors caused by bias.

(5) power spectrum analysis

The power spectrum of each sleep-wake state was calculated for each part after dividing the recording by 30 minutes from 60 minutes before photostimulation to 60 minutes after photostimulation. To assess sleep intensity, delta waves (0.5-2.5 Hz) were obtained from the power spectrum of the slow-wave sleep phase during stimulation.

< Example 1> In inducing sleep VLPO Astrocyte relevance

In order to confirm whether astrocytes in the VLPO site can induce sleep, the present inventors used a photogenetic technique capable of selectively controlling astrocyte activity without direct influence on adjacent neurons. First, channelrhodopsin-2 (ChR2), which is a light-sensitive cationic channel, was selectively expressed in cultured astrocytes through a viral vector (Ad-ChR2 or AAV-ChR2). Photostimulation (473 nm wavelength) through the optical fiber induced a direct internal membrane current in the cultured astrocytes, indicating that ChR2 is functionally expressed in the astrocytes. In ChR2-expressing astrocytes, photostimulation induced a direct inner membrane current in an intensity dependent manner, and the maximum amplitude through photostimulation ranged from 1.4 to 1.7 mW. Accordingly, in all subsequent experiments, the intensity used to stimulate ChR2-expressing astrocytes was 1.7 mW. The photostimulation-induced membrane current was greatly reduced in Na ⁺ -free (replaced by NMDG ⁺ with equivalent molar concentration) external solution, but further decreased as extracellular Ca ^{2 +} was removed, which ChR2 penetrated Ca ^{2 +} It supports that you can do it. Light stimulation was inducing a momentary increase in intracellular Ca ^{2 +} concentrations the cells were maintained for Ca ²⁺ vibrations (oscillations).

Next, the present inventors injected Ad-ChR2 or AAV-ChR2 into the VLPO site of the rat using a stereotaxic system. Seven days after virus injection, ChR2-expressing astrocytes were delivered into the VLPO site through optical stimulation by optical fiber (Figs. 1A-C), and an electroencephalogram (EEG) from the animal was performed to analyze the sleep patterns of the animals. Recorded. After the completion of each experiment, not only the astrocyte expression of ChR2 but also the spatial expression profile of ChR2 in the VLPO site was confirmed (Figs. 1A-C). When the animals were in the sleep phase, photostimulation (1 Hz, 500 ms duration, 120 min) did not significantly change the sleep and/or arousal period (FIG. 1D ). In contrast, when the animals were in awake state, photostimulation significantly increased the duration of slow-wave sleep and paradoxical sleep, and decreased the sleep latency (p <0.05, unpaired t-test, FIGS. 1E-F). The above effect was not observed in the rats injected with the control virus vector (Ad-eGFP or AAV-eYFP). Although photostimulation increased sleep duration, it did not increase delta waves during slow-wave sleep (Fig. 1Eb). In addition, photostimulation of the hippocampus, which is known not to be related to sleep regulation, did not induce sleep, indicating a consistent result that the sleep-inducing effect of VLPO photostimulation was specific. According to a previous study, it was reported that the level of c-Fos expression in VLPO is proportional to the duration of sleep, so the present inventors confirmed whether photostimulation of astrocyte ChR2 affects the expression of c-Fos protein in VLPO neurons. The expression of c-Fos in neurons was higher in the photostimulation group than in the control group without photostimulation.

< Example 2> VLPO Sleep-regulating neuroactive substances within the site ( gliotransmitters ) Of

The result that photostimulation of ChR2-expressing astrocytes increases c-Fos expression in VLPO cells, including galanin-positive neurons, supports the possibility of interaction between astrocytes and neurons at the VLPO site. Since neuron-astrocytic interaction can be mediated by neuroactive substances (gliotransmitters), the present inventors confirmed whether neuroactive substances (gliotransmitters) are released from VLPO astrocytes. After 7 days of injection of Ad-ChR2 into the VLPO site of a living rat, photostimulation was performed on VLPO astrocytes, and then extracellular fluid was obtained using microdialysis technology in the VLPO site (Fig. 2A). As a result of additional bioluminescence analysis of microdialysates collected during 120 minutes of photostimulation, it was found that the ATP concentration in the extracellular fluid was significantly higher in the ChR2-expressing experimental group compared to the experimental group without ChR2 expression (6.1 ± 0.6 nM in the control and experimental groups, respectively. And 54.0 ± 8.5 nM, mean and standard deviation, p <0.01, n = 3, Figure 2B). The increase in ATP concentration induced by photostimulation in the microdialysate was greatly reduced when pretreatment with α-aminoadipate (L-α-AA, 10 nM), an inhibitor of metabolism of astrocytes, was greatly reduced. Astrocytes are the main source of ATP production in cells in an in vivo experiment. However, the concentration of other known neuroactive substances (gliotransmitters) such as glutamate, D-serine and glycine in the microdialysate obtained at the VLPO site during photostimulation did not increase (Fig. 2C). ). In addition, in an in vitro experiment using a primary astrocyte culture without neurons, the present inventors confirmed that photostimulation increased the ATP concentration in the culture medium transfected with ChR2 (1.4 ± in the control group and the experimental group, respectively. 0.4 nM and 21.7 ± 0.8 nM, mean ± standard deviation, p <0.01, n = 6, Figure 2D), which supports that optogenetic stimulation induces ATP release from astrocytes. The increase in extracellular ATP concentration induced by photostimulation was significantly reduced by treatment with 100 nM Brilliant Blue G (BBG), a P2X7 receptor antagonist, or 3 μM carbenoxolone (CBX), a hemichannel blocker (Fig. 2E), which showed that ATP was P2X7. It supports that it can be released through receptors or hemichannels.

< Example 3> VLPO In the excitability of neurons Astrocyte Effect of activation

Judging that astrocytes have a high possibility of releasing ATP as gliotransmitters during photostimulation, the present inventors used whole-cell patch-clamp technology in organotypic section culture including VLPO sites. Potential mechanisms indicative of cell-neuron interactions were identified. In the above culture, ChR2 was expressed at the VLPO site through Ad-ChR2 infection. The input resistance measured from ChR2-expressing astrocytes in section culture was 180.4 ± 55.5 MΩ (29.4 to 592.9 MΩ, n = 10). The remaining membrane potential of the astrocytes was -81.5 ± 1.5 mV (-74.0 to -89.4 mV, n = 10), and photostimulation increased the membrane potential of the astrocytes to 5.8 ± 1.6 mV (1.9 to 16.2 mV, n = 10). Was depolarized. Although the photostimulation-induced membrane currents recorded from astrocytes were smaller in the organotypic section culture than in the primary culture, the current densities were significantly different (3.2 ± 0.5 pA/pF respectively in the section and primary cultures). And 2.5 ± 0.6 pA, n = 10, p = 0.40, one-way ANOVA).

Two types of GABAergic neurons at the VLPO site have been identified, for example, sleep-induced projection neurons and local interneurons. The neurons differ from each other in reactivity to norepinephrine (NE), membrane properties including spike thresholds (low-threshold spike [LTS] type versus non-LTS type), and galanie expression properties. First, the present inventors confirmed whether photostimulation at different frequencies (0.5 to 5 Hz, 5 min train) changes the excitability of VLPO neurons. In both types of VLPO neurons, the AP frequency most effectively affected the 1 Hz photostimulation frequency. In addition, the present inventors analyzed the effect of the spatial relationship between VLPO astrocytes and neurons on the regulation of neural activity. In both types of VLPO neurons, the AP frequency response to photostimulation was related to the soma-soma distance between neurons and ChR2-expressing astrocytes (p <0.01). Thus, all subsequent electrophysiological results were recorded from VLPO neurons within 100-μm intervals from ChR2-expressing astrocytes, and photostimulation was applied to the patched neurons at a frequency of 1 Hz (500 ms duration). In addition, in order to confirm the morphology of VLPO neurons, NE reactivity and spike thresholds were checked, and electrophysiological results of VLPO neurons indicating inconsistency in the two characteristics were removed. For the neurons (41 of 283 neurons), photostimulation reduced the AP frequency to 81.3 ± 10.8% of the control group (n = 41, p <0.01). In a subset of experiments, electrophysiological records were obtained from VLPO neurons of acute sections infected with AAV-ChR2-eYFP. The changes in the excitability of neurons due to photostimulation were similar in the culture of acute and tracheal sections (FIGS. 3 to 6 ). Therefore, the results were collected from both sections during the analysis, but the related cases were separated and shown.

For local GABAergic intermediate neurons (hereinafter referred to as "NE-DP" neurons) exhibiting the characteristics of the non-LTS form and depolarized with 100 μM NE, photostimulation (500 ms duration, 1 Hz, 5 min) is the AP frequency Was reduced to 58.5 ± 5.4% of the control group, and the membrane potential was slightly hyperpolarized to -55.3 ± 0.8 mV to -58.6 ± 0.9 mV (n = 24, p <0.01, FIGS. 3A-C). The photostimulation-mediated inhibitory response in the GABAergic intermediate neuron is equivalent to increasing neuronal excitability in the GABAergic projection neuron. In NE-DP neurons, both water bath-applied ATP (100 μM) and adenosine (100 μM) decreased the excitability of NE-DP neurons (Fig. 3D-E). Adenosine-induced decrease in excitability in these neurons was found to be 2MeCCPA (1 μM, selective A ₁ receptor activator), CGS21680 (1 μM), BAY 60-6583 (1 μM) and HEMADO (1 μM, selective A), which did not affect AP frequency. ₃ receptor activator). Thus, the present inventors confirmed whether the photostimulation response observed in NE-DP neurons is dependent on the adenosine A ₁ receptor. Photostimulation-induced hyperpolarization (-54.3 ± 0.9 mV and -56.9 ± 0.8 mV, n = 16, p <0.01, respectively, in the control and photostimulation conditions) was reduced by 1 μM DPCPX (in the control and photostimulation conditions, respectively- 54.5 ± 0.9 mV and -54.5 ± 0.9 mV, n = 16, p = 0.67, Figure 3D). In addition, the photostimulation failed to decrease the AP frequency in the presence of 1 μM DPCPX (98.5 ± 6.8% of DPCPX conditions, n = 16, p = 0.12, Fig. 3E). The above results show that photostimulation of astrocytes ChR2 reduces the neuronal excitability of GABAergic intermediate neurons in an adenosine A ₁ receptor-dependent manner, and ATP released from astrocytes is ectonucleoside triphosphate diphosphohydrolase (NTPDase). ) And/or tissue-nonspecific alkaline phosphatase (TNAP), including ATP-degrading enzymes.

Since TNAP is highly expressed in the brain and is responsible for extracellular ATP hydrolysis in several brain regions, the present inventors confirmed the effect of TNAP on photostimulation-induced excitatory changes in NE-DP neurons. In these neurons, photostimulation-induced hyperpolarization (-54.3 ± 1.0 mV and -57.4 ± 1.7 mV, respectively, n = 9, p <0.01 in the control and photostimulation conditions) was obtained by a 10 μM TNAP inhibitor (TNAP-I). Reduced (-53.3 ± 0.8 mV and -53.4 ± 0.7 mV, n = 9, p = 0.77, Fig. 3D, respectively in control and photostimulated neurons), and photostimulation reduced AP frequency in the presence of 10 μM TNAP-I. It also failed (115.1 ± 15.2% of the TNAP-I condition, n = 9, p = 0.95, Figure 3E). However, the present inventors have confirmed that ARL67156 (30 μM), an NTPDase blocker in NE-DP neurons, does not change the photostimulation-induced membrane potential and AP frequency. Consistent with the above results, direct perfusion of 3 μl TNAP-I (10 mg/ml) into the VLPO site in vivo significantly increased waking time. In addition, the present inventors have confirmed that the photostimulation response in NE-DP neurons is completely reduced by 3 μM BBG or 100 μM CBX, which is caused by photogenetic stimulation of ATP release through the P2X7 receptor and/or hemichannel. It supports previous findings that it may be possible. Taken together, the above results show that ATP released from astrocytes after photostimulation can be rapidly hydrolyzed to adenosine by the specific enzyme TNAP, and that adenosine acts on the A ₁ receptor to reduce neuronal excitability of GABAergic intermediate neurons. Show.

In GABAergic projection neurons (hereinafter referred to as "NE-HP" neurons) exhibiting LTS-type characteristics and depolarized with 100 μM NE, photostimulation increased the AP frequency to 161.7 ± 11.3% of the control group (n = 22 , p <0.01), the membrane potential was slightly hyperpolarized from -56.6 ± 0.8 mV to -53.5 ± 1.0 mV (n = 22, p <0.01, FIGS. 4A-C). The excitatory response in GABAergic projection neurons by photostimulation may also inhibit neuronal excitability in the arousal center by increasing GABAergic inhibitory release. However, water bath-applied ATP (100 μM) and adenosine (100 μM) decreased the excitability of NE-HP neurons (Fig. 4D), and the photostimulation-induced increase in neuronal excitability was PPADS (20 μM, non-selective P2 receptor. Antagonists), but support that the P2 receptor is not involved in the increase in neuronal excitability induced by photostimulation. In addition, none of the adenosine receptor agonists tested did increase the excitability of NE-HP neurons. Similar to the results in NE-DP neurons, photostimulation-induced depolarization in NE-HP neurons (-58.5 ± 1.2 mV and -55.7 ± 1.3 mV, n = 12, p <0.01, respectively, in control and photostimulation conditions) It was reduced by 1 μM DPCPX (-58.4 ± 1.3 mV and -58.5 ± 1.3 mV, respectively, in control and photostimulation conditions, n = 12, p = 0.65, Fig. 4D). On the other hand, the photostimulation failed to increase the AP frequency in the presence of 1 μM DPCPX (131.5 ± 29.9% of DPCPX condition, n = 12, p = 0.75, Fig. 4E). In addition, the present inventors confirmed the effect of TNAP-I on photostimulation-induced changes in the excitability of NE-HP neurons. Photostimulation-induced depolarization (-56.3 ± 0.8 mV and -52.9 ± 1.2 mV, n = 7, p <0.01, respectively, in control and photostimulation conditions) was reduced by 10 μM TNAP-I (in control and photostimulation conditions, respectively). -55.9 ± 1.5 mV and -54.9 ± 2.1 mV, respectively, n = 7, p = 0.23, Figure 4D), photostimulation also failed to increase the AP frequency in the presence of 10 μM TNAP-I (TNAP-I condition 104.2 ± 4.0%, n = 7, p = 0.40, Figure 4E).

< Example 4> VLPO Differential neurons at the site- Astrocyte Analysis of potential mechanisms indicative of interactions

The results show that the photostimulation-induced excitatory response in NE-HP neurons is indirectly based on neural circuits in VLPO by reducing the neuronal excitability of NE-DP neurons, which is believed to be able to inhibit NE-HP neurons. It indicates that it can be mediated by a mechanism. The present inventors confirmed that SR95531, a selective GABA _A receptor blocker, increases the neuronal excitability of NE-HP neurons in a concentration-dependent manner. Through the above results, it was tested whether photostimulation of astrocyte ChR2 affects GABAergic synaptic transmission in NE-HP. The present inventors used high concentration-Cl ^- internal solution (110 mM pipette Cl ^- concentration), and recorded GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) at a holding potential of -60 mV. I did. The introverted sIPSCs completely disappeared in the presence of 1 μM SR95531. Under these conditions, optogenetic stimulation of astrocytes significantly reduced GABAergic sIPSC frequency (73.3 ± 4.1% of the control group, n = 9, p <0.01) without the influence of the current amplitude (102.7 ± 1.7% of the control group, n = 9). , p = 0.16, Figs. 5A-B). In addition, the cumulative distribution of the inter-event intervals shifted to the right, which is consistent with the decrease in sIPSC frequency (Fig. 5B). The photostimulation-induced decrease in sIPSC frequency was reduced by 1 μM DPCPX (106.0 ± 29.2% of DPCPX condition, n = 9, p = 0.54, Figure 5C-D). These results support that the photostimulation-induced decrease in sIPSC frequency is dependent on the adenosine A ₁ receptor. Even with this in mind, it is unclear how astrocyte-derived ATP inhibits NE-DP neurons without direct effect on NE-HP neurons at the VLPO site. For this differential response, it is possible to explain that even though VLPO astrocytes release ATP around the two types of VLPO neurons, the released ATP is selectively hydrolyzed near NE-DP neurons. The present inventors confirmed that the TNAP immunoreactivity at the VLPO site overlaps with GAD67, but does not overlap with the galanine immunoreactivity (FIGS. 5E-F). These results support that at the VLPO site, TNAP is expressed in GABAergic intermediate neurons rather than in sleep-induced projection neurons. In addition, according to the immunohistochemical results as well as the ATP concentration measurement results in primary astrocyte culture, TNAP did not seem to be expressed in VLPO astrocytes.

Considering that optogenetic stimulation of astrocytes at the VLPO site excites NE-HP VLPO neurons, which are GABAergic projection neurons, in the end, photostimulation of VLPO astrocytes is an arousal-related brain including tuberomammillary nucleus (TMN). GABAergic synaptic transmission to the site can be increased. To confirm this point, the present inventors performed organotype cultivation of filamentous brain slices containing VLPO and TMN, and it was confirmed that activation of VLPO astrocytes affects GABAergic delivery to TMN histaminergic neurons. Histaminergic neurons are identified by regular firing patterns and membrane properties such as the presence of hyperpolarization- and cyclic nucleotide-activated cationic channel sag potentials and the expression of a specific histaminergic marker, histidine decarboxylase. Became. The present inventors confirmed that optogenetic stimulation of VLPO astrocytes in TMN histaminergic neurons significantly increased the sIPSC frequency (150.2 ± 10.4% of the control group, n = 9, p <0.01) without the effect of the current amplitude (control group). 102.1 ± 4.8%, n = 9, p = 0.48, Figures 6A-B). In addition, the cumulative distribution of the internal-event interval shifted to the left (p <0.01, K-S test), which is consistent with the increase in sIPSC frequency (Fig. 6B). The photostimulation-induced increase in sIPSC frequency was reduced by 1 μM DPCPX (98.5 ± 10.8% of DPCPX condition, n = 9, p = 0.68, Fig. 6C-D). Finally, the present inventors confirmed whether optogenetic stimulation of VLPO astrocytes changes the excitability of TMN neurons. In the above experiment, the VLPO site of the rat was infected with AAV-ChR2-eYFP, and electrophysiological recording was performed from TMN histaminergic neurons using an acute filamentous section. The present inventors found that optogenetic stimulation of VLPO astrocytes in TMN histaminergic neurons significantly reduced the AP frequency (77.2 ± 7.2% of the control group, n = 6, p <0.05), and hyperpolarized the membrane potential to 2.8 ± 0.7 mV. Was confirmed (n = 6, p <0.05, Fig. 6E-F). The above results indicate that optogenetic stimulation of VLPO astrocytes increases GABAergic transmission, thereby reducing excitability of TMN histaminergic neurons, thereby inducing sleep (FIG. 7).

Claims (11)

1) Astrocytes in the ventrolateral preoptic nucleus (VLPO) of animals other than humans are transformed with an expression vector containing a light-activated ion channel gene, and the photoactivated ions Expressing the channel; And 2) photostimulating astrocytes in the VLPO. delete The method of claim 1, wherein the light-activated ion channel gene is channelrhodopsin-2 (ChR2), which is a light-sensitive cation channel. The method of claim 1, wherein the expression vector is an adenovirus vector or an adeno-associated viral vector. The method of claim 1, wherein the step of photostimulating astrocytes in the VLPO comprises photostimulating a light source of 450 to 480 nm for 30 to 120 minutes, 1 to 3 times. The method of claim 1, 3 to 5, wherein the animal is a rat. By transforming astrocytes in the ventrolateral preoptic nucleus (VLPO) of the brain with an expression vector containing a photoactivated ion channel gene, expressing the photoactivated ion channel, and photostimulating astrocytes in the VLPO A sleep-inducing animal model other than the produced human. delete The sleep-inducing animal model other than humans according to claim 7, wherein the animal is a rat. Administering a sleep control drug candidate to the sleep-inducing animal model according to claim 7 or 9; And
A method for screening a sleep control drug comprising the step of selecting a drug candidate material exhibiting a sleep control effect compared to the control substance administered animal group. The method of claim 10, wherein the sleep control drug is a narcolepsy treatment, a stimulant, or a sleep agent.

Images