JP2006217866A - Neurocyte to which photosensitivity is newly imparted - Google Patents
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Abstract
Description
本発明は、光感受性を新たに賦与した神経細胞に関する。より詳細には、本来光感受性を有しない神経細胞に、光受容体チャネルをコードする遺伝子を遺伝子工学的に導入・発現させることにより、光感受性を賦与した神経細胞に関する。 The present invention relates to a nerve cell newly imparted with photosensitivity. More specifically, the present invention relates to a neuron that has been given photosensitivity by introducing and expressing a gene encoding a photoreceptor channel into a neuron that originally has no photosensitivity.
ヒトの網膜にある光受容細胞は、可視光に応答して膜電位が変化し、明状態でマイナス（過分極）側に、暗状態でプラス（脱分極）側に変位する。すなわち、光の明暗情報を電気的な情報に変換する機能を担っている。脊椎動物の光受容体細胞では、ロドプシン→Gタンパク質→フォスフォジエステラーゼ→サイクリックGMP→サイクリックGMP受容体チャネルの分子の連鎖反応により、光によりロドプシン分子に引き起こされた構造変化がイオンチャネルに伝えられ、電気的な信号に変換される。これに対し、ヒトやその他の動物の中枢および末梢神経系にある神経細胞の多くは、光に対する応答性を有しない。多くの神経細胞は、他の神経細胞からのみ情報を受け取っており、その主要なメカニズムは、シナプスにおける伝達物質の放出と伝達物質の化学受容である。 Photoreceptive cells in the human retina change in membrane potential in response to visible light and are displaced to the minus (hyperpolarization) side in the bright state and to the plus (depolarization) side in the dark state. That is, it has a function of converting light brightness information into electrical information. In vertebrate photoreceptor cells, the rhodopsin → G protein → phosphodiesterase → cyclic GMP → cyclic GMP receptor channel molecular chain reaction causes light-induced structural changes to the rhodopsin molecule in the ion channel. It is transmitted and converted into an electrical signal. In contrast, many nerve cells in the central and peripheral nervous systems of humans and other animals do not respond to light. Many neurons receive information only from other neurons, and the main mechanism is the release of transmitters at the synapse and chemoreception of transmitters.
しかし、何らかの原因で脳・神経系における情報の伝達が障害を受けることがある。たとえば、網膜の光受容体細胞が変性すると、眼球に入った光情報が脳に伝えられなくなり、失明する。交通事故などで脊髄を損傷すると、脳において生じた運動指令が脊髄の運動神経に伝えられなくなり、下半身が不随になる。これらの機能障害は、損傷された情報伝達経路を他の手段によりバイパスすることにより軽減されることが期待される。このようなバイパス手段により機能障害を軽減する試みとして、神経細胞の電気刺激などに応答する特性を利用して、電気刺激法などにより、感覚や運動の障害を改善することが検討されてきた。また、脳・神経系を用いた基礎・応用研究においては、神経細胞を直接電気刺激し、その出力応答を計測することにより、脳・神経系の機能が解析されてきた。しかし、電気刺激法などの従来の神経細胞刺激法は、時間、空間または両方において高い分解能の得られない問題があった。また、多くの場合、神経細胞やその周辺の組織を傷つける（侵襲的である）という欠点があった。 However, transmission of information in the brain / nervous system may be impaired for some reason. For example, when the photoreceptor cells of the retina are degenerated, light information that has entered the eyeball cannot be transmitted to the brain, resulting in blindness. When the spinal cord is damaged due to a traffic accident or the like, motor commands generated in the brain cannot be transmitted to the motor nerves of the spinal cord, and the lower body becomes involuntary. These functional impairments are expected to be mitigated by bypassing the damaged information transmission pathway by other means. As an attempt to alleviate dysfunction by using such bypass means, it has been studied to improve sensory and motor disorders by an electrical stimulation method or the like using characteristics of nerve cells that respond to electrical stimulation. In basic and applied research using the brain and nervous system, the functions of the brain and nervous system have been analyzed by directly stimulating nerve cells and measuring their output responses. However, conventional nerve cell stimulation methods such as electrical stimulation have a problem that high resolution cannot be obtained in time, space, or both. Further, in many cases, there is a drawback that nerve cells and surrounding tissues are damaged (invasive).
ここで、生物界に広く目を向けてみると、生物はさまざまな方法で光を感知し、行動を制御していることが知られる。生物界に分布する光感受性のタンパク分子の多くは、ロドプシンファミリーに属するが、そのシグナル伝達機序は多様である（非特許文献１参照）。したがって、光感受性のタンパク分子を、遺伝子工学的に神経細胞に導入・発現させることにより、光感受性を賦与された神経細胞を脳・神経系に組み込むことが、原理的に可能である。しかし、現在に至るまで、実用的なものは得られていない。Zemelman等は、ショウジョウバエ由来のアレスチン-2、ロドプシン、Gqaを培養神経細胞に共発現させることにより、光感受性を賦与された神経細胞が得られることを報告している（ChARGe法）（非特許文献２参照）。 Here, if you look closely at the living world, it is known that living organisms sense light and control behavior in various ways. Many of the photosensitive protein molecules distributed in the living world belong to the rhodopsin family, but their signal transduction mechanisms are diverse (see Non-Patent Document 1). Therefore, it is possible in principle to incorporate light-sensitive nerve cells into the brain / nervous system by introducing and expressing light-sensitive protein molecules in nerve cells by genetic engineering. However, until now, no practical one has been obtained. Zemelman etc., from Drosophila arrestin -2 rhodopsin, by coexpressing G q a to cultured neurons, nerve cells endowed with photosensitivity is reported that the resulting (CHARGE method) (Non Patent Document 2).
しかし、3種類の遺伝子を導入する方法は、安定した結果を得ることが難しいとともに光応答速度が遅いという欠点がある。また、Banghart等は電位感受性カリウムチャネルを化学修飾することにより、光感受性を賦与された神経細胞が得られることを報告している（SPARK法）（非特許文献３参照）。 However, the method of introducing three types of genes has the disadvantages that it is difficult to obtain stable results and that the light response speed is slow. Banghart et al. Have reported that photosensitized neurons can be obtained by chemically modifying a voltage-sensitive potassium channel (SPARK method) (see Non-Patent Document 3).
しかし、生きた動物の脳にある神経細胞を化学修飾することには困難がある。また、光応答速度が秒オーダーと遅いので、その適用には限界がある。 However, there are difficulties in chemically modifying nerve cells in the brain of living animals. Moreover, since the optical response speed is as slow as a second order, its application is limited.
淡水池沼に普通に生息しているクラミドモナスは、葉緑体を持ち、光合成をする単細胞真核生物である緑藻類に属している。クラミドモナスは、眼点と呼ばれる特殊な膜領域で光を受容し、鞭毛運動を制御することにより、光に集まる習性（走光性）を有している。クラミドモナスの一種Chlamydomonas reinhardtiiの眼点に分布しているロドプシンファミリータンパク質の遺伝子配列が解明され、公開されている（非特許文献４参照）。
このタンパク質は、可視光に応答してイオンを透過させ、膜電位を制御しているので、生物界における光受容体チャネルの事例である（非特許文献５、６参照）。したがって、この分子単独で膜電位を変化させている特徴がある。クラミドモナスにおいては、2種類の光受容体チャネル、チャネルオプシン1とチャネルオプシン2がある。チャネルオプシン1は、カエル（ゼノパス）の卵母細胞に発現させると、光に応答してH+を透過させる性質をもっている（非特許文献５参照）。
Chlamydomonas normally inhabiting freshwater ponds and marshes belong to the green alga, a unicellular eukaryote that has chloroplasts and photosynthesizes. Chlamydomonas has the habit of gathering in light (phototaxis) by receiving light in a special membrane region called an eye point and controlling flagellar movement. The gene sequence of rhodopsin family proteins distributed in the eye spot of Chlamydomonas reinhardtii, a kind of Chlamydomonas, has been elucidated and published (see Non-Patent Document 4).
This protein is an example of a photoreceptor channel in the biological world because it transmits ions in response to visible light and controls the membrane potential (see Non-Patent Documents 5 and 6). Therefore, this molecule alone has a feature of changing the membrane potential. In Chlamydomonas, there are two types of photoreceptor channels, channel opsin 1 and channel opsin 2. Channel opsin 1 has the property of transmitting H + in response to light when expressed in frog (Xenopus) oocytes (see Non-Patent Document 5).
チャネルオプシン2は、Na+などの陽イオン透過性もあわせもち、培養哺乳類細胞（HEK293、 BHK）に発現させることで、400-500 nmの青色光に応答した脱分極が引き起こされることが報告されている（非特許文献６）。チャネルオプシンは、光受容体チャネルの一種であり、単一の分子で、光感受性とイオンチャネルの機能をあわせ持っているので、現在得られるもののなかで最も応用の可能性の高いものであるが、神経細胞に発現した報告はない。 Channel opsin 2 is also permeable to cations such as Na + and is reported to cause depolarization in response to 400-500 nm blue light when expressed in cultured mammalian cells (HEK293, BHK). (Non-Patent Document 6). Channel opsin is a type of photoreceptor channel, and it is a single molecule that has both photosensitivity and ion channel functions. There are no reports of expression in neurons.
網膜にある光受容細胞が変性して視力を失った場合に、網膜の神経節細胞は健常であるので、光受容細胞の役割を他の方法にて実現できれば視力を回復できる可能性がある。そのような試みの一つとして、光感受性を有する有機化学材料を用いて、光情報を電気信号に変換し、視神経を電気刺激する方法が知られている（特許文献１参照）。また、眼球に入った光を増幅し、画像を受信し、そのデータを電気信号として微少な電極に伝え、網膜神経を直接電気刺激することにより、視覚を回復するいわゆる人工網膜の試みもなされている（特許文献２参照）。 When photoreceptor cells in the retina degenerate and lose visual acuity, the ganglion cells of the retina are healthy, so that if the role of the photoreceptor cells can be realized by other methods, there is a possibility that the visual acuity can be recovered. As one of such attempts, a method is known in which optical information is converted into an electrical signal and an optic nerve is electrically stimulated using an organic chemical material having photosensitivity (see Patent Document 1). Attempts have also been made for so-called artificial retina that restores vision by amplifying the light that enters the eyeball, receiving images, transmitting the data as electrical signals to minute electrodes, and directly stimulating the retinal nerves. (See Patent Document 2).
しかしながら、これらの方法では空間解像度に限界があるし、人工網膜の場合は眼球やその近くに埋め込む手術が必要で、長期使用に耐えられないといった問題点が残されている。
本発明は、何らかの原因で脳・神経系における情報の伝達が障害を受け、それ故に視覚障害や運動障害といった色々な機能障害が生じた場合に、損傷を受けた情報伝達経路を他の手段によりバイパスすることで軽減できる有効な手段を提供することを課題とする。 In the present invention, when the transmission of information in the brain / nervous system is impaired for some reason, and therefore various functional disorders such as visual impairment and movement disorder occur, the damaged information transmission path is determined by other means. It is an object to provide an effective means that can be reduced by bypassing.
上記課題は、神経細胞に新たに光感受性を賦与することで達成できることを本発明者等は見いだした。光感受性は、光受容体チャネルを遺伝子工学的に神経細胞に導入・発現させることにより、賦与することができる。より詳細には、緑色藻類のクラミドモナスその他の生物に由来する光受容体チャネルおよびその改変タンパク分子を、遺伝子工学的に神経細胞に導入・発現させることにより、光照射に応答し膜電位をプラス（脱分極）方向またはマイナス（過分極）方向に変位させる神経細胞を作製することができる。本明細書において、光受容体チャネルとは、光受容体としての機能とイオンチャネルの機能を単一の分子においてあわせもつ分子をいう。 The present inventors have found that the above problem can be achieved by newly imparting photosensitivity to nerve cells. Photosensitivity can be imparted by introducing and expressing a photoreceptor channel in a nerve cell by genetic engineering. More specifically, by introducing and expressing photoreceptor channels derived from the green alga Chlamydomonas and other organisms and their modified protein molecules into neurons by genetic engineering, membrane potential is increased in response to light irradiation ( Neurons that are displaced in the direction of depolarization or minus (hyperpolarization) can be produced. In the present specification, a photoreceptor channel refers to a molecule having both a function as a photoreceptor and a function of an ion channel in a single molecule.
本発明者らは、遺伝子工学的方法を用いて、緑色藻類のクラミドナスに由来するチャネルオプシンをラットの内分泌細胞および神経細胞に発現させ、青色発光ダイオードによる光パルスにより、十分大きな脱分極が引き起こされ、神経細胞に新たな活動が引き起こされることを確認した。 Using genetic engineering methods, the present inventors expressed channel opsin derived from the green alga Chlamydonus in rat endocrine cells and nerve cells, and light pulses from blue light-emitting diodes caused sufficiently large depolarization. , Confirmed that new activity is caused in nerve cells.
今後、クラミドモナスのチャネルオプシン以外のさまざまな光受容体チャネルが発見されることが予想される。これらは、光の強さや波長に対する応答特性、チャネルとしてのキネティクスやイオン透過性の特性において異なることが予想される。また、これらの生物由来の光受容体チャネルを修飾・改変することにより、さまざまな特性を有する光受容体チャネルを作り出すことができる。すなわち、本発明によれば、光受容体チャネルを遺伝子工学的に神経細胞に導入・発現させることにより、より詳細には、生物由来の光受容体チャネルおよびその改変タンパク分子の情報を含む遺伝子が人工的に組み込まれることにより、新たに光感受性を獲得し、強さや波長の異なるさまざまな光刺激に応答し、膜電位をプラス（脱分極）側またはマイナス（過分極）側に変位させるような神経細胞を得ることができる。このような神経細胞を用いることで、機能障害を受けた脳・神経系の情報伝達の代替をさせることができる。 In the future, various photoreceptor channels other than Chlamydomonas channel opsin are expected to be discovered. These are expected to differ in response characteristics to light intensity and wavelength, kinetics as a channel, and ion permeability characteristics. In addition, by modifying and altering the photoreceptor channels derived from these organisms, it is possible to create photoreceptor channels having various characteristics. That is, according to the present invention, by introducing and expressing a photoreceptor channel into a nerve cell by genetic engineering, more specifically, a gene containing information on an organism-derived photoreceptor channel and its modified protein molecule is obtained. By artificially incorporating it, newly gaining photosensitivity, responding to various light stimuli with different strengths and wavelengths, and shifting the membrane potential to the plus (depolarization) side or minus (hyperpolarization) side Neurons can be obtained. By using such a nerve cell, it is possible to substitute for information transmission in the brain / nervous system that has suffered a functional disorder.
また本発明は、光受容体チャネルの遺伝情報を組み込んだDNAおよびRNAウィルスベクターを介して、新たに光感受性を獲得した神経細胞、光受容体チャネルの遺伝情報が組み込まれた遺伝子改変動物を作成して得られる、新たに光感受性を獲得した神経細胞、光受容体チャネルの遺伝情報が組み込まれた幹細胞を作成して得られる、新たに光感受性を獲得した神経細胞なども提供することで、広範囲の機能障害への対応を可能とする。 In addition, the present invention creates a genetically modified animal in which genetic information of a photoreceptor channel or a newly acquired neuronal cell or photoreceptor channel is newly obtained through DNA and RNA viral vectors incorporating the genetic information of the photoreceptor channel. By providing newly obtained nerve cells that have acquired photosensitivity, stem cells into which genetic information of photoreceptor channels has been incorporated, newly obtained nerve cells that have acquired photosensitivity, etc. Enables a wide range of functional failures.
本発明によれば、遺伝子工学的に光感受性を付加した神経細胞を既存の神経回路に組み込む効果があるので、高い空間解像度で、細胞特異的に、これを傷つけることなく（非侵襲的に）、光情報を神経回路にインプットするという効果が得られる。遺伝子導入された神経細胞は、恒常的に光感受性を付加され、また、特に大掛かりな装置を必要としないで光刺激することができるので、コストパフォーマンスに優れた効果が得られる。また、ウィルスベクター法、ジーンガン法、トランスジェニック法などと併用することにより、生きているヒトや動物の脳・神経系に光感受性を新たに獲得した神経細胞を組み込むことができるので、インビボでこれらの光感受性神経細胞を介して、長期間にわたり非侵襲的に情報や信号を脳・神経性にインプットできる効果がある。 According to the present invention, since there is an effect of incorporating a nerve cell to which photosensitivity has been added in genetic engineering into an existing neural circuit, it is highly spatial resolution, cell-specific, without damaging it (non-invasively) The effect of inputting optical information to the neural circuit is obtained. Since the nerve cell into which the gene has been introduced is constantly added with photosensitivity, and can be stimulated with light without requiring a particularly large device, an effect excellent in cost performance can be obtained. In addition, when used in combination with viral vector methods, gene gun methods, transgenic methods, etc., neurons that have newly acquired photosensitivity can be incorporated into the brain and nervous system of living humans and animals. It has the effect of being able to input information and signals to the brain and nerves non-invasively over a long period of time through light sensitive neurons.
以下、本発明の実施の形態について図面を参照しながら説明する。本発明はこの実施例に限定されるものではない。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to this embodiment.
（１）Chop2-Venus発現プラスミドの作製
チャネルオプシンは、7回膜貫通型のロドプシン様構造を有していると推定される。N末の細胞外ドメインにレチナール結合部位があり、光子反応により、レチナールが光学異性
体変換することによる構造の変化により、チャネルが開口し、H+やNa+の陽イオンが透過し、膜電位がプラス変位する。図1Aは、チャネルオプシンの一種のチャネルオプシン２(chop-2)のアミノ酸一次配列のN末315残基である。膜貫通部位と推定されるドメインを太字で示した。チャネルオプシン２などのタンパク質は、DNAやRNAなどの核酸による遺伝子配列情報にもとづいて、細胞において合成されている。本実施例におけるチャネルオプシン-2 (Chop2) のcDNAはドイツ・マックス-プランク研究所のNagel博士から分与された。このプラスミドは、chop2のアミノ酸１〜315残基の塩基配列がpBK-CMV-Δ[1098-1300] ベクターのBamHI/HindIIIサイトにクローニングされたもので、このプラスミドベクターをDH5αコンピテント細胞に導入し、カナマイシンで選抜した後、プラスミドDNAを精製した。このプラスミドを鋳型にして２種類の合成プライマー (5’- gcgaattccaccatggattatggaggcgccctg-3’、 5’- gcggatccttgccggtgcccttgttgaccg-3’) を用いてPCR反応を行い、両端にEcoRIとBamHI部位を持つchop2 cDNAフラグメントを調製した。このcDNAフラグメントをpVenus-N1（Clontech社のpDsRed2-N1のDsRed2を蛍光タンパク質の一種であるVenusのDNA配列に置換したもので発明者らが以前に作製した）のEcoRI/BamHIにサブクローニングして、chop2とVenusの融合タンパク質 (chop2-Venus) を発現させるプラスミドベクターを作製した。この結果、チャネルオプシン２遺伝子DNA配列の3’末をVenusに置き換えた配列からなるDNA鎖 (chop-2-Venusコンストラクト)を組みこんだプラスミドベクターを得た。図1B、 Cは、それぞれ、chop-2-VenusコンストラクトのDNA配列情報にもとづいて、遺伝子導入された細胞において合成されるタンパク質の１次構造と３次構造を推定したものである。レチナールは、生体内に普遍的に存在しており、自然にタンパク質に組み込まれる。図1Cには、このタンパク質と細胞膜の位置関係図も示した。
(1) Preparation of Chop2-Venus expression plasmid
Channel opsin is presumed to have a seven-transmembrane rhodopsin-like structure. There is a retinal binding site in the extracellular domain of the N-terminal, and the photon reaction causes a structural change due to optical isomer conversion of the retinal, resulting in the opening of the channel, the permeation of H + and Na + cations, and the membrane potential. Is displaced positively. FIG. 1A shows the N-terminal 315 residues of the primary amino acid sequence of channel opsin 2 (chop-2). Domains presumed to be transmembrane sites are shown in bold. Proteins such as channel opsin 2 are synthesized in cells based on gene sequence information based on nucleic acids such as DNA and RNA. The channel opsin-2 (Chop2) cDNA in this example was provided by Dr. Nagel of the Max-Planck Institute in Germany. In this plasmid, the nucleotide sequence of amino acids 1 to 315 of chop2 was cloned into the BamHI / HindIII site of the pBK-CMV-Δ [1098-1300] vector. This plasmid vector was introduced into DH5α competent cells. After selection with kanamycin, plasmid DNA was purified. Using this plasmid as a template, PCR was performed using two kinds of synthetic primers (5'-gc gaattc caccatggattatggaggcgccctg-3 ', 5'-gc ggatcc ttgccggtgcccttgttgaccg-3'), and chop2 cDNA with EcoRI and BamHI sites at both ends Fragments were prepared. Subclone this cDNA fragment into EcoRI / BamHI of pVenus-N1 (prepared previously by the inventors by replacing DsRed2 of Clontech's pDsRed2-N1 with the DNA sequence of Venus, a kind of fluorescent protein) A plasmid vector for expressing a fusion protein of chop2 and Venus (chop2-Venus) was prepared. As a result, a plasmid vector incorporating a DNA strand (chop-2-Venus construct) composed of a sequence in which the 3 'end of the channel opsin 2 gene DNA sequence was replaced with Venus was obtained. FIGS. 1B and 1C show the primary structure and tertiary structure of the protein synthesized in the transfected cells based on the DNA sequence information of the chop-2-Venus construct, respectively. Retinals are ubiquitous in the body and are naturally incorporated into proteins. FIG. 1C also shows the positional relationship between this protein and the cell membrane.
（２）PC12細胞の培養
ラット副腎髄質細胞由来のPC12細胞はコラーゲンコート処理を施した 60 mm プラスチックディッシュ上で培養し維持した。培養液には85％ RPMI1640培地、10％ ウマ血清、5％ ウシ胎仔血清、 25 units/ml ペニシリン、25 mg/ml ストレプトマイシンを用いた。遺伝子導入操作を行う前にポリ-L-リジンコート処理を施した12 mm丸形カバーガラス上に細胞を播き、30-50%コンフルエントになった時点で、遺伝子導入処理を行った。遺伝子導入処理の後3~5日経ったものを機能検定に用いた。培養液は3日に一度、半量だけ交換した。継代は週に一度、コンフルエントになる前に行った。
(2) Culture of PC12 cells PC12 cells derived from rat adrenal medulla cells were cultured and maintained on 60 mm plastic dishes treated with collagen. 85% RPMI1640 medium, 10% horse serum, 5% fetal calf serum, 25 units / ml penicillin, 25 mg / ml streptomycin was used for the culture solution. Prior to the gene transfer operation, cells were seeded on a 12 mm round cover glass that had been subjected to a poly-L-lysine coating treatment, and when the cells became 30-50% confluent, the gene transfer treatment was performed. What was passed 3 to 5 days after the gene transfer treatment was used for the functional test. Only half of the culture medium was changed once every three days. Passaging was done once a week before becoming confluent.
（３）大脳皮質神経細胞の培養
大脳皮質神経細胞は、凍結大脳皮質を用いて調製した。凍結大脳皮質は、妊娠18日齢のSplague-Dawley系ラットの胎仔の脳から大脳皮質を取り出し、凍結用培地（60% MEM、20%
ハンクス平衡塩溶液、15% ウシ胎仔血清、5% ウマ血清、40 mM グルコース、8% ジメチルスルホキシド、2 mM グルタミン）にて液体窒素中で凍結保存した。この凍結大脳皮質を解凍し、0。6% グルコースを含むハンクス平衡塩溶液で５分間×1回、培地（60% MEM、
20% ハンクス平衡塩溶液、15% ウシ胎児血清、5% ウマ血清、30 mMグルコース、2 mMグルタミン、1 mMピルビン酸ナトリウム）で５分間×２回の洗浄後、SUMILON社の神経細胞分散液を用いて大脳皮質を分散させた。分散した細胞はポリ-L-リジンコート処理を施した12 mm丸形カバーガラス上に３×105個の密度で播き、37℃、5% CO2条件下で培養し、培養７〜10日後に遺伝子導入処理を行った。遺伝子導入後さらに一週間ほど培養した神経細胞を、機能検定に用いた。培養液は作製の翌日に全量交換し、その後は三日ごとに半量交換した。
(3) Culture of cerebral cortical neurons Cerebral cortical neurons were prepared using frozen cerebral cortex. Frozen cerebral cortex was obtained by removing cerebral cortex from fetal brains of 18-day-old Splague-Dawley rats and using freezing medium (60% MEM, 20%
Hank's balanced salt solution, 15% fetal calf serum, 5% horse serum, 40 mM glucose, 8% dimethyl sulfoxide, 2 mM glutamine) was stored frozen in liquid nitrogen. The frozen cerebral cortex is thawed, and the medium (60% MEM, 60% MEM, once with a Hanks balanced salt solution containing 0.6% glucose for 5 min.
20% Hank's balanced salt solution, 15% fetal bovine serum, 5% horse serum, 30 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate) for 5 minutes x 2 times, and then the SUMILON neuron dispersion Used to disperse cerebral cortex. Dispersed cells were seeded at a density of 3 × 10 5 on 12 mm round cover glass treated with poly-L-lysine coating, cultured at 37 ° C. under 5% CO 2 , and cultured for 7 to 10 days. Later, gene transfer treatment was performed. Neurons cultured for about one week after gene transfer were used for functional tests. The whole amount of the culture solution was changed on the next day after the production, and thereafter half the amount was changed every three days.
（４）遺伝子導入
PC12細胞あるいは大脳皮質神経細胞へのchop2-Venus遺伝子の導入にはQiagen社のEffectene(R) Transfection Reagentを用いて、メーカー指定の方法にて遺伝子導入を行った。遺伝子導入には12 mm丸形カバーガラス１枚辺り0。2〜0。4 μgのchop2-Venus発現プラスミドを用いた。
(4) Gene transfer
For introduction of the chop2-Venus gene into PC12 cells or cerebral cortical neurons, Qiagen's Effectene (R) Transfection Reagent was used to introduce the gene by the method specified by the manufacturer. For gene transfer, 0 to 2 to 0. 4 μg of chop2-Venus expression plasmid was used per 12 mm round cover glass.
（５）実験装置
レチナールを結合したチャネルオプシン2は400-500 nmの光の波長に反応するので、細胞を顕微鏡下まで運ぶ際や、明視野で観察する際に青色光の含まれない環境を作った。実験室の照明には黄色の蛍光灯（パナソニックFL 40S。Y-F）を用い、520 nm以下の波長の光を遮断した。また、CRTモニターの背景色を黄色にした。以下の装置はすべて、エアーテーブル上に設置し、振動の影響を除去した。また、暗幕内に設置し、室内光の影響を除去した。主要な装置の概略を図2に示した。
・ 正立型落射蛍光顕微鏡（オリンパスBH2-RFC）：細胞観察時の透過光はGIFフィルターを介し、波長490 nm以下の光のを遮断した。落射光源には、キセノンランプと青色発光ダイオードを用い、両者は光路の途中においたチョッパー（オリンパスOSP-EXCH）により切り替えた。キセノンランプ光は、Venusの励起・観察に用い、光のON-OFFは、電磁シャッターにより制御した。フィルターおよびダイクロイックミラーの組み合わせにより、励起光波長 450-490 nm、 蛍光波長 >515 nmによりChop2-Venusを発現している細胞を同定した。対物レンズには、60倍水浸レンズ（オリンパスLUMplanPl/IR60x）を用いた。
・ 青色発光ダイオード（OSUB5111A; OptoSupply）：電気刺激装置（日本光電SEN-7203）により作成した矩形波パルス電流により点灯した。青色光はダイクロイックミラーを介して、顕微鏡対物レンズに導入した。
・ 顕微鏡用XY駆動装置（メディカル・エイジェントOXY-1）：後述の3次元マイクロマニピュレーターを固定部に設置し、前述の正立型落射蛍光顕微鏡を駆動部に設置した。マイクロメーターにより、X-Y2軸方向の駆動をそれぞれ微細に制御した。
・ 3次元マイクロマニピュレータ（ナリシゲMX-1R）：後述のパッチ電極のホルダーをX-Y-Zの三軸方向に駆動した。それぞれの駆動軸には、粗動と微動の両様式を併用した。
・ 測定用チェンバー：透明アクリル板を加工し、低部にカバーグラスを貼り付けた。ぺリスタポンプにより、ガス交換チューブ（95% O2、5% CO2混合ガスを通気させる）を介し、チェンバーに人工脳脊髄液（表1）を循環させた。チェンバー内に培養細胞を付着させたカバーグラスを置いた。
・ フォトダイオード（シャープBS500B）：チェンバーの真下に受光部を置き、微小電極用増幅器に接続した。
(5) Channel Opsin 2 coupled with experimental retinal responds to light wavelengths of 400-500 nm, so it can be used in environments that do not contain blue light when carrying cells under the microscope or observing in bright field. Had made. For the laboratory lighting, a yellow fluorescent lamp (Panasonic FL 40S. YF) was used to block light with a wavelength of 520 nm or less. The background color of the CRT monitor was changed to yellow. The following devices were all installed on an air table to eliminate the effects of vibration. In addition, it was installed in a dark screen to eliminate the effects of room light. An outline of the main equipment is shown in FIG.
Erecting epifluorescence microscope (Olympus BH2-RFC): Transmitted light during cell observation was blocked through GIF filters for light with a wavelength of 490 nm or less. The incident light source was a xenon lamp and a blue light emitting diode, and both were switched by a chopper (Olympus OSP-EXCH) in the middle of the optical path. Xenon lamp light was used for excitation and observation of Venus, and ON / OFF of the light was controlled by an electromagnetic shutter. By combining a filter and a dichroic mirror, cells expressing Chop2-Venus with an excitation light wavelength of 450-490 nm and a fluorescence wavelength of> 515 nm were identified. A 60 × water immersion lens (Olympus LUMplanPl / IR60x) was used as the objective lens.
Blue light-emitting diode (OSUB5111A; OptoSupply): Lighted by a square wave pulse current created by an electrical stimulator (Nihon Kohden SEN-7203). Blue light was introduced into the microscope objective lens via a dichroic mirror.
Microscope XY drive device (Medical Agent OXY-1): A three-dimensional micromanipulator described later was placed on the fixed part, and the above-mentioned upright epifluorescence microscope was placed on the drive part. The micrometer controlled each drive in the X-Y2 axis direction finely.
・ Three-dimensional micromanipulator (Narishige MX-1R): A patch electrode holder, which will be described later, was driven in the three axis directions of XYZ. Each drive shaft used both coarse and fine movement modes.
-Measuring chamber: A transparent acrylic plate was processed, and a cover glass was attached to the lower part. Artificial cerebrospinal fluid (Table 1) was circulated through the chamber by a peristaltic pump through a gas exchange tube (aerated with 95% O 2 and 5% CO 2 mixed gas). A cover glass with cultured cells attached was placed in the chamber.
• Photodiode (Sharp BS500B): A light receiving part was placed directly under the chamber and connected to a microelectrode amplifier.
（６）計測・記録装置およびデータ解析
PC12細胞および培養神経細胞の膜電位と膜電流を同時計測した。また、照射光の強さをフォトダイオード電流として同時計測した。これらは、アナログ−ディジタル変換し、コンピューターに記録した。計測には以下の装置を用いた。
・ パッチクランプ増幅器（アキソンAXOPATCH200A）：パッチ電極を介して、フィードバックにより膜電位を一定に維持したときの膜電流を計測した（電位固定実験）。また、フィードバックにより膜電流を一定にしたときの膜電位を計測した（電流固定実験）。
・ 微小電極用増幅器（日本光電MEZ-8201）：フォトダイオードに20 nAの逆電流を通電しながら、光電流を計測した。計測値は、青色発光ダイオードの最大出力にたいする相対値であらわした。
・ アナログ−ディジタル変換装置（アキソンDIGIDATA1200）：パッチクランプ増幅器の電流出力および電圧出力、微小電極用増幅器の電圧出力をアナログ−ディジタル変換し、コンピューターに出力した。
・ コンピューター（デルOptiplex GXi）：データ取得ソフトウェア（アキソンAxoScope
Ver。8。2）を用いてデータを取得した。データの解析には、Clamp-fit Ver。8。2（アキソン）とMicrosoft Excelを用いた。
(6) Measuring / recording device and data analysis
The membrane potential and membrane current of PC12 cells and cultured neurons were measured simultaneously. Moreover, the intensity of irradiated light was measured simultaneously as a photodiode current. These were analog-to-digital converted and recorded on a computer. The following apparatus was used for the measurement.
-Patch clamp amplifier (Axon AXOPATCH200A): The membrane current was measured when the membrane potential was kept constant by feedback via the patch electrode (potential fixation experiment). Also, the membrane potential when the membrane current was made constant by feedback was measured (current fixation experiment).
・ Microelectrode amplifier (Nihon Kohden MEZ-8201): Photocurrent was measured while applying a 20 nA reverse current to the photodiode. The measured value was expressed as a relative value to the maximum output of the blue light emitting diode.
Analog-digital converter (Axon DIGIDATA1200): The current output and voltage output of the patch clamp amplifier and the voltage output of the microelectrode amplifier were converted from analog to digital and output to a computer.
• Computer (Dell Optiplex GXi): Data acquisition software (Axon AxoScope
Ver. Data was acquired using 8.2). Clamp-fit Ver for data analysis. 8.2 (Axon) and Microsoft Excel were used.
（７）ホールセルパッチクランプ法
チャネルオプシン２タンパク質を発現しているPC12細胞が光感受性を新たに獲得したか否かをホールセルパッチクランプ法により確認した。すなわち、直径1。5 mmの内芯入り
ガラス管（ハーバード・アパラータスGC150F）をパッチピペットプラー（ナリシゲPC-10）を用いて加熱牽引し、先端径2-2。5 mmのガラス管ピペット（パッチピペット）を作製し、先端をマイクロフォージ（ナリシゲMF-830）にて加熱研磨した。パッチピペットの先端までパッチピペット内液（表1）を満たしパッチ電極を作成した。パッチ電極に、陰圧・陽圧チューブ、電線を取り付け、マニピュレーターに固定した。正立落射蛍光顕微鏡下にVenusの蛍光によりChop2-Venusを発現している細胞を同定した。パッチ電極に陽圧をかけながら、細胞に接近させ、細胞表面に押し当ててディンプル（くぼみ）をつくり、陰圧に切り替えて細胞膜をガラス管に付着させ、さらに強い陰圧をかけてガラス管に付着した部位の膜を破り、細胞内部とガラス管内部を電気的に導通させた（ホールセルモード、図3）。ホールセルモードで電位固定実験および電流固定実験をおこなった。
(7) Whole cell patch clamp method It was confirmed by the whole cell patch clamp method whether or not PC12 cells expressing channel opsin 2 protein newly acquired photosensitivity. That is, a 1.5 mm diameter cored glass tube (Harvard Appalata GC150F) is heated and pulled using a patch pipette puller (Narishige PC-10), and a 2 mm diameter glass tube pipette (patch). Pipette) was prepared, and the tip was heated and polished with a microforge (Narishige MF-830). A patch electrode was prepared by filling the solution in the patch pipette (Table 1) to the tip of the patch pipette. A negative / positive pressure tube and an electric wire were attached to the patch electrode and fixed to the manipulator. Cells expressing Chop2-Venus were identified by Venus fluorescence under an upright epifluorescence microscope. While applying positive pressure to the patch electrode, approach the cell, press against the cell surface to create dimples, switch to negative pressure to attach the cell membrane to the glass tube, and apply stronger negative pressure to the glass tube. The membrane of the attached site was broken, and the inside of the cell and the inside of the glass tube were electrically connected (hole cell mode, Fig. 3). The potential fixation experiment and the current fixation experiment were conducted in the whole cell mode.
（８）遺伝子導入したPC12細胞の形態
図4Aは、PC12細胞にchop-2-Venusコンストラクトを遺伝子導入し、タンパク分子を発現させたものである。チャネルオプシン2タンパク質を発現している細胞をVenusの蛍光により同定した。共焦点顕微鏡で観察するとチャネルオプシン２タンパク質は、PC12細胞の周囲に限局して分布しており（図4B）、期待どおり細胞膜選択的に発現していることが示唆される。
(8) Morphology of the PC12 cell into which the gene was introduced FIG. 4A shows a protein molecule expressed by introducing a chop-2-Venus construct into a PC12 cell. Cells expressing channel opsin 2 protein were identified by Venus fluorescence. When observed with a confocal microscope, the channel opsin 2 protein is localized around the PC12 cells (FIG. 4B), suggesting that it is selectively expressed in the cell membrane as expected.
（９）光受容体チャネルを導入したPC12細胞の光感受性の確認
ホールセルモードで電流固定実験をおこなった結果を図5に示す。青色発光ダイオードの光パルス照射にともない速やかに膜電位がプラス方向へ変位する脱分極応答が認められた（図5A）。図5Aに示した例では、約40 mVプラス変位した。しかし、チャネルオプシン２を発現していない細胞には、電流の発生や膜電位の応答は認められなかった（図5B）。すなわち、光感受性を新たに獲得したPC12細胞を得たことを確認した。光照射による膜電位の変位は、PC12のCa2+チャネルを活性化し、分泌を引き起こすのに十分な大きさである。
(9) Confirmation of Photosensitivity of PC12 Cells Introduced with Photoreceptor Channels The results of current fixation experiments in whole cell mode are shown in FIG. A depolarization response in which the membrane potential rapidly shifted in the positive direction with the light pulse irradiation of the blue light-emitting diode was observed (FIG. 5A). In the example shown in FIG. 5A, the displacement was about 40 mV plus. However, no current generation or membrane potential response was observed in cells that did not express channel opsin 2 (FIG. 5B). That is, it was confirmed that PC12 cells that newly acquired photosensitivity were obtained. The displacement of the membrane potential due to light irradiation is large enough to activate the Ca 2+ channel of PC12 and cause secretion.
ホールセルモードで電位固定実験をおこなった結果を図6に示す。膜電位を-60 mVに固定したときにイオンチャネルを流れる電流を測定したところ、光パルス照射にともない、強さ依存的に細胞外から内へ向かう電流の発生が観測された（図6A）。図6Aおよび以後の図においては、細胞内から外へ向かう電流をプラス、細胞外から内へ向かう電流をマイナスで表示している。光照射により誘発される電流は、ミリ秒オーダーで最大になり、不活性化し、不活性化の遅い定常電流に移行する（図6A）。また、光照射の終了にともない、ミリ秒オーダーで０に戻った。電流のピーク値および定常値の大きさは、光の強さに依存していた（図6B）。定常電流の大きさは、強い光に対して頭打ちになる傾向が認められたが、ピーク電流の大きさは、光が強ければ強いだけ大きな値を示し、本実験で用いたもっとも強い光に対しても、頭打ちになる傾向は認められなかった。すなわち、光照射に対し、大きなダイナミックレンジで、ミリ秒オーダーの速やかな応答を示すPC12細胞を得たことを確認した。 Figure 6 shows the results of a potential fixation experiment in the whole cell mode. When the current flowing through the ion channel was measured when the membrane potential was fixed at -60 mV, the generation of an inward current from the outside of the cell was observed with light pulse irradiation (FIG. 6A). In FIG. 6A and the subsequent figures, the current flowing from the inside of the cell to the outside is indicated by plus, and the current flowing from the outside of the cell to the inside is indicated by minus. The current induced by light irradiation becomes maximum in the order of milliseconds, deactivates, and shifts to a steady-state current that is slowly deactivated (FIG. 6A). Moreover, it returned to 0 in the millisecond order with the end of light irradiation. The magnitude of the peak value of the current and the steady-state value depended on the light intensity (FIG. 6B). The steady-state current tended to reach a peak for strong light, but the peak current showed as large a value as the light was strong, which was higher than the strongest light used in this experiment. However, there was no tendency to reach a peak. That is, it was confirmed that PC12 cells showing a rapid response in the millisecond order with a large dynamic range to light irradiation were obtained.
（１０）光受容体チャネルを導入したPC12細胞のイオン透過性に生じた光感受性の確認
人工脳脊髄液（表1）のNa+濃度は153 mMになるよう設定している。電位固定実験において、光照射により活性化される電流の大きさは、電位固定膜電位に依存し、0 mV付近でほぼ０になり、陽性保持電位で外向きに反転した（図 7A）。この特性は、神経細胞のシナプス下膜にあるグルタミン酸受容体チャネルの性質に極めて類似している。グルタミン酸受容体チャネルの場合、シナプス前終末から遊離されたグルタミン酸が結合することにより、陽イオン非特異的なチャネルが開口し、Na+やK+が細胞内外のそれぞれのイオン濃度や細胞内外の電位差に依存して受動的に移動することにより、膜電流や膜電位を発生する。光照射により同様の応答が引き起こされたかを検討する目的で、細胞外液を低Na+緩衝液（表1）に置き換えて、電位固定実験をおこなった（図 7B）。この溶液は、Na+の一部
を非透過性陽イオンのN-メチル-D-グルカミンに置換し、26 mMに減少させているが、他のイオン濃度は人工脳脊髄液と同じである。ピーク電流については、大きさが減少するとともに、反転電位が約40 mV陰性変位した（図 7C）。同様の膜電位およびイオン依存性が定常電流にも認められた（図 7D）。したがって、光照射に応答し、Na+、 K+などの陽イオンを透過させる性質を有するPC12細胞を得た。この性質は、アセチルコリン受容体やグルタミン酸受容体を介する興奮性シナプス応答にきわめてよく似ているので、チャネルオプシン２を導入・発現することにより光感受性を獲得した細胞では、光刺激が興奮性シナプス入力の代替手段として用いられることがわかった。これらの性質は、光感受性を有する神経細胞を作製する素材として、チャネルオプシン２が非常に適している分子であることを意味している。
(10) Confirmation of photosensitivity generated in ion permeability of PC12 cell introduced with photoreceptor channel The Na + concentration of artificial cerebrospinal fluid (Table 1) is set to 153 mM. In the potential fixation experiment, the magnitude of the current activated by light irradiation was dependent on the potential fixation membrane potential, and was almost 0 around 0 mV, and reversed outward at the positive holding potential (FIG. 7A). This property is very similar to the nature of glutamate receptor channels in neuronal subsynaptic membranes. In the case of glutamate receptor channels, glutamate released from the presynaptic terminal binds to open a non-cation-specific channel, and Na + and K + have different ion concentrations inside and outside the cell and potential differences inside and outside the cell. Membrane current and membrane potential are generated by moving passively depending on. In order to investigate whether a similar response was caused by light irradiation, an extracellular solution was replaced with a low Na + buffer (Table 1), and a potential fixation experiment was performed (FIG. 7B). This solution replaces a part of Na + with the non-permeable cation N-methyl-D-glucamine and reduces it to 26 mM, but other ion concentrations are the same as artificial cerebrospinal fluid. As for the peak current, the magnitude decreased and the reversal potential was negatively displaced by about 40 mV (FIG. 7C). Similar membrane potential and ion dependence was observed in the steady-state current (Fig. 7D). Therefore, PC12 cells having the property of transmitting cations such as Na + and K + in response to light irradiation were obtained. This property is very similar to the excitatory synaptic response mediated by acetylcholine receptor and glutamate receptor. Therefore, in cells that have acquired photosensitivity by introducing and expressing channel opsin 2, light stimulation is an excitatory synaptic input. It was found to be used as an alternative to These properties mean that channel opsin 2 is a very suitable molecule as a material for producing light-sensitive nerve cells.
（１１）光受容体チャネルを導入した神経細胞の光感受性の確認
図8Aは、ラット胎児の大脳皮質から得られた神経細胞を分散し、培養したものである。神経細胞は、突起を伸ばし、互いにシナプスを作り、ネットワークを形成している。このような培養神経細胞にchop-2-Venusコンストラクトを遺伝子導入した。図8Bのように、Venusの蛍光の存在を手がかりにして、チャネルオプシン２タンパク分子を発現した神経細胞を同定することができる。チャネルオプシン２は、神経細胞の細胞体、突起、終末部に広く分布し、局在は認められなかった。チャネルオプシン２を発現している培養神経細胞にホールセルパッチクランプ法を適用した。電流固定下における静止膜電位は、-60〜-75
mVの範囲にあり、チャネルオプシンを発現していないコントロールの培養神経細胞と差異は認められなかった。チャネルオプシン２を発現している神経細胞に光刺激を行うと、強さ依存的に脱分極し（図 8C）、活動電位が引き起こされた（図8D）。しかし、チャネルオプシン２を発現していない神経細胞においては、光刺激に応答する膜電位の変化は認められなかった。すなわち、chop-2-Venusコンストラクトを遺伝子導入・発現させることにより、新たに光応答性を獲得した神経細胞を得たことを確認した。このような神経細胞は、光刺激に応答して活動電位を引き起こすことを確認した。
(11) Confirmation of Photosensitivity of Neurons Introduced with Photoreceptor Channels FIG. 8A shows a dispersion and culture of neurons obtained from rat cerebral cortex. Nerve cells stretch their processes, make synapses with each other, and form a network. The chop-2-Venus construct was introduced into such cultured neurons. As shown in FIG. 8B, a neuron expressing a channel opsin 2 protein molecule can be identified based on the presence of Venus fluorescence. Channel opsin 2 was widely distributed in the cell body, process, and terminal part of nerve cells, and no localization was observed. The whole cell patch clamp method was applied to cultured neurons expressing channel opsin 2. Resting membrane potential under fixed current is -60 to -75
There was no difference from control cultured neurons that were in the mV range and did not express channel opsin. When a nerve cell expressing channel opsin 2 was subjected to light stimulation, it was depolarized in an intensity-dependent manner (FIG. 8C) and an action potential was induced (FIG. 8D). However, changes in membrane potential in response to light stimulation were not observed in neurons that did not express channel opsin 2. That is, it was confirmed that a nerve cell that newly acquired photoresponsiveness was obtained by introducing and expressing a chop-2-Venus construct. It was confirmed that such nerve cells cause action potentials in response to light stimulation.
（１２）神経ネットワークへの光情報の導入
したがって、チャネルオプシン2を発現することにより光感受性を獲得した神経細胞がネットワークを形成している場合、部位選択的な光照射をおこなうことにより、その神経細胞に活動を引き起こし、ネットワークを機能させることが可能になった（図9A）。また、タンパク質の情報の情報を含む遺伝子DNAの5’側にある非転写型DNA配列領域（プロモーター領域）の特性により、固有の特徴を有する神経細胞に限局して、チャネルオプシン2を発現させることができる。このような場合、光照射に空間的な選択性がなくても、その神経細胞を選択的に刺激することが可能になる（図9B）。このことから、ウィルスベクター法、ジーンガン法、トランスジェニック法などを用いて、チャネルオプシン2遺伝子を導入・発現させ、新たに光感受性を獲得させた神経細胞を生体の脳に作り出すことにより、光照射により、脳をパタン刺激する展望が容易に開かれる。このようなパタン刺激は、脳に情報を直接送信する手段となる。
(12) Introduction of optical information into a neural network Therefore, when nerve cells that have acquired photosensitivity by expressing channel opsin 2 form a network, by performing site-selective light irradiation, the nerve cell It was possible to cause cell activity and make the network function (Figure 9A). In addition, due to the characteristics of the non-transcribed DNA sequence region (promoter region) on the 5 'side of gene DNA containing protein information, channel opsin 2 can be expressed exclusively in neurons with unique characteristics. Can do. In such a case, the nerve cells can be selectively stimulated even if the light irradiation has no spatial selectivity (FIG. 9B). From this, light irradiation is achieved by introducing and expressing the channel opsin 2 gene in the brain of the living body by introducing and expressing the channel opsin 2 gene using viral vector method, gene gun method, transgenic method, etc. This makes it easy to open the prospect of pattern stimulation of the brain. Such pattern stimulation provides a means for transmitting information directly to the brain.
また、本発明に用いる光受容体チャネルとしては、チャネルオプシン２に限らず、クラミドモナス類やその他の生物に見出される光受容体チャネルおよびその遺伝子改変体であるならば、特に限定されるものではない。とくにその細胞内ドメインに神経細胞の初節やランビエ絞輪に集積する性質を有する構造を付加することにより、光パルスによる脱分極により活動電位を引き起こす効率を高めた改変体の利用も十分可能である。イオン透過性の異なる改変体、たとえば、Ca2+透過性を高めた改変体や、K+選択的、あるいは陰イオン選択的な透過性を有し、光刺激により膜電位をマイナス変位（過分極）させるような改変体の利用も考えられる。また、吸収スペクトルの異なる改変体の利用もあり得る。遺伝子導入法は、とくに限定されるものではなく、ウィルスベクター法、ジーンガン法、トランスジェニック法などを利用することができる。光刺激に用いる光源は、そのチャネルオプシンの構造変化を引き起こすものであるならば、特に限定されるものではない。 In addition, the photoreceptor channel used in the present invention is not limited to channel opsin 2, and is not particularly limited as long as it is a photoreceptor channel found in Chlamydomonas or other organisms and a gene variant thereof. . In particular, by adding a structure that has the property of accumulating in the first node of the neuron and the Lambier diaphragm to the intracellular domain, it is possible to use a variant with enhanced efficiency that causes action potential by depolarization by a light pulse. is there. Variants with different ion permeability, for example, variants with enhanced Ca 2+ permeability, K + selective or anion selective permeability, and negative displacement of membrane potential by hyperstimulation (hyperpolarization) It is also possible to use a modified product such as In addition, it is possible to use a variant having a different absorption spectrum. The gene transfer method is not particularly limited, and a virus vector method, a gene gun method, a transgenic method and the like can be used. The light source used for light stimulation is not particularly limited as long as it causes a structural change of the channel opsin.
本発明に関わる光感受性を獲得した神経細胞は、医療、福祉・介護、情報通信等のさまざまな分野に利用できる。また、さまざまな機器と脳・神経系との接合部に利用できる。 The nerve cells that have acquired photosensitivity according to the present invention can be used in various fields such as medical treatment, welfare / nursing care, and information communication. It can also be used at the junction between various devices and the brain / nervous system.
たとえば、ヒトの眼球においては、網膜にある受容体細胞で光が感知され、双極細胞を経て、神経節細胞に活動が伝えられる。神経節細胞の軸索突起が視神経として、脳に神経を送っている。網膜色素変性症や加齢にともなう黄斑変性症などにおいては、受容体細胞が変性することにより、視力を失う。前者は、遺伝的な背景のあるもので、比較的若い時に失明するが、世界中に150万人の患者がいるといわれる。後者は、６５歳以上における
失明の最大要因である。このような患者では、網膜の神経節細胞は健常である。そこで、網膜の双極細胞や神経節細胞は、本来光感受性を有しないが、本発明を用いて、遺伝子工学的方法により、光受容体チャネルを導入・発現させることにより、光感受性を獲得した双極細胞や神経節細胞を網膜に組み込むことができる。この結果、網膜の光受容体細胞が変性していても、双極細胞や神経節細胞が光情報を受容し、膜電位変位の信号に変換し、脳に情報を伝えるようになる。この方法を用いれば、ビデオカメラやフォトダイオードなどの光受容装置、刺激用電極などを用いることなく、まったく非侵襲的に高い解像度の視覚を回復し、これら網膜変性疾患による視覚障害者の生活能力を向上させることが期待される。
For example, in the human eyeball, light is sensed by receptor cells in the retina, and the activity is transmitted to ganglion cells via bipolar cells. The neurites of ganglion cells send nerves to the brain as optic nerves. In retinitis pigmentosa and macular degeneration associated with aging, visual acuity is lost due to degeneration of receptor cells. The former has a genetic background and is blinded at a relatively young age, but is said to have 1.5 million patients worldwide. The latter is the biggest cause of blindness in people over the age of 65. In such patients, retinal ganglion cells are healthy. Therefore, retinal bipolar cells and ganglion cells originally have no photosensitivity, but using the present invention, a bipolar cell that has acquired photosensitivity by introducing and expressing a photoreceptor channel by a genetic engineering method. Cells and ganglion cells can be incorporated into the retina. As a result, even if the photoreceptor cells of the retina are degenerated, bipolar cells and ganglion cells receive light information, convert it into a signal of membrane potential displacement, and transmit information to the brain. By using this method, high resolution vision can be restored non-invasively without using a video camera, a photo-receptive device such as a photodiode, a stimulating electrode, etc., and the living ability of visually impaired people due to these retinal degenerative diseases Is expected to improve.
本発明を応用する方法は、光を介するので、網膜、神経、脳などの組織に対しまったく非侵襲的である。また、時間および空間解像度において従来の方法よりも優れている。また、遺伝子工学的手法を応用するので、適当なプロモーターを選択することにより、脳においてある特定の機能を有する神経細胞のみを選択的に刺激できる特徴がある。したがって、産業上においてきわめて有用な方法である。 The method to which the present invention is applied is completely non-invasive to tissues such as retina, nerves and brain since it is mediated by light. It is also superior to conventional methods in time and space resolution. In addition, since genetic engineering techniques are applied, only a nerve cell having a specific function in the brain can be selectively stimulated by selecting an appropriate promoter. Therefore, it is a very useful method in industry.
Claims (14)
An apparatus for photostimulating the photoreceptor channel according to claim 2 inside and outside a living body.
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