Name: 著者スポットライト:上腺における神経コーディングと視覚処理のメカニズムの解明
Uploaded: 2023-04-21T15:00:00
Description: Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

この研究は、中国国家自然科学基金会(32271060)の支援を受けています。Y.-t.L.は、研究をデザインし、実験を行い、データを解析し、原稿を書きました。Z.L.とR.W.が実験を行った。

すべての実験手順は、動物福祉ガイドラインに従って実施され、北京の中国脳研究所のIACUCによって承認されました。
注:このプロトコルのタイムラインは次のとおりです:1)吸盤を作ります。2)ウイルスを注入する。3)ヘッドプレートを埋め込みます。4)3週間後、プラグを埋め込みます。5)トレッドミルで~3日間の回復と慣れの後、2光子/広視野イメージングを実行します。<...

上丘における2光子および広視野カルシウムイメージング

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T., Turan, Z., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+motion+direction+in+the+mouse+superior+colliculus.">Functional architecture of motion direction in the mouse superior colliculus.</a> Current Biology. 30 (17), 3304-3315 (2020).</li><li>Gribizis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+cortex+gains+independence+from+peripheral+drive+before+eye+opening.">Visual cortex gains independence from peripheral drive before eye opening.</a> Neuron. 104 (4), 711-723 (2019).</li><li>Inayat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+in+the+most+superficial+lamina+of+the+mouse+superior+colliculus+are+highly+selective+for+stimulus+direction.">Neurons in the most superficial lamina of the mouse superior colliculus are highly selective for stimulus direction.</a> The Journal of Neuroscience. 35 (20), 7992-8003 (2015).</li><li>Barchini, J., Shi, X., Chen, H., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+encoding+of+motion+contrast+in+the+mouse+superior+colliculus.">Bidirectional encoding of motion contrast in the mouse superior colliculus.</a> eLife. 7, 35261 (2018).</li><li>Savier, E. L., Chen, H., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+locomotion+on+visual+responses+in+the+mouse+superior+colliculus.">Effects of locomotion on visual responses in the mouse superior colliculus.</a> The Journal of Neuroscience. 39 (47), 9360-9368 (2019).</li><li>Schr&#246;der, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arousal+modulates+retinal+output.">Arousal modulates retinal output.</a> Neuron. 107 (3), 487-495 (2020).</li><li>Ge, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+waves+prime+visual+motion+detection+by+simulating+future+optic+flow.">Retinal waves prime visual motion detection by simulating future optic flow.</a> Science. 373 (6553), (2021).</li><li>Chen, H., Savier, E. 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J., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+evidence+for+stereotypical+direction+columns+in+the+mouse+superior+colliculus.">Lack of evidence for stereotypical direction columns in the mouse superior colliculus.</a> The Journal of Neuroscience. 41 (3), 461-473 (2021).</li><li>Kasai, M., Isa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+light+isoflurane+anesthesia+on+organization+of+direction+and+orientation+selectivity+in+the+superficial+layer+of+the+mouse+superior+colliculus.">Effects of light isoflurane anesthesia on organization of direction and orientation selectivity in the superficial layer of the mouse superior colliculus.</a> The Journal of Neuroscience. 42 (4), 619-630 (2022).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+apical+complex+couples+cell+fate+and+cell+survival+to+cerebral+cortical+development.">The apical complex couples cell fate and cell survival to cerebral cortical development.</a> Neuron. 66 (1), 69-84 (2010).</li><li>Kaifosh, P., Zaremba, J. D., Danielson, N. B., Losonczy, A. S. I. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Python+software+for+analysis+of+dynamic+fluorescence+imaging+data.">Python software for analysis of dynamic fluorescence imaging data.</a> Frontiers in Neuroinformatics. 8, 80 (2014).</li><li>Pnevmatikakis, E. A., Giovannucci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NoRMCorre:+An+online+algorithm+for+piecewise+rigid+motion+correction+of+calcium+imaging+data.">NoRMCorre: An online algorithm for piecewise rigid motion correction of calcium imaging data.</a> Journal of Neuroscience Methods. 291, 83-94 (2017).</li><li>Kerlin, A. M., Andermann, M. L., Berezovskii, V. K., Reid, R. 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プロトコルの重要なステップ 最も重要なステップは、ステップ5.2と5.3の開頭手術です。まず、ラムダの後0.5mmの骨は太く、内部に血管があるため、穴あけプロセス中に出血する可能性があります。出血を止めるために適切なジェルフォームを準備する必要があります。.第二に、横洞のすぐ上の骨を取り除くと、血管性動脈硬化症の可能性が高くなります。トラブルシューティ?...

上結腸(SC)は、すべての脊椎動物において重要な視覚中枢です。哺乳類では、網膜および視覚野1から直接入力を受ける。光記録は皮質2,3,4,5に広く適用されているが、SCでの応用は光アクセスの悪さによって妨げられている6,7,8,9,10,11,12,13,14,15,16,17 、18,19。このプロトコルの目的はSCの神経活動の光学記録のための2つの相補的な方法についての細部を提供することである。
SCは皮質と横洞の下に位置し、結腸ニューロンへの光アクセスを制限します。この制限を克服する1つのアプローチは、重なり合う皮質を吸引し、前外側SC 7,9,10,13,14,19を露出させることです。しかし、SCは皮質入力を受け取るため、このような操作はSCニューロンが視覚刺激にどのように反応するかに影響を与える可能性があります。この制限を克服するために、皮質をそのまま残しながら、シリコンプラグで後内側SCの表層を画像化する代替プロトコルをここで詳しく説明します8,11。具体的には、単一細胞の分解能を達成するために、2光子顕微鏡を適用して、野生型マウスの後内側SCにおけるカルシウム応答を画像化しました。さらに、広い範囲をカバーするために、広視野顕微鏡を適用して、後皮質が発達していない変異マウスのSC全体を画像化しました20。
このプロトコルで説明されている2つの方法は、互いに補完的です。皮質を切除しない2光子カルシウムイメージングは、皮質入力が無傷の単一細胞分解能で神経活動を記録するのに適しています。広視野カルシウムイメージングは、空間分解能を犠牲にしながら、SC全体の神経活動を記録するのに適しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-mm lens</td>
			<td>Computar</td>
			<td>M5018-MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mm coverslip</td>
			<td>Warner instruments</td>
			<td>CS-5R</td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filter</td>
			<td>Chroma Technology</td>
			<td>HQ575/250 m-2p</td>
			<td></td>
		</tr>
		<tr>
			<td>butyl cyanoacrylate</td>
			<td>Vetbond, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera for monitoring pupil</td>
			<td>FLIR</td>
			<td>BFS-U3-04S2M-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>camera for widefield imaging</td>
			<td>Basler</td>
			<td>acA2000-165&#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>corona treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic</td>
			<td>Chroma Technology</td>
			<td>T600/200dcrb&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>galvanometers</td>
			<td>Cambridge Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bead sterilizer</td>
			<td>RWD</td>
			<td>RS1502</td>
			<td></td>
		</tr>
		<tr>
			<td>microdrill</td>
			<td>RWD</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>QUAD</td>
			<td></td>
		</tr>
		<tr>
			<td>photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>R3896</td>
			<td></td>
		</tr>
		<tr>
			<td>rotory encoder</td>
			<td>USdigital</td>
			<td>MA3-A10-125-N</td>
			<td></td>
		</tr>
		<tr>
			<td>self-curing dental adhesive resin cement&#160;</td>
			<td>SuperBond C&#38;B, Sun Medical Co, Ltd. Moriyama, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>thermostatic heating pad&#160;</td>
			<td>RWD</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai Tai HP DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>translucent silicone adhesive&#160;</td>
			<td>Kwik-Sil, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>treadmill</td>
			<td>Xinglin Biology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Virus Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV2/9-hsyn-Gcamp6m</td>
			<td>Vector Core at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild type</td>
			<td>Laboratory Animal Resource Center at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emx1-Cre</td>
			<td>The Jackson Laboratory&#160;</td>
			<td>5628</td>
			<td></td>
		</tr>
		<tr>
			<td>Pals1flox/wt</td>
			<td>Christopher A. Walsh Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH Image</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium imaging in mouse superior colliculus

すべての脊椎動物で進化的に保存された中脳構造である上結腸(SC)は、大脳皮質が出現する前の最も洗練された視覚中枢です。~30種類の網膜神経節細胞(RGC)から直接入力を受け、それぞれが特定の視覚的特徴をコード化しています。SCが単に網膜の特徴を継承するのか、それとも追加の潜在的に de novo プロセシングがSCで起こるのかは、とらえどころのないままです。SCにおける視覚情報の神経コーディングを明らかにするために、ここでは、覚醒マウスの2つの相補的な方法で視覚応答を光学的に記録するための詳細なプロトコルを提供します。1つは、2光子顕微鏡を用いて、重なり合う皮質を切除することなく、1細胞の分解能でカルシウム活性を画像化する方法であり、もう1つは、広視野顕微鏡を用いて、皮質がほとんど発達していない変異マウスのSC全体を画像化する方法です。このプロトコルでは、動物調製、ウイルス注入、ヘッドプレート移植、プラグ移植、データ収集、データ解析など、これら2つの方法を詳しく説明します。代表的な結果は、2光子カルシウムイメージングが1細胞分解能で視覚的に誘発された神経応答を明らかにし、広視野カルシウムイメージングがSC全体の神経活動を明らかにすることを示しています。この2つの方法を組み合わせることで、異なるスケールでのSCの神経コードを明らかにすることができ、そのような組み合わせは他の脳領域にも適用できます。

The superior colliculus (SC) is an important visual center in all vertebrates. In mammals, it receives direct inputs from the retina and the visual cortex1. While optical recording has been widely applied to the cortex2,3,4,5, its application in the SC is hindered by poor optical accesss6,7,8,9,10,11,12,13,14,15,16,17,18,19. The goal of this protocol is to provide details about two complementary methods for optical recording of the neural activity in the SC.
The SC is located beneath the cortex and transverse sinus, which limits optical access to the collicular neurons. One approach to overcome this limitation is to aspirate the overlaying cortex and expose the anterior-lateral SC7,9,10,13,14,19. However, because the SC receives cortical inputs, such an operation might affect how the SC neurons respond to visual stimuli. To overcome this limitation, we detail here an alternative protocol to image the superficial layer of the posterior-medial SC with a silicon plug, while leaving the cortex intact8,11. Specifically, to achieve single-cell resolution, we applied two-photon microscopy to image calcium responses in the posterior-medial SC of wild-type mice. In addition, to achieve broad coverage, we applied wide-field microscopy to image the entire SC of a mutant mouse whose posterior cortex has not developed20.
The two methods described in this protocol are complementary to each other. The two-photon calcium imaging without ablating the cortex is appropriate for recording neural activity at&#160;single-cell resolution with intact cortical inputs. The wide-field calcium imaging is appropriate for recording neural activity in the entire SC while sacrificing spatial resolution.

著者スポットライト:上腺における神経コーディングと視覚処理のメカニズムの解明 

マウス上結腸におけるカルシウムイメージング

My research focuses on neural coding and mechanisms for visual processing. We are trying to answer how various information is encoded in the brain, especially in the superior colliculus under the underlying neuro-mechanisms. To understand visual processing, scientists combine various approaches, including neuronal morphology, antegrade and retrograde tracing, IL sequencing, neuro modeling, machine learning, is understood well.The challenge is to remove the skull over the SSA, cutting and left in the bone in one larger piece, is likely to fail because the bone is attached to the dura. By combining two photo on a wide field calcium image, our recent work revealed the function architecture of motion direction in the mouse SC at both single cell resolution and a global scale. We found that neurons with similar preference form patches up to 500 micrometer under global to the upward on the nasal motion.With our protocol, we are addressing two research gaps. First, researchers can perform long-term cast imaging in mouse SC at a single cell resolution without breaking the cortex. Secondly, researchers can record the neuroactivity across the entire SC using widefield microscoping.Our protocol provides a measure to image the posterior medial SC at single cell resolution with a intact cortex in mice. Also, we use a biocompatible plug to expose the SC, which reduces infection for chronic imaging. Our results provide a way to study the neural coding of visual information across a large visual field.Combined with optogenetics, one can study half inputs from different brain regions modulating the neuroactivity in SC.

図1A、B は、吸盤とプラグの作り方をそれぞれ示しています。 図2 は、プラグを正常に埋め込む方法を示しています。プラグを埋め込んだ後、 図2Dに示すように、後内側SCが露出します。 図3 は、2光子顕微鏡を用いて画像化した野生型マウスの例から得られたSCニューロンのカルシウム応答?...

Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

このプロトコルは野生型マウスの皮質をそのまま残している間2光子の顕微鏡検査との単一ニューロンの活動をイメージ投射すること、および部分皮質の突然変異体のマウスの広視野顕微鏡検査が付いている全体のSCをイメージ投射することを含む目がさめているマウスの優秀なcolliculus (SC)のカルシウム応答を、イメージ投射するためのプロシージャを詳述する。

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

私の研究テーマは、神経符号化と視覚処理のメカニズムです。私たちは、脳内、特に神経機構の根底にある上結腸において、さまざまな情報がどのようにコード化されているのかを解明しようとしています。視覚処理を理解するために、科学者は、ニューロンの形態、順行性および逆行性追跡、ILシーケンシング、神経モデリング、機械学習など、さまざまなアプローチを組み合わせてよく理解されています。課題は、骨が硬膜に付着しているため、SSA上の頭蓋骨を切除し、骨を1つの大きな部分に残して骨に残すことです。広視野カルシウム画像上に2枚の写真を組み合わせることで、マウスSCの運動方向の機能構造を、単一細胞分解能と地球規模の両方で明らかにしました。その結果、同様の嗜好を持つニューロンは、鼻の動きで上向きに500マイクロメートルまでパッチを形成することがわかった。私たちのプロトコルでは、2つの研究ギャップに対処しています。まず、研究者は、皮質を壊すことなく、単一細胞分解能でマウスSCの長期キャストイメージングを行うことができます。第二に、研究者は広視野顕微鏡を使用してSC全体の神経活動を記録することができます。私たちのプロトコルは、マウスの無傷の皮質を持つ単一細胞分解能で後部内側SCを画像化する手段を提供します。また、生体適合性プラグを使用してSCを露出させることで、慢性画像診断の感染を軽減します。本研究成果は、広い視野における視覚情報の神経符号化を研究する方法を提供するものである。光遺伝学と組み合わせることで、SCの神経活動を調節するさまざまな脳領域からの入力の半分を研究することができます。

calcium-imaging-in-mouse-superior-colliculus

Research

JoVE Journal

Neuroscience

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

神経科学

Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse

Begin the process of creating a suction cup by depositing a drop of PBS in an acrylic dish. Use a 21 gauge flat needle to fill the tip of the needle with PBS by capillarity. Cover the tip of the needle with a translucent silicone adhesive, and set it for approximately 10 minutes.Cut the silicone adhesive with scissors to a disc with a two millimeter diameter. To prepare the plugs, take a 0.75 millimeter thick plastic shim stock and cut out a one by one centimeter square in the center. Clean the shim stock and two acrylic blocks with alcohol, and wipe them with lens paper.Place the shim stock on one acrylic block, and deposit silicone adhesive into the center to fill 90%of the opening. Add another acrylic block. Apply approximately one kilogram of force, and wait 20 minutes.Under a surgical microscope, cut the silicone adhesive with a scalpel into one by 1.5 millimeter triangular prisms. Remove any dust from the triangular prisms with sticky plastic tape, and place them on a glass cover slip. Place the cover slip with the triangular prisms under a corona treater and turn it on.Bring the corona treater closer to the cover slip until lightning appears, and hold it in that position for a few minutes until the prisms and cover slip are attached. After placing the plugs in a Petri dish, incubate at 70 degrees Celsius overnight.

マウスにおけるカルシウムイメージングのための吸盤とプラグの作製

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

Begin by securing the anesthetized mouse onto a stereotaxic frame with ear bars. Apply ophthalmic ointment on the mouse's eyes to prevent drying, and maintain the body temperature of the mouse at 37 degrees Celsius with a thermostatic heating pad. After shaving the fur and sterilizing the surgical site, inject lidocaine, tilidine, and meloxicam.Once the skin over the SC is removed, clean the skull with a cotton swab. Adjust the stereotaxic frame until the skull surface is flat. Using a drill, create a whole 0.5 millimeter lateral, and 0.42 millimeter anterior to the lambda, until the dura is exposed.Inject an adeno-associated virus, or AAV, expressing GCaMP6m, at depths of one millimeter and 1.6 millimeters from the Lambda using a beveled glass micro-pipette. After the injection, slowly withdraw the injector and proceed with the implantation of the head plate.

カルシウムイメージングのためのマウス上結腸へのウイルスコンストラクト(AAV)注射

Implantation of the Headplate in Mouse for Calcium Imaging 

After injecting the viral construct into the mouse brain, clean the muscles and tissues over the skull, and scratch the skull with a blade. Prepare the dental adhesive resin cement by mixing 3/4 spoonful of polymer, three drops of monomer, and one drop of catalyst in a ceramic bowl on ice. Apply the mixture to the head plate and the skull.Attach them and wait for the adhesive to set for five minutes. Finally, inject antibiotics to prevent infections. After removing the mouse from the stereotaxic frame, place it on a heating pad for recovery, and return it to the home cage after recovering from anesthesia.

カルシウムイメージングのためのマウスへのヘッドプレートの移植 

Implanting the Plug in the Mouse for Calcium Imaging

Three weeks after the viral infection, anesthetize and fix the mouse with a head plate on a stereotaxic frame. Soak a gel foam in saline to stop potential bleeding. Use a micro-drill to create a three by two millimeter oval, centered at 0.5 millimeter posterior to the lambda.Thin the boundary of the oval until it cracks, then thin the skull before the oval recording window to close the cover slip to the SC.Using fine forceps, take off the bone flaps. Make a cut down the dura, posterior to the transverse sinus and remove the piece of dura. Dry the skull and the recording window with gel foam and cotton swabs.Then deposit a drop of silicone adhesive into the recording window. Use the suction cup to hold the plug with negative pressure, and use a motorized micro-manipulator to move the suction cup and lower the plug into the silicone adhesive until the cover slip touches the skull. Once the head plate is cleaned, apply butyl cyanoacrylate and resin cement around the boundary of the plug to fix it to the head plate and proceed with imaging.

カルシウムイメージングのためのマウスへのプラグの埋め込み

Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons

After implanting the head plate and plug, secure the animal on a rotating treadmill with its head plate. Place the treadmill beneath the two-photon microscope and adjust the height to an appropriate position. Place a black aluminum cone between the objective and the head plate to prevent light contamination from the monitor for visual stimulation.To perform two-photon imaging, adjust the laser power at the sample plane between 20 and 80 milliwatts. Scan a 600-by-600 micrometer field of view at 4.8 hertz with a spatial resolution of 2.4 micrometers per pixel and image neural activity up to 350 micrometers in depth. Using a rotary encoder, record the mouse's locomotion on the treadmill.Use a camera with a 50 millimeter lens to record its pupil size and position. Lastly, synchronize these recordings, and image acquisitions to visual stimulation by recording triggers sent by the stimulation computer. The calcium responses of SC neurons from a wild type mouse and the standard deviation projection of the fluorescence across images are presented.

マウス上結腸ニューロンの2光子カルシウムイメージング

Wide-Field Calcium Imaging of Mouse Superior Colliculus Neurons

To begin, inject 100 nanoliters of AAV expressing GCaMP6m at a depth of 200 micrometers at the center of each side of the SC to the mutant mouse whose posterior cortex has failed to develop. After approximately three weeks, implant a five millimeter diameter cover slip over the SC.After fixing the cover slip, acquire images using a CMOA camera after passing an emission filter at 10 hertz. The calcium responses of SC neurons from a partial cortex mutant mouse were imaged using wide-field microscopy.The acquired image was down sampled to one quarter of the original size, and visually evoked calcium responses are shown.