ونحن نشكر مختبر كوشي كامل لإجراء مناقشات مفيدة. نشكر باتي Jansma وجامعة ولاية اريزونا وزارة العلوم العصبية للحصول على المشورة والمساعدة في التصوير. كما نشكر مختبر Porreca لاستخدام Vibratome بهم. وأيد هذا البحث من قبل المعاهد الوطنية الأميركية للصحة (NIH NS065116، AAK).

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الكتاب ليس لديهم ما يكشف.

وبالنظر إلى أن التغيرات الخلوية في الخلايا المضيفة المصابة وقد تم ربط لنتائج المرض في العدوى مع الكائنات الحية داخل الخلايا الأخرى مثل فيروس نقص المناعة البشرية، وداء الكلب، والكلاميديا ​​18،19، قمنا بتطوير تقنية من شأنها أن تسمح لنا لدراسة التفاعلات الحميمة ...

A correction was made to <a href="http://www.jove.com/video/52237">"3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii"</a>. The ordering of the authors was inverted. The order has been updated from:
 Anita A. Koshy, Carla M. Cabral
to:
Carla M. Cabral, Anita A. Koshy

A correction was made to <a href="http://www.jove.com/video/52237">"3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii"</a>.

Erratum: 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

ويتمثل الهدف العام من هذه الطريقة هو الحصول على ارتفاع القرار، وصور ثلاثية الأبعاد من الخلايا العصبية الفردية التي تقوم إصابة بطفيل التوكسوبلازما داخل الخلايا إلزام. غالبا ما تعتبر التوكسوبلازما واحدة من الطفيليات الأكثر نجاحا بسبب مجموعة مضيفه كبير وسيطة، والتي تشمل البشر والقوارض. في كل من البشر والقوارض، بعد العدوى الحادة من خلال تناول طعام أو ماء ملوث، التوكسوبلازما غير قادرة على التسبب في العدوى المستمرة للجهاز العصبي المركزي عن طريق تحويل من شكله تتكاثر بسرعة (على tachyzoite) لالبطيء الطويل تكرار وencysting النموذج (المتباطئة ). في الأفراد مناعيا، ويعتقد أن هذا المرض الجهاز العصبي المركزي الكامنة لتكون أعراض نسبيا، ولكن في الأفراد المناعة مثل مرضى الإيدز أو متلقى، تجدد الطفيلي يمكن أن يؤدي إلى التهاب الدماغ بالمقوسات قاتلة 1،2. وبالإضافة إلى ذلك، الدراسات الحديثة هكتارلقد أظهرت أن العدوى الكامنة مع التوكسوبلازما يمكن أن يؤدي إلى تغيرات سلوكية في القوارض 3،4، على الرغم من أن آلية لا تزال مجهولة. والمثير للدهشة أنه بالرغم من هذه البيانات تسليط الضوء على أهمية التفاعل CNS- التوكسوبلازما، والقليل نسبيا هو معروف عن هذه العلاقة، وخاصة على المستوى الخلوي والجزيئي. وقد أعاق القدرة على دراسة الجوانب حتى بسيطة للتفاعل الدماغ الطفيلي في جزء من القيود التكنولوجي. على سبيل المثال، فإن الغالبية من العمل تبين أن الخلايا العصبية هي الخلايا التي لا تزال قائمة الخراجات تم القيام به مع المجهر الإلكتروني (EM) 5،6. على الرغم من EM يعطي ارتفاع القرار، وذلك هو مضيعة للوقت، والعمل المكثف، ومكلفة. وقد تم مؤخرا استخدام المناعي (IF) المقايسات بالتزامن مع المجهري متحد البؤر لتأكيد العمل الذي قام به EM 7. IF المقايسات سهلة من الناحية الفنية لأداء وغير مكلفة نسبيا، ولكن باستخدام هذه التقنيات لالفضوليةTAND العلاقة المكانية بين الكيس والخلايا العصبية المصابة تتطلب إعادة الإعمار المسلسل، والتي تستغرق وقتا طويلا، من الصعب من الناحية الفنية، وربما يؤدي إلى فقدان معلومات قيمة. وهكذا، قمنا بتطوير طريقة التي يمكن استخدامها مع نموذج الفأر من الجهاز العصبي المركزي داء المقوسات ويسمح لنا الصورة بكاملها من الخلايا العصبية المصابة دون EM أو المناعية (IHC). من خلال تطوير هذه التقنية، يمكننا أن نبدأ لاستكشاف العلاقة الخلوية بين الخلايا المصابة والكيس بطريقة سريعة وغير مكلفة نسبيا. طريقة ضعنا يجمع بين التقنيات الحديثة للمقاصة بصريا والتصوير أقسام الدماغ سميكة بواسطة المجهر متحد البؤر 8 مع نظام الذي يصادف في خلايا الجسم الحي التي تم حقنها مع البروتينات الطفيلي 9،10. في هذا النظام، ونحن تصيب الفئران لجنة المساواة العرقية-مراسل التي تعبر عن بروتين الفلورية الخضراء (GFP) إلا بعد لجنة المساواة العرقية بوساطة إعادة التركيب 11 مع التوكسوبلازما </em&gt; السلالات التي تعبر عن البروتين الفلوري الأحمر (RFP) وحقن لجنة المساواة العرقية recombinase في الخلايا المضيفة 9. هذا المزيج يسمح لنا حصاد مخ الفأر المصاب بعد ثبوت العدوى CNS، وقطع أقسام الدماغ سميكة، وبسرعة تحديد المجالات ذات الصلة إلى الصورة من خلال إيجاد RFP + الخراجات. ومن المهم أن نلاحظ أنه مع التعبير الخلية المضيفة من GFP يعتمد فقط على حقن لجنة المساواة العرقية عن الطفيليات، وليس على العدوى، وعدد من الخلايا GFP + لا تحتوي على طفيليات 10. وبما أن الهدف من هذا البروتوكول هو أن تكون قادرة على الصورة كاملة الخلايا العصبية المصابة، ويتم التركيز فقط على GFP + الخلايا العصبية التي تحتوي أيضا على طلب تقديم العروض + الكيس، ولكن يمكن أيضا أن تستخدم بروتوكول لصورة GFP + / RFP - الخلايا العصبية. مرة واحدة يتم حصاد الدماغ المصاب ومقطوع، ويتم تقديم الأقسام شفافة عن طريق مسح الجلسرين. ثم يتم تصويرها المناطق المناسبة من المقاطع مع المجهر متحد البؤر، ALخوار التصور غير مسبوق من الخلايا المضيفة المصابة والطفيليات متكيسة في مجملها. هنا نقدم بروتوكول كامل لتحديد وإزالة بصريا، والتصوير إصابة الخلايا العصبية.

<table border="0" cellpadding="0" cellspacing="0" width="1877">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Name of Reagent/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:1008px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Series 1000 Sectioning System</td>
			<td>Technical Products International, Inc.</td>
			<td></td>
			<td>Other vibratomes are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#1.5 Coverslips</td>
			<td>VWR</td>
			<td>48393 251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond Scriber</td>
			<td>VWR</td>
			<td>52865-005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM 510 Meta confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Ketaject&#174; Ketamine HCl Inj., USP 100mg/ml</td>
			<td>Western Medical Supply, Inc.</td>
			<td>4165</td>
			<td></td>
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			<td height="21" style="height:21px;">AnaSed&#174; Injection Xylazine 20mg/ml</td>
			<td>Lloyd Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZsGreen Mice</td>
			<td>Jackson Laboratories</td>
			<td>7906</td>
			<td>B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical equipment</td>
			<td></td>
			<td></td>
			<td>Thumb forceps; Fine scissors-angled to side, sharp-sharp; Sharp-sharp scissors; Kelly hemostats; Mayo scissors; Micro spatula.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Foreskin Fibroblasts (HFF) cells</td>
			<td></td>
			<td></td>
			<td>These are primary cells from human foreskins.&#160; We make these in-house but they may be purchased from outside vendors.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s High Glucose Modified Eagles Medium (DMEM)</td>
			<td>HyClone</td>
			<td>SH30081.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Penicillin Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">200mM L-alanyl-L-glutamine</td>
			<td>Corning</td>
			<td>25-015-Cl</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:469px;">25cm2 Canted neck flask</td>
			<td>Fisher Scientific</td>
			<td>1012639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 1X Without Calcium and Magnesium</td>
			<td>VWR</td>
			<td>45000-446</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 10X, USP Sterile Ultra Pure Grade</td>
			<td>amresco</td>
			<td>K813-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Fetal Bovine Serum</td>
			<td style="width:265px;">Gibco</td>
			<td style="width:135px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Bright-Line Hemocytometer</td>
			<td style="width:265px;">Sigma-aldrich</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Brain Slicer Matrix</td>
			<td>Zivic Instruments</td>
			<td>BSMAS005-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td style="width:135px;">BP358-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td style="width:265px;">Sigma-aldrich</td>
			<td style="width:135px;">H3393-100KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Paraformaldehyde</td>
			<td style="width:265px;">Fisher Scientific</td>
			<td style="width:135px;">O4042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">20ml Disposable Scintillation Vials</td>
			<td style="width:265px;">Fisher Scientific</td>
			<td style="width:135px;">FS74500-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol, Ethyl, 95%, 190 Proof</td>
			<td style="width:265px;">In-house</td>
			<td>17212945</td>
			<td>This product is purchased from an in-house stockroom.&#160; Other companies are compatible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Imaris Software</td>
			<td style="width:265px;">Bitplane</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Clear nail polish</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Other brands are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">10ml Syringe with Luer-Lok</td>
			<td style="width:265px;">VWR</td>
			<td>BD309604</td>
			<td>Other syringes are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Three-way Stopcock</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Hypodermic needle</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible - used to pin down mouse.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cell Scraper</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G x 12&#34; Tubing, Safety Blood Collection Set, with Luer Adapter</td>
			<td style="width:265px;">Greiner Bio-One</td>
			<td style="width:135px;">450099</td>
			<td>Other brands are compatible</td>
		</tr>
	</tbody>
</table>

3-d imaging and analysis of neurons infected in vivo with toxoplasma gondii

التوكسوبلازما هو إلزام، الطفيليات داخل الخلايا مع مجموعة المضيف واسع، بما في ذلك البشر والقوارض. في كل من البشر والقوارض، التوكسوبلازما يضع العدوى المستمرة مدى الحياة في الدماغ. بينما الإصابة في الدماغ وهذا هو دون أعراض في معظم الناس مناعيا، في وضع الجنين أو الأفراد المناعة مثل متلازمة نقص المناعة (الإيدز) من المرضى، وهذا ميل للوالمثابرة في الدماغ يمكن أن يؤدي إلى مرض عصبي مدمر. وبالتالي، فمن الواضح أن التفاعل التوكسوبلازما brain- أمر حاسم لهذا المرض أعراض التي تنتجها التوكسوبلازما، ولكن لدينا القليل من الفهم والتفاعل الخلوي أو الجزيئية بين خلايا الجهاز العصبي المركزي (CNS) والطفيليات. في نموذج الفأر من الجهاز العصبي المركزي داء المقوسات وقد كان من المعروف لأكثر من 30 عاما أن الخلايا العصبية هي الخلايا التي استمرت الطفيلي، ولكن القليل من المعلومات متاح حول أيجزء من الخلايا العصبية مصاب عموما (سوما، التغصنات، محور عصبي)، وإذا هذه العلاقة الخلوية يتغير بين السلالات. في جزء منه، هذا النقص هو الثانوي لصعوبة التصوير وتصور الخلايا العصبية المصابة كلها من حيوان. ان مثل هذه الصور عادة ما تتطلب باجتزاء المسلسل وخياطة من الأنسجة تصويرها بواسطة المجهر الإلكتروني أو المجهري متحد البؤر بعد المناعية. من خلال الجمع بين عدة تقنيات، الطريقة الموصوفة هنا يمكن استخدام أبواب سميكة (160 ميكرون) لتحديد والصورة كاملة الخلايا التي تحتوي على الخراجات، والسماح التصور والتحليل من الفردية، والخلايا العصبية المصابة بشكل مزمن ثلاثي الأبعاد دون الحاجة لالمناعية، المجهر الإلكتروني ، أو باجتزاء المسلسل وخياطة. باستخدام هذه التقنية، يمكننا أن نبدأ في فهم العلاقة بين الطفيل الخلوية والخلايا العصبية المصابة.

ويشمل الرقم 7 صور تمثيلية اثنين من GFP + الخلايا العصبية من اثنين مختلفة 160 ميكرون أبواب سميكة، فضلا عن قياس التمثيل في المسافة من الكيس الى خلية الجسم عن الشكل 7B. أرقام التي تحتوي على كيس 7 A و B توضيح أن هذا جديدة بروتوكول يسمح ل?...

Watch this Scientific Journal Video about 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii at JoVE.com

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

باستخدام هذا البروتوكول، كنا قادرين على الصورة 160 ميكرون أقسام الدماغ سميكة من الفئران المصابة بالطفيلي التوكسوبلازما، والتي تمكن التصور وتحليل العلاقة المكانية بين الطفيلي encysting والخلايا العصبية المصابة.

التصوير 3-D وتحليل الخلايا العصبية المصابة<em&gt; في فيفو</em&gt; مع<em&gt; التوكسوبلازما</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

3-d-imaging-analysis-neurons-infected-vivo-with-toxoplasma

Research

JoVE Journal

Neuroscience

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

11.4K Views.  University of Arizona.  باستخدام هذا البروتوكول، كنا قادرين على الصورة 160 ميكرون أقسام الدماغ سميكة من الفئران المصابة بالطفيلي التوكسوبلازما، والتي تمكن التصور وتحليل العلاقة المكانية بين الطفيلي encysting والخلايا العصبية المصابة. 

Video: 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

تحجيم Fluidic صالحة للحقن الفيروسية استهداف سليمة دوائر الدماغ 3 - D

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

التصوير الضوئي للخلايا العصبية في العقدة كراب فموي معدي مع الجهد حساسة للملونات

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

الفلورسنت وصفها الوراء يسمح لخارج الخلية المستهدفة واحدة وحدة التسجيل من الخلايا العصبية التي تم تحديدها<em&gt; في الجسم الحي</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

تصوير الأعصاب وظيفية عن طريق الموجات فوق الصوتية تعطيل حاجز الدم في الدماغ والتصوير بالرنين المغناطيسي المنغنيز معززة لل

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

في الجسم الحي ثنائي الفوتون التصوير من الخبرة التي تعتمد على التغيرات الجزيئية في الخلايا العصبية القشرية

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

تعديل لColliculo-مهادية قشرية ماوس الدماغ شريحة، دمج الطباعة 3-D من مكونات الغرفة ومتعدد المقاييس التصوير الضوئي

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

في فيفو التصوير المتعدد الوسائط، وتحليل للماوس المستحثة بالليزر طراز نيوفاسكولاريزيشن تشورويدال

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

في فيفو اثنين-فوتون تصوير الخلايا العصبية القشرية في الفئران حديثي الولادة

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

توليد الجيل من 3D الكولاجين القائم على Hydrogels لتحليل النمو المحوري والسلوك أثناء تطوير الجهاز العصبي

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.