وأيد OG - P بواسطة CONACYT ق منحة (CB - 2008-101476) وFRABA (686/10). AQ - H بدعم من المعهد الوطني للصحة ، ومعهد هوارد هيوز الطبي ، ومؤسسة روبرت وود جونسون ومؤسسة الخلايا الجذعية ولاية ماريلاند.

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل الإبر الزجاج قبل الجراحة الماوس : <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع أنبوب زجاجي الشعرية في مجتذب micropipette. </li><li style=";text-align:right;direction:rtl"> الحرارة منتصف أنبوب زجاجي لتليين أنبوب زجاجي في منطقة المترجمة. </li><li style=";text-align:right;direction:rtl"> تمديد أنبوب زجاجي على طول محورها الطولي من قبل مسافة كافية للتسبب الأولية انخفاضا في قطر أنبوب زجاجي في منطقة المترجمة. </li><li style=";text-align:right;direction:rtl"> إبقاء تمتد أنبوب زجاجي حتى ينكسر. بهذه الطريقة ، يتم الحصول على متطابقتين الإبر واحد للبرميل. </li></ol></li><li style=";text-align:right;direction:rtl"> وضع الإبرة الزجاج في microforge. زاوية 30 درجة تكفي لشطبة غيض. </li><li style=";text-align:right;direction:rtl"> الاختيار تحت المجهر أن كل إبرة والقطر الداخلي الصحيح. بالنسبة لمعظم العقاقير ماء قطرها ميكرون 30-50 الداخلية مثالية (الشكل 1). </li><li style=";text-align:right;direction:rtl"> تملأ الإبرة مع الزيوت المعدنية من أوسع نهاية الإبرة الزجاج. اسمحوا ان الزيوت المعدنية (MSDS ، القط. M7700) يدخل من الشعرية حتى تصل ~ 50 ٪ من طول الأنبوب الشعرية. وضعت عالية فراغ الشحوم (داو كورننغ ، القط. AB - 05054) حول المكبس لختم أوسع نهاية الإبرة. </li><li style=";text-align:right;direction:rtl"> إدراج بواسطة المكبس أوسع نهاية الإبرة المعدنية ودفع النفط برفق حتى ينظر إلى قطرة صغيرة من زيت الخروج من رأس الإبرة الزجاج. </li><li style=";text-align:right;direction:rtl"> تأمين الزجاج في إبرة صاحب microinjector. </li><li style=";text-align:right;direction:rtl"> تخدير الفأر مع آفيرتين 2.5 ٪ (2،2،2 - ثلاثي بروم إيثانول + كحول أميلي ثالثي ، 01:01 ث / ت). جرعة من 25 غرام 30μL في داخل الصفاق. </li><li style=";text-align:right;direction:rtl"> وضع وسادة التدفئة على الجهاز للحفاظ على درجة حرارة المجسم الحيوان الجسم عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> مكان آمن ورئيس جهاز الماوس في المجسم باستخدام قضبان الأذن وحامل الأسنان. </li><li style=";text-align:right;direction:rtl"> تنظيف رأس الفأر مع 0.1 ٪ كلورهكسيدين غلوكونات الحل ، تليها الايثانول 70 ٪ و 0.1 ٪ جلوكونات الكلورهيكسيدين للمرة الثانية. </li><li style=";text-align:right;direction:rtl"> شق الجلد مع شفرة جراحية # 15 من آذان الفأر إلى خط الجمجمة لامبا. </li><li style=";text-align:right;direction:rtl"> تلميع الجمجمة مع القطن المبادلة لتجف بها وسحب الجلد خارج الحقل الجراحية. </li><li style=";text-align:right;direction:rtl"> استخدام غيض من الزجاج لإبرة نقطة في قمة الرأس للخياطة Bregma. يوجد لديك لضبط الموقف المبدئي من حاقن (في تنسيق &quot;صفر&quot;). </li><li style=";text-align:right;direction:rtl"> تعيين نقطة لحفر عن طريق تحريك &quot;X&quot; و &quot;Y&quot; محور الجهاز المجسم على الجمجمة. </li><li style=";text-align:right;direction:rtl"> حفر ثقوب دقيقة جدا في التنسيق المحدد. وقد سبق تعقيم الأدوات المعدنية وMicrodrills أخرى عند 250 درجة مئوية لمدة 60 ثانية مع حبة تعقيم الزجاج الجافة (كول بالمر ، القط. EW - رقم 10779-00). </li><li style=";text-align:right;direction:rtl"> ملقط مع غرامة إزالة بعناية أعمق طبقة من العظام وكسر الغشاء duramatter (سوف نرى بعض السائل الدماغي الشوكي تسرب). </li><li style=";text-align:right;direction:rtl"> تأكد من أن الإبرة يمكن أن تذهب من خلال الثقوب المحفورة. </li><li style=";text-align:right;direction:rtl"> 1 ميكرولتر ماصة للدواء الذي تريد حقن ووضعها على قطعة صغيرة من parafilm. </li><li style=";text-align:right;direction:rtl"> وضع parafilm على الجمجمة. </li><li style=";text-align:right;direction:rtl"> امتصاص الدواء من خلال الدوران على العجلات microinjector ، في حين كنت الاختيار تحت المجهر أن السائل هو الصعود إلى الإبرة الزجاج. </li><li style=";text-align:right;direction:rtl"> إزالة parafilm وإعادة تعيين تنسيق صفر. </li><li style=";text-align:right;direction:rtl"> تحرك الإبرة إلى إحداثيات المحدد ، أسفل حامل حتى غيض من الزجاج إبرة اللمس برفق على سطح الدماغ ، وتعيين &quot;Z&quot; تنسيق في صفر. </li><li style=";text-align:right;direction:rtl"> إدخال الإبرة في الزجاج لحمة على عمق الدماغ المحدد. </li><li style=";text-align:right;direction:rtl"> حقن الدواء ببطء بمعدل NL 1 ~ / ثانية. </li><li style=";text-align:right;direction:rtl"> عندما يتم حقن حجم الرغبة ، والسماح للنشر المخدرات في لحمة لمدة 2 دقيقة. ثم الشروع في إزالة الإبرة بسلاسة وببطء. </li><li style=";text-align:right;direction:rtl"> الصمغ على الجرح الجراحي وإزالة الحيوان من الجهاز المجسم ووضع الحيوانات في قفص قبل تحسنت تحتوي على سرير نظيف للانتعاش التخدير. </li><li style=";text-align:right;direction:rtl"> لتوفير ما بعد الجراحة تلقى تسكين جميع الحيوانات 5 ملغ / كغ تحت الجلد Ketorolac كل ساعة 12 لمدة 24 ساعة. </li></ol> ممثل النتائج : عند اتباع هذا البروتوكول ، ويتم الحصول على حقن دقيقة جدا ومسار الإبرة ضيقة جدا تقلل من إصابات الدماغ. نتيجة ممثل هذا الأسلوب ، نحن حقن lysophosphatidyl الكولين (lysolecithin) في الجسم الثفني التي تنتج من إزالة الميالين مساحات المادة البيضاء 1-4. للحد من إصابات الدماغ التي تنتجها الإبرة الزجاج ، وحقن نحن فقط من 20 NL lysolecithin في الجسم الثفني ، ولكن يمكن حقن كميات أكبر إذا كان ذلك مطلوبا بقدر 200 NL مع نفس الأسلوب. تم الكشف عن إزالة الميالين بواسطة الغائب التعبير بروتين النخاعين الأساسي في مساحات المادة البيضاء (الشكل 2). <img src="/files/ftp_upload/2082/2082fig1.jpg" alt="الشكل 1" /> الشكل 1. إبرة الزجاجية التي يبلغ قطرها 50 ميكرومتر. المسافة قصيرة بين اثنين من القرادفي جدول يمثل طول 50 ميكرومتر. <img src="/files/ftp_upload/2082/2082fig2.jpg" alt="الشكل 2" /> حقن Lysolecithin الرقم 2. في الجسم الثفني. ويرد إزالة الميالين كتعبير المايلين لا البروتين الأساسي (منطقة منقط). لاحظ صغر حجم الجهاز إبرة الزجاج (الأسهم). بار = 100 ميكرومتر

<ol>
	<li>Menn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+oligodendrocytes+in+the+subventricular+zone+of+the+adult+brain.">Origin of oligodendrocytes in the subventricular zone of the adult brain.</a> J Neurosci. 26, 7907-7918 (2006).</li><li>Gonzalez-Perez, O., Romero-Rodriguez, R., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Growth+Factor+Induces+the+Progeny+of+Subventricular+Zone+Type+B+Cells+to+Migrate+and+Differentiate+into+Oligodendrocytes.">Epidermal Growth Factor Induces the Progeny of Subventricular Zone Type B Cells to Migrate and Differentiate into Oligodendrocytes.</a> Stem Cells. 27, 2032-2043 (2009).</li><li>Hall, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+aspects+of+remyelination+after+demyelination+produced+by+the+intraneural+injection+of+lysophosphatidyl+choline.">Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidyl choline.</a> J Cell Sci. 13, 461-477 (1973).</li><li>Webster, G. R., Thompson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+presence+of+lysolecithin+in+nervous+tissues.">Observations on the presence of lysolecithin in nervous tissues.</a> Biochim Biophys Acta. 63, 38-45 (1962).</li><li>Doetsch, F., Caille, I., Lim, D. A., Garc&#237;a-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subventricular+Zone+Astrocytes+Are+Neural+Stem+Cells+in+the+Adult+mammalian+Brain.">Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult mammalian Brain.</a> Cell. 97, 1-20 (1999).</li><li>Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+converts+transit-amplifying+neurogenic+precursors+in+the+adult+brain+into+multipotent+stem+cells.">EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells.</a> Neuron. 36, 1021-1034 (2002).</li><li>Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+seizures+by+transplantation+of+cortical+GABAergic+interneuron+precursors+into+Kv1.1+mutant+mice.">Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.</a> Proc Natl Acad Sci U S A. 106, 15472-15477 (2009).</li><li>Richardson, R. M., Barbaro, N. M., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+cell+transplantation+for+temporal+lobe+epilepsy.">Developing cell transplantation for temporal lobe epilepsy.</a> Neurosurg Focus. 24, E17-E17 (2008).</li><li>Alvarez-Dolado, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J Neurosci. 26, 7380-7389 (2006).</li></ol>

الإعلان عن أي تضارب في المصالح.

وأظهرت الأسلوب في هذا الفيديو هو مفيد جدا لتقديم معظم العقاقير أو ناقلات فيروسية في أماكن دقيقة جدا في الدماغ. بعض المزايا الرئيسية لهذه التقنية هي موثوقية نقطة الاستهداف ، ودقة من الحقن وصغر حجم المخ ، والضرر الآفة المسالك 1 ، 2 ، 5 ، 6. يجب مرة واحدة موحدة هي تقني...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Capillary glass tube</td>
<td>&nbsp;</td>
<td>Wiretrol I</td>
<td>5-000-1001</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model 730</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>Model 48000</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>MSDS</td>
<td>M7700</td>
<td>&nbsp;</td>
<tr>
<td>High-vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>05054-AB</td>
<td>&nbsp;</td>
<tr>
<td>Anesthesia: 2.5% Avertin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2,2,2-tribromoethanol + tert-amyl alcohol, 1:1 w/v</td>
</tr>
<tr>
<td>Heater pad</td>
<td>&nbsp;</td>
<td>Mastex</td>
<td>Model 900</td>
<td>&nbsp;</td>
<tr>
<td>Stereotactic device</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model Kopf-900</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scalpel blade # 15</td>
<td>&nbsp;</td>
<td>Medi-Cut</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro driller</td>
<td>&nbsp;</td>
<td>Ideal Micro Drill</td>
<td>67-1000</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Fine Scientific Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-&mu;L Micropipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm M.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Vasiorkop with Contraves system</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>Model MO-10</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants

Microinjections في لحمة الدماغ هي الاجراءات الهامة لتسليم المخدرات ، وناقلات فيروسية أو زرع الخلايا. آفة الدماغ التي تنتج إبرة حقن خلال مساره هو مصدر قلق كبير خصوصا في مخ الفأر ليست فقط في الدماغ هو صغير ولكن أيضا في بعض الأحيان هناك حاجة لحقن متعددة. نعرض هنا طريقة لإنتاج الزجاج الإبر الشعرية مع التجويف 50 ميكرون مما يقلل بشكل كبير من تلف في الدماغ ويسمح لاستهداف دقيقة في الدماغ القوارض. هذا الأسلوب يسمح للتسليم كميات صغيرة (2-10 NL) ، ويقلل من مخاطر النزيف ، ويقلل من انتشار المخدرات السلبي في لحمة الدماغ. باستخدام مختلفة الحجم من أنابيب الزجاج الشعرية ، أو تغيير تجويف الإبرة ، يمكن حقنه عدة أنواع من المواد والخلايا. Microinjections مع أنبوب زجاجي الشعرية تمثل تحسنا كبيرا في تقنيات الحقن العميق للمخ واستهداف مع الحد الأدنى من الأضرار الجانبية في القوارض الصغيرة.

Watch this Scientific Journal Video about Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants at JoVE.com

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

في هذه المقالة ، وتبين لنا طريقة لجعل الإبر الزجاج الشعرية مع التجويف 50 ميكرومتر. هذا الأسلوب يقلل بشكل ملحوظ من تلف في الدماغ ويقلل من انتشار السلبية للمخدرات ويسمح باستهداف دقيق في الدماغ القوارض.

استهداف المنشآت الدماغ العميق مع Microinjections لتسليم المخدرات وناقلات فيروسية ، أو زرع الخلايا

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

targeting-deep-brain-structures-with-microinjections-for-delivery

Research

JoVE Journal

Neuroscience

12.5K Views.  University of Colima. في هذه المقالة ، وتبين لنا طريقة لجعل الإبر الزجاج الشعرية مع التجويف 50 ميكرومتر. هذا الأسلوب يقلل بشكل ملحوظ من تلف في الدماغ ويقلل من انتشار السلبية للمخدرات ويسمح باستهداف دقيق في الدماغ القوارض.

Video: Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

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 ...

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

داخل القحف حقن الغدة المرتبطة ناقلات فيروسية

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

تحفيز الدماغ العميق مع الرنين المغناطيسي الوظيفي في وقت واحد في القوارض

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

غير زجرية-EEG القياسات اللاسلكية: فوق الجافية والعميق داخل المخ EEG التجسيمي الكهربائي التنسيب

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

الإبر مخصصة لميكروينجيكشنز في الدماغ القوارض

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

استكشاف الفضاء السحيق-كشف تشريح لهياكل تلين للكشف عن البطينات الجانبية للدماغ البشري

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

بيولوجيا الخلية كمية من نيوروديجينيريشن في المورفولوجية من خلال تحليل غير منحازة للبروتينات فلوريسسينتلي المعلمة باستخدام إيماجيج

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

في فيفو إعادة برمجة مباشرة من الخلايا الغلية المقيمة في الخلايا العصبية المشتركة عن طريق الحقن داخل الدماغ من ناقلات الفيروسية

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Brain Morphology of Cannabis Users With or Without Psychosis: A Pilot MRI Study

Cannabis is the illicit drug most commonly used worldwide, and its consumption can both induce psychiatric symptoms in otherwise healthy subjects and unmask a florid psychotic picture in patients with a prior psychotic risk. Previous studies suggest that chronic and long-term cannabis exposure may exert significant negative effects in brain areas enriched with cannabinoid receptors. However, whether brain alterations determined by cannabis dependency will lead to a clinically significant phenotype or to a psychotic outbreak at some point of an abuser&#8217;s life remains unclear. The aim of this study was to investigate morphological brain differences between chronic cannabis users with cannabis-induced psychosis (CIP) and non-psychotic cannabis users (NPCU) without any psychiatric conditions and correlate brain deficits with selective socio-demographic, clinical and psychosocial variables.
3T magnetic resonance imaging (MRI) scans of 10 CIP patients and 12 NPCU were acquired. The type of drug, the frequency, and the duration, as well socio-demographic, clinical and psychosocial parameters of dependency were measured. CIP patients had extensive grey matter (GM) decreases in right superior frontal gyrus, right precentral, right superior temporal gyrus, insula bilaterally, right precuneus, right medial occipital gyrus, right fusiform gyrus, and left hippocampus in comparison to chronic cannabis users without psychosis. Finally, in CIP patients, the results showed a negative correlation between a domain of the Brief Psychiatric Rating Scale (BPRS), BPRS-Activity, and selective GM volumes. Overall, the results suggest that cannabis-induced psychosis is characterized by selective brain reductions that are not present in NPCU. Therefore, neuroimaging studies may provide a potential ground for identifying putative biomarkers associated with the risk of developing psychosis in cannabis users.

This is a 3T magnetic resonance imaging study aiming to investigate grey matter volume differences between cannabis-induced psychosis patients and non-psychotic chronic cannabis users.

مورفولوجيا الدماغ من متعاطي القنب مع أو بدون الذهان: دراسة التصوير بالرنين المغناطيسي التجريبية

Cannabis is the illicit drug most commonly used worldwide, and its consumption can both induce psychiatric symptoms in otherwise healthy subjects and ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.