نود أن نشكر الدكتور ديفين غاري لبصيرته وردود الفعل على استخدام خلايا GRP.

<ol>
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وقد تم تمويل هذه الدراسة من قبل المعهد الوطني للصحة [P30HD024061 معاهد الصحة القومية (AWP)، NINDS K08NS063956 (AF)، وR01NS028208 (MVJ)]، ومؤسسة الطفل الأعصاب. محتويات هذا التقرير هي الجهة الوحيدة المسؤولة عن المؤلفين ولا تمثل بالضرورة وجهة النظر الرسمية للمعهد الوطني للصحة، DHHS. الكتاب ليس لديهم تضارب المصالح ذات الصلة لهذه الدراسة.

<p class="jove_content"&gt; اضطرابات في الجهاز العصبي المركزي المسألة أبيض وتشمل عددا كبيرا من مسببات، بما في ذلك أسباب وراثية التهابات، ونقص تروية والسامة<sup&gt; 19،21،22،23،24</sup&gt;. بينما التصلب المتعدد وvasculopathies هي السبب الرئيسي للمرض المادة البيضاء في البالغين، وتلين بيضاء حول البطينات الدماغية المرتبطة الخداج هو السبب الاكثر شيوعا للاصابة في المادة البيضاء في عدد السكان في مرحلة الطفولة. من أجل تحديد المزيد من آليات المرض إلى مزيد من الدراسة من نسب الخلايا ofoligodendrocytic، التي تتأثر كثيرا من جراء إهانة فترة ما حول الولادة، هو المطلوب. تم عزل GRPS من الحبل الشوكي القوارض الجنينية وتبين أن tripotential في الطبيعة، مما أدى إلى oligodendrocytes واثنين من السكان نجمية متميزة، ولكن لا تفرق في الخلايا العصبية<sup&gt; 2،25،26</sup&gt;. كذلك، ثبت عمليا أن الخلايا السليفة oligodendrocyte (OPC) مشتقة من العصب البصري والفئران الجنينية في وقت متأخر أو مبكر دماغ الفئران بعد الولادة إلى أن تنضج في oligodendrocytes يعمل في ظروف مناسبة وسائل الاعلام<sup&gt; 27،28</sup&gt;. وبالإضافة إلى ذلك، تم الجنين أو الكبار OPC حقوق الإنسان مستمد التلاعب بها الى مزيد من الظواهر oligodendrocyte أكثر نضجا<sup&gt; 29،30</sup&gt;.</p&gt;<p class="jove_content"وقد تم عزل&gt; GRP خلايا من الحبل الشوكي في كل من الفئران وE13.5 E12-13.5 في الفئران. في الخلافات الفئران تم الإبلاغ بين الخلايا GRP ظهري وبطني المشتق في استجابتها للظروف التي تعزز من جيل oligodendrocytes، أو الخلايا النجمية مع بطني المشتقة GRPS واظهار قدر أكبر من الميل إلى التفريق في الظواهر أكثر نضجا<sup&gt; 25،26،28</sup&gt;. هذا قد يكون راجعا إلى بطني على ظهري الهجرة من الخلايا الوليدة GRP، التي يمكن أن تكون أقل تقبلا لمنبهات التمايز. Immunopanning يسمح لsubpopulation التي تعبر عن A2B5 إلى أن تكون معزولة والتي تم جمعها لأغراض الصيانة في الثقافة.</p&gt;<p class="jove_content"&gt; يتم اشتقاق لدينا الماوس الثقافات GRP من مجموع الأنسجة النخاع الشوكي الذي ينتج غير المتجانسة، وسكان المجمع من الخلايا من الخلايا التي يتم اختيارها من قبل GRP الكيميائية (وسط) والتقنيات القائمة على الأجسام المضادة. في حين أن دراستنا قد تستخدم والحبل الشوكي الخلايا المشتقة الماوس GRP، وأكثر مؤخرا أنه أفيد اشتقاق خلايا مخ الفأر سلف oligodendrocyte المشتقة التي هي أيضا قادرة على تطوير في oligodendrocytes ناضجة<sup&gt; 31</sup&gt;. هنا يتم تربيتها في البداية الخلايا المشتقة كما oligospheres ذلك باستخدام مستنبت مناسب يتم معالجته الى PDGFαR المخصب + سكان OPC،<sup&gt; 31</sup&gt;. وقد ركزت دراستنا على تأثير نموذج لدينا من نقص الأوكسجين الاقناري الإهانة على الخلايا في الطيف نسب oligodendrocyte من A2B5 +، PDGFαR-GRP السلائف للبروتين النخاعين الأساسي معربا عن (MBP +) oligodendrocytes ناضجة. في الواقع لدينا<em&gt; في المختبر</emوقد أظهرت&gt; توصيف هذه الخلايا غير الناضجة GRP ناضجة الى خلايا MBP التعبير سواء في الزراعات الأحادية أو في الثقافات بالتعاون مع العصبونات القشرية. يمكن التلاعب بها هذه الخلايا في الثقافة في مسارات محددة التمايز لدراسة الآليات التي تشارك في فترة ما حول الولادة إصابة المادة البيضاء. هناك نماذج عدة من أمراض الماوس المادة البيضاء التي تنطوي على خلايا من نسب oligodendrocyte وتوفر هذه الفئة من السكان يسمح للتحقيق شامل في الآليات التي تتسبب في الاصابة وكذلك لاستكشاف جدوى الخيارات الممكنة العلاج بالخلايا.</p

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th></tr><tr><td> DMEM / F12 01:01 </td><td> إينفيتروجن </td><td> 11320033 </td></tr><tr><td> B27 </td><td> إينفيتروجن </td><td> 17504044 </td></tr><tr><td> N2 </td><td> إينفيتروجن </td><td> 17502048 </td></tr><tr><td> RH-FGF-الأساسية </td><td> إينفيتروجن </td><td> PHG0026 </td></tr><tr><td> التربسين (0.05٪) EDTA </td><td> جودة البيولوجي، المؤتمر الوطني العراقي. </td><td> 118-087-721 </td></tr><tr><td> بولي-L-يسين HBR </td><td> سيغما </td><td> P1274-25MG </td></tr><tr><td> Laminin </td><td> سيغما </td><td> L2020-1MG </td></tr><tr><td> الدناز 1 </td><td> سيغما </td><td> DN25 </td></tr></tbody></table>

derivation of glial restricted precursors from e13 mice

هذا هو بروتوكول لاشتقاق الخلايا الدبقية المقيد (GRP) مقدمة من الحبل الشوكي لأجنة الفأر E13. هذه الخلايا هي السلائف في وقت مبكر ضمن النسب الخلية oligodendrocytic. في الآونة الأخيرة، وقد تم دراسة هذه الخلايا كمصدر محتمل لعلاجات الأمراض التصالحية في المادة البيضاء. تلين بيضاء الدماغ المحيطة بالبطينات (PVL) هو السبب الرئيسي للمرض غير وراثية المادة البيضاء في مرحلة الطفولة ويؤثر على ما يصل الى 50٪ من الأطفال الخدج للغاية. وتشير هذه البيانات إلى حساسية متزايدة من الدماغ النامية من نقص التروية، نقص الأكسجة، الاكسدة والتي تستهدف بشكل انتقائي excitotoxicity المادة البيضاء الوليدة. السلائف المقيد الدبقية (GRP)، الخلايا الاولية oligodendrocyte (OPC) وoligodendrocytes غير ناضج (preOL) ويبدو أن اللاعبين الرئيسيين في تطوير PVL وتخضع للدراسات مستمرة. وعلاوة على ذلك، وقد حددت الدراسات السابقة مجموعة فرعية من أنسجة الجهاز العصبي المركزي والتي زادت القابلية للغلوتامات excitotoxicity كمافضلا عن نمط النمو في هذه الحساسية. مختبرنا يقوم حاليا بالتحقيق في دور الأسلاف oligodendrocyte في الخلايا PVL واستخدامها في مرحلة GRP للتنمية. نحن الاستفادة من هذه الخلايا المشتقة GRP في نماذج تجريبية عدة لاختبار استجابتهم لتحديد الضغوط بما يتفق مع PVL. يمكن التلاعب بها الخلايا في المختبر GRP إلى OPCs وpreOL للتجارب زرع مع نماذج PVL الماوس ونماذج في المختبر من PVL مثل السب بما في ذلك نقص التروية، نقص الأكسجة. باستخدام الخلايا المزروعة والدراسات في المختبر انه لن يتم تخفيض التباين بين التجارب التي يسهل تفسير البيانات. الخلايا المستزرعة كما يسمح لتخصيب من السكان GRP مع التقليل من تأثير تلوث خلايا غير GRP النمط الظاهري.

Watch this Scientific Journal Video about Derivation of Glial Restricted Precursors from E13 mice at JoVE.com

Derivation of Glial Restricted Precursors from E13 mice

هذا البروتوكول يحدد الاشتقاق من السلائف المقيد الغروية من النخاع الشوكي الجنين والمحافظة عليها في المختبر إما للزراعة أو لدراسة نسب oligodendrocytic.

الاشتقاق من السلائف المقيد الغروية من E13 الفئران

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

derivation-of-glial-restricted-precursors-from-e13-mice

Research

JoVE Journal

Neuroscience

11.2K Views.  Johns Hopkins University. هذا البروتوكول يحدد الاشتقاق من السلائف المقيد الغروية من النخاع الشوكي الجنين والمحافظة عليها في المختبر إما للزراعة أو لدراسة نسب oligodendrocytic.

Video: Derivation of Glial Restricted Precursors from E13 mice

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

كفاءة اشتقاق أسلاف الإنسان متعلق بالخلايا العصبية والخلايا العصبية من افرة القوة الخلايا الجذعية الجنينية البشرية التعريفي مع جزيء صغير

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

الاشتقاق الثقافات Oligodendrocyte المخصب وOligodendrocyte نيورون / Myelinating المشارك من الثقافات في مرحلة ما بعد الولادة مناديل الفئران

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

حمل اللدونة من نجمي المستقبلات بواسطة التلاعب أسعار اطلاق النار متعلق بالخلايا العصبية

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

خالية من التغذية اشتقاق الخلايا العصبية كريست السلف من افرة القوة البشرية الخلايا الجذعية

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

إعداد الثقافات الدبقية المختلطة الابتدائية من الكبار ماوس النخاع الشوكي الأنسجة

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

الاشتقاق الثقافات السحايا الرقيقة يزدرع من تشريح الجهات المانحة الدماغ البشري

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

تمايز الخلايا الجذعية الجنينية الماوس في السلائف إينتيرنيورون القشرية

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

غرس القنية إلى ماجنا تشيستيرنا من القوارض

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

تسجيل مكانياً مقيد التذبذبات في قرن آمون التصرف الفئران

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

زرع هوموتشرونيك إينتيرنيورون السلائف في أوائل العقول الماوس بعد الولادة

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...