وأيد هذا العمل يا CIRM زمالة predoctoral (EDK)، NARSAD جائزة الباحث الصغير (DK)، وجائزة أدمغة NIMH (R01MH087495) (DK).

<ol>
	<li>Kirby, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+amygdala+regulation+of+adult+hippocampal+neurogenesis+and+fear-related+activation+of+newborn+neurons.">Basolateral amygdala regulation of adult hippocampal neurogenesis and fear-related activation of newborn neurons.</a> Mol. Psychiatry. , (2011).</li><li>Goosens, K. A., Maren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contextual+and+auditory+fear+conditioning+are+mediated+by+the+lateral,+basal,+and+central+amygdaloid+nuclei+in+rats.">Contextual and auditory fear conditioning are mediated by the lateral, basal, and central amygdaloid nuclei in rats.</a> Learn Mem. 8, 148-155 (2001).</li><li>Korz, V., Frey, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+modulation+of+hippocampal+long-term+potentiation+under+stress+and+no-stress+conditions+in+basolateral+amygdala-lesioned+and+intact+rats.">Bidirectional modulation of hippocampal long-term potentiation under stress and no-stress conditions in basolateral amygdala-lesioned and intact rats.</a> J. Neurosci. 25, 7393-7400 (2005).</li><li>Bhatnagar, S., Vining, C., Denski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chronic+stress-induced+changes+in+hypothalamic-pituitary-adrenal+activity+by+basolateral+amygdala.">Regulation of chronic stress-induced changes in hypothalamic-pituitary-adrenal activity by basolateral amygdala.</a> Ann. N.Y. Acad. Sci. 1032, 315-319 (2004).</li><li>Ikegaya, Y., Saito, H., Abe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+hippocampal+long-term+potentiation+in+basolateral+amygdala-lesioned+rats.">Attenuated hippocampal long-term potentiation in basolateral amygdala-lesioned rats.</a> Brain Res. 656, 157-164 (1994).</li><li>Ikegaya, Y., Saito, H., Abe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basomedial+and+basolateral+amygdaloid+nuclei+contribute+to+the+induction+of+long-term+potentiation+in+the+dentate+gyrus+in+vivo.">The basomedial and basolateral amygdaloid nuclei contribute to the induction of long-term potentiation in the dentate gyrus in vivo.</a> Eur. J. Neurosci. 8, 1833-1839 (1996).</li><li>Carter, R. N., Pinnock, S. B., Herbert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+amygdala+modulate+adaptation+to+repeated+stress.">Does the amygdala modulate adaptation to repeated stress.</a> Neurosciene. 126, 9-19 (2004).</li><li>Roozendaal, B., McGaugh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+amygdala+lesions+block+the+memory-enhancing+effects+of+glucocorticoid+administration+in+the+dorsal+hippocampus+of+rats.">Basolateral amygdala lesions block the memory-enhancing effects of glucocorticoid administration in the dorsal hippocampus of rats.</a> Eur. J. Neurosci. 9, 76-83 (1997).</li><li>Roesler, R., Roozendaal, B., McGaugh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+amgdala+lesions+block+the+memory-enhancing+effect+of+8-Br-cAMP+infused+into+the+entorhinal+cortex+of+rats+after+training.">Basolateral amgdala lesions block the memory-enhancing effect of 8-Br-cAMP infused into the entorhinal cortex of rats after training.</a> Eur. J. Neurosci. 15, 905-910 (2002).</li><li>Roozendaal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoid+effects+on+memory+consolidation+depend+on+functional+interactions+between+the+medial+prefrontal+cortex+and+basolateral+amygdala.">Glucocorticoid effects on memory consolidation depend on functional interactions between the medial prefrontal cortex and basolateral amygdala.</a> J. Neurosci. 29, 14299-14308 (2009).</li><li>Kaufer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restructuring+the+neuronal+stress+response+with+anti-glucocorticoid+gene+delivery.">Restructuring the neuronal stress response with anti-glucocorticoid gene delivery.</a> Nat. Neurosci. 7, 947-953 (2004).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain: in stereotaxic coordinates-the new coronal set. , (2004).</li><li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+Stereotaxic+Surgery+in+Rodents.">Survivable Stereotaxic Surgery in Rodents.</a> J. Vis. Exp. (20), e880 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

طريقة التجسيمي المقدمة هنا يسمح لآفة excitotoxic من مناطق الدماغ محددة عن طريق ضخ NMDA. ويمكن تكييف الطرق الأساسية التجسيمي الى بث مجموعة متنوعة من وكلاء الدوائية والبيولوجية بطريقة مواقع محددة. يمكن أيضا أن يتم تكييفها لاستهداف مجموعة متنوعة من مناطق الدماغ، التي يحددها إ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> ن ميثيل مد الاسبراجسي حمض 98٪ </td><td> فيشر العلمية </td><td> AC32919-0500 </td><td></td></tr><tr><td> المعيار المزدوج مختبر التجسيمي w/45 درجة. الأذن البارات </td><td> Stoelting </td><td> 51653 </td><td> البديل بائع: Kopf * ملاحظة تحذيرية: ضمان التوافق من الملحقات التجسيمي إذا شراء من بائعين متعددين </td></tr><tr><td> 10μl SYR الخاصة (* / * / *) </td><td> هاميلتون </td><td> 701SN </td><td></td></tr><tr><td> DREMEL موتو، أداة </td><td> Stoelting </td><td> 58600 </td><td> البديل بائع: Kopf </td></tr><tr><td> كربيد الأزيز، قبضة HP، حجم 2 </td><td> شين الأسنان </td><td> 2284578 </td><td></td></tr><tr><td> Stoelting 6 برنامج الحقنةmable مضخة </td><td> Stoelting </td><td> 53140 </td><td> البديل بائع: Kopf </td></tr><tr><td> غير القابل للصدأ تحت الجلد 316 الفولاذ العادي الحائط أنابيب قياس 30 0.0123 &quot;OD X 0.00625&quot; معرف X 0.003 &quot;وول (إبرة الحقن) </td><td> أجزاء صغيرة </td><td> HTXX-30R، 06-05 </td><td></td></tr><tr><td> Intramedic PE 20 أنابيب (أنابيب التسريب) </td><td> VWR </td><td> 63019-025 </td><td></td></tr><tr><td> كليب لا ارادي، 9MM، غير معقمة </td><td> كينت العلمية كورب </td><td> INS500346 </td><td> البديل بائع: أدوات العلم الجميلة </td></tr><tr><td> منعكس مطبق كليب لقطات 9MM </td><td> كينت العلمية كورب </td><td> 12031-09 </td><td> البديل بائع: أدوات العلم الجميلة </td></tr><tr><td> منحني هارتمان مرقئ </td><td> أدوات العلم غرامة </td><td> 13003-10 </td><td></td></tr><tr><td> لندن ملقط </td><td> أدوات الالات غرامة </td><td> 11080-02 </td><td></td> &lt;/ TR&gt; <tr><td> 2٪ الكلورهيكسيدين حل </td><td> Allivet </td><td> 30159 </td><td> البديل بائع: PetSolutions </td></tr><tr><td> 10٪ حل البوفيدون اليود </td><td> CVS </td><td> SKU # 739575 </td><td></td></tr><tr><td> حار حبة معقم </td><td> هارفارد جهاز </td><td> 610183 </td><td></td></tr></tbody></table> الجدول رقم 1. جدول محدد الكواشف والمعدات.

stereotaxic surgery for excitotoxic lesion of specific brain areas in the adult rat

وتتوسط العديد من الوظائف السلوكية في الثدييات، بما في ذلك القوارض والبشر، وبصورة رئيسية من قبل مناطق الدماغ منفصلة. وهناك طريقة مشتركة لوظيفة المميزين من مناطق الدماغ المختلفة للسلوك أو النتائج التجريبية الأخرى هو تنفيذ الاجتثاث المترجمة من وظيفة. في البشر، وغالبا ما تتم دراسة السكان المريض مع آفات الدماغ المترجمة للعجز، أملا في الكشف عن وظيفة الكامنة وراء المنطقة المتضررة. في القوارض، يمكن للمرء أن تحفز تجريبيا آفات من مناطق محددة في الدماغ. ويمكن تحقيق آفة في عدة طرق. يمكن للآفات كهربائيا تسبب أضرارا موضعية ولكن سيضر مجموعة متنوعة من أنواع الخلايا، وكذلك الألياف العبور من مناطق أخرى من الدماغ الذي يحدث ليكون بالقرب من موقع الآفة. محرض قد تقنيات وراثية باستخدام خلية من نوع المروجين محددة تمكن أيضا في مواقع محددة الاستهداف. هذه التقنيات معقدة وليس من العملي دائما اعتمادا على المنطقة المستهدفة في المخ. Excitotآفة أوضاع مؤكسدة تستخدم جراحة التجسيمي، على النقيض من ذلك، هو واحد من الأساليب الأكثر موثوقية والعملية لlesioning الخلايا العصبية مثير دون الإضرار الخلايا الدبقية المحلية أو العبور الألياف. هنا، نقدم بروتوكولا للتسريب التجسيمي للexcitotoxin، ن ميثيل مد اسبارتاتي (NMDA)، في مجمع اللوزة basolateral. باستخدام مؤشرات التشريحية، ونحن نطبق إحداثيات التجسيمي لتحديد موقع لدينا هدف المنطقة من المخ وخفض إبرة حقن في مكان أعلى بقليل من الهدف. نحن لبث ثم excitotoxin لدينا في الدماغ، مما يؤدي إلى موت الخلايا العصبية excitotoxic قريب. بينما موضوعنا التجريبية من الاختيار هو فأر، يمكن تطبيق نفس الأساليب إلى غيرها من الثدييات، مع التعديلات المناسبة في المعدات والإحداثيات. ويمكن استخدام هذه الطريقة على مجموعة متنوعة من مناطق الدماغ، بما في ذلك اللوزة basolateral 1-6، وغيرها من اللوزة نوى 6، 7، 8 الحصين، والأنف والحنجرةorhinal قشرة (9) وقشرة الفص الجبهي 10. ويمكن أيضا استخدامه للبث المركبات البيولوجية مثل ناقلات فيروسية 1 و 11. ويمكن أيضا هذه التقنية الأساسية التجسيمي يمكن تكييفها لزرع المزيد من مضخات التناضحي دائم، مما يسمح للمزيد من التعرض لفترات طويلة الى مجمع المصالح.

Watch this Scientific Journal Video about Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat at JoVE.com

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

يوصف الاجتثاث استهدفت منطقة محددة في الدماغ (ق) عن طريق التسريب من excitotoxin باستخدام الإحداثيات التجسيمي. ويمكن أيضا هذه التقنية يمكن تكييفها للتسريب من غيرها من المواد الكيميائية في الدماغ الفئران.

التجسيمي جراحة الآفات Excitotoxic من مناطق محددة في الدماغ في الجرذ الكبار

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

stereotaxic-surgery-for-excitotoxic-lesion-specific-brain-areas-adult

Research

JoVE Journal

Neuroscience

35.2K Views.  University of California Berkeley. يوصف الاجتثاث استهدفت منطقة محددة في الدماغ (ق) عن طريق التسريب من excitotoxin باستخدام الإحداثيات التجسيمي. ويمكن أيضا هذه التقنية يمكن تكييفها للتسريب من غيرها من المواد الكيميائية في الدماغ الفئران.

Video: Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

جراحة القوارض التجسيمي والتحسينات نتائج الخيرية لرعاية الحيوان العلوم العصبية السلوكية

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

توسيع الجنينية والكبار الخلايا الجذعية العصبية من قبل<em&gt; في الرحم</em&gt; الصعق الكهربائي أو حقن التجسيمي الفيروسية

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

المجهرية من التعبير الجيني في الدماغ اسماك الزرد الكبار عن طريق حقن مكروي Cerebroventricular [أليغنوكليوتيد] Morpholino

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

الدماغ شريحة Biotinylation: و<em&gt; فيفو السابقين</em&gt; النهج لقياس منطقة محددة بلازما بروتين غشاء الاتجار الكبار الخلايا العصبية

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

باستخدام الإسفار خلية المنشط الفرز لفحص الخلية من نوع محدد التعبير الجيني في الأنسجة دماغ الفئران

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

تسريب التجسيمي من بلازميدة قليلة القسيمات اميلويد بيتا في الحصين ماوس

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

جراحة ستيريوتاكسيك للتلاعب بالجينات في خلايا أدمغة الفأر الولدان سترياتال

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

في فيفو الحقن المجسمة داخل الدماغ لتحفيز البصريات من المدخلات بعيدة المدى في شرائح الدماغ الماوس

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

تقييم الحث علي الاكتئاب علي المدى الطويل في شرائح البالغين المخيخار

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).