אנו מודים לד&quot;ר מייקל ווטן על עזרתו בהכנת כתב היד. עבודה זו נתמכה על ידי NIH 2RO1NS033661 (MWW).

<ol>
	<li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126, 397-442 (1977).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li><li>Brewer, G. 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A., Myers, C., Meeter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortico-hippocampal+interaction+and+adaptive+stimulus+representation:+a+neurocomputational+theory+of+associative+learning+and+memory.">Cortico-hippocampal interaction and adaptive stimulus representation: a neurocomputational theory of associative learning and memory.</a> Neural Netw. 18, 1265-1279 (2005).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li><li>Mao, L., Wang, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gliogenesis+in+the+striatum+of+the+adult+rat:+alteration+in+neural+progenitor+population+after+psychostimulant+exposure.">Gliogenesis in the striatum of the adult rat: alteration in neural progenitor population after psychostimulant exposure.</a> Brain Res. Dev. Brain Res. 130, 41-51 (2001).</li><li>Mao, L., Wang, J. 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J., Federoff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+-free+media+for+neural+cell+cultures.">Serum -free media for neural cell cultures.</a> Protocols for Neural Cell Culture. , (2001).</li><li>Shen, J., Watanabe, S., Kaneko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+dissociation+with+papain+reduces+the+density+of+cGMP-activated+channels+of+the+retinal+rod.">Cell dissociation with papain reduces the density of cGMP-activated channels of the retinal rod.</a> Jpn. J. Physiol. 45, 151-164 (1995).</li><li>Stratmann, G., Sall, J. W., May, L. D., Loepke, A. W., Lee, M. 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אין ניגוד עניינים הצהיר.

תרבויות בהיפוקמפוס נעשה שימוש בביולוגיה מולקולרית של יותר מ -20 שנה. אמנם באופן עקרוני, תרבויות עצביים יכול להתבצע מכל חלק של המוח, בהיפוקמפוס תרבויות הוכיחו להיות פופולרי ביותר בשל הארכיטקטורה הפשוטה יחסית של האוכלוסייה תא העצב בהיפוקמפוס 7. תרבויות בהיפוקמפו?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> מוכר </td><td> מספר קטלוגי </td></tr><tr><td> זנב עכברוש קולגן 1 </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> פולי-D-ליזין פתרון </td><td> Chemicon </td><td> A-003-E </td></tr><tr><td> הנקס תמיסת מלח מאוזן </td><td> Invitrogen </td><td> 14175-095 </td></tr><tr><td> טריפסין פתרון (1X) 0.25%, נוזל </td><td> Invitrogen </td><td> 15050-065 </td></tr><tr><td> בינוני NeuroBasal (1X) נוזלי </td><td> Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 מוסף (50X) נוזלי </td><td> Invitrogen </td><td> 17504-044 </td></tr><tr><td> L-גלוטמין 200 מ&quot;מ (100x) נוזלי </td><td> Invitrogen </td><td> 25030-149 </td></tr><tr><td> פניצילין (10,000 יחידות למ&quot;ל) / סטרפטומיצין (10,000 מיקרוגרם / מ&quot;ל) </td><td> Invitrogen </td><td> 15140-148 </td></tr><tr><td> HI-תורמים סוס סרום </td><td> אטלנטה ביולוגיות </td><td> S12150H </td></tr></tbody></table>

isolation and culture of hippocampal neurons from prenatal mice

תרבויות העיקריות של עכברוש נוירונים בהיפוקמפוס Murine נמצאים בשימוש נרחב כדי לחשוף את המנגנונים התאיים בנוירוביולוגיה. על ידי בידוד הולך וגדל נוירונים בודדים, החוקרים מסוגלים לנתח מאפיינים הקשורים בסחר נייד, מבנה נייד ולוקליזציה חלבון הפרט באמצעות מגוון של שיטות ביוכימיות. תוצאות ניסויים אלה הם קריטיים לבדיקת תיאוריות העוסקות הבסיס העצבי של הזיכרון והלמידה. עם זאת, תוצאות ברורות של צורות אלה של ניסויים מבוססות על היכולת לגדול תרבויות עצביים עם זיהום מינימלי של סוגי תאים אחרים במוח. בפרוטוקול זה, אנו משתמשים בתקשורת ספציפיים המיועדים הצמיחה נוירון ו דיסקציה זהירה של הרקמה העוברית בהיפוקמפוס כדי לייעל צמיחה של נוירונים בריאים תוך מזעור סוגי תאים מזהמים (האסטרוציטים IE). עוברי רקמות העכבר ההיפוקמפוס יכול להיות קשה יותר לבודד מאשר רקמת מכרסם דומה בשל גודל המדגם עבור dissection. אנחנו מראים טכניקות הנתיחה מפורטים של ההיפוקמפוס מ גורים ביום 19 (E19) עובריים של עכברים. לאחר רקמת ההיפוקמפוס הוא מבודד, ניתוק עדין של תאים עצביים מושגת עם ריכוז מדוללת של טריפסין הפרעה מכנית נועד תאים נפרדים של רקמת חיבור, תוך מתן נזק מינימלי תאים בודדים. תיאור מפורט של איך להכין בטפי להחדרת נוזלים לשמש הפרעה כלול. צפיפות ציפוי אופטימליים ניתנים חיסונית הקרינה פרוטוקולים על מנת למקסם את תרבית תאים מוצלח. הפרוטוקול מספק שיטה מהירה (כ 2 שעות) ויעיל התרבות של תאים מרקמות העכבר בהיפוקמפוס.

Watch this Scientific Journal Video about Isolation and Culture of Hippocampal Neurons from Prenatal Mice at JoVE.com

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

אנו מספקים פרוטוקול תרבות מטוהרים מאוד נוירונים בהיפוקמפוס של המוח עכבר טרום לידתי ללא שימוש שכבת תא גלייה מזין.

בידוד והתרבות של נוירונים בהיפוקמפוס מעכברים טרום לידתי

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

isolation-and-culture-of-hippocampal-neurons-from-prenatal-mice

Research

JoVE Journal

Neuroscience

70.2K Views.  Auburn University. אנו מספקים פרוטוקול תרבות מטוהרים מאוד נוירונים בהיפוקמפוס של המוח עכבר טרום לידתי ללא שימוש שכבת תא גלייה מזין.

Video: Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection והתרבות של נוירונים Commissural מחוט השדרה עובריים

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection ותרבות ראשונית של נוירונים בהיפוקמפוס עכבר עובריים קליפתיים

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

סידן פוספט Transfection של ראשוני נוירונים בהיפוקמפוס

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

בידוד והתרבות של ניתק עצב סנסורי מאפרוח עוברים

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

בידוד, תרבות והטווח ארוך תחזוקה של יסודיים mesencephalic דופאמין נוירונים ממוח עוברי מכרסם

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

בהיפוקמפוס ראשי בצפיפות נמוכה תרבות Neuron

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

נוירונים חוט השדרה בידוד ותרבות מ עכברים ניאונטליים

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

התרבות הראשית של הנוירונים מבודדים מתוך עכבר ממוח עובריים

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

בידוד ותרבות של והעושות, טרוליר, ושדרה מוטוריים נוירונים מ בטרום לידתי : עכברים הטרנסגניים

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.