وأيد هذا العمل عن طريق منح المعاهد الوطنية للصحة لNS052535 شكيمة وEY014061 وEY014997 لARL ، فضلا عن جائزة UL1DE019583 - 02 الكونسورتيوم.

للحيلولة دون تحيز ، وينبغي المجرب أداء التقييمات لم يكن لديك معرفة التركيب الوراثي للحيوان. وسجل التدابير الفردية على مقياس من 0-3 ، مع 0 يمثل غياب النمط الظاهري و 3 ذات الصلة التي تمثل مظهرا أشد. يتم تنفيذ كل اختبار عدة مرات للتأكد من أن النتيجة هي استنساخه. وسوف يعقد السمنة تفسير جميع التدابير وصفها. قد يرغب المحقق لوزن الفئران التالية التهديف النمط الظاهري لتقييم دور محتمل للسمنة في النتائج. اختبار الحافة الاختبار هو قياس الحافة مباشرة من التنسيق ، وهو ضعف في الترنحات المخيخ والعديد من الاضطرابات العصبية الأخرى. هذا الإجراء هو الأكثر مقارنة مباشرة الى علامات الإنسان للرنح مخيخي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رفع الماوس من القفص ووضعه على الرف في القفص. سوف الفئران عادة المشي على طول الحافة ومحاولة لينزل مرة أخرى إلى القفص. </li><li style=";text-align:right;direction:rtl"> مراقبة الماوس كما يمشي على طول الحافة القفص ويخفض نفسه في قفصه. وفأر من النوع البري عادة المشي على طول الحافة دون أن تفقد توازنها ، وسوف يخفض نفسه مرة أخرى في قفص بأمان ، وذلك باستخدام الكفوف. يتم تعيين هذه النتيجة 0. إذا كان الماوس يفقد مكانته في حين أن المشي على طول الحافة ، ولكن من ناحية أخرى يبدو منسقة ، فإنه يحصل على درجة من 1. إذا كان لا يستخدم فعليا رجليه الخلفيتين ، أو الأراضي على رأسه بدلا من الكفوف عند النزول الى داخل القفص ، فإنه يحصل على النتيجة من 2. إذا كان يقع قبالة الحافة ، أو يكاد ، في حين أن المشي أو محاولة لخفض نفسه ، أو يهز وترفض التحرك على كافة على الرغم من التشجيع ، ويتلقى على 3 نقاط. وسوف تتطلب بعض الفئران دفع لطيف لتشجيعهم على السير على طول الحافة أو النزول الى داخل القفص. </li><li style=";text-align:right;direction:rtl"> سجل نتيجة اختبار الحافة. </li></ol> Hindlimb الشبك Hindlimb الشبك هي علامة على تطور المرض في عدد من نماذج الماوس من التنكس العصبي ، بما في ذلك بعض الترنحات المخيخ [1]. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فهم بالقرب من قاعدة الذيل ورفع الماوس واضح للجميع الأجسام المحيطة بها. </li><li style=";text-align:right;direction:rtl"> مراقبة الموقف hindlimb لمدة 10 ثانية. إذا كنت على الدوام hindlimbs مفلطحة الخارج ، بعيدا عن البطن ، ويتم تعيينه على درجة من 0. إذا تم سحب one hindlimb نحو البطن لأكثر من 50 ٪ من الوقت مع وقف التنفيذ ، فإنه يتلقى بنتيجة 1. إذا تراجع جزئيا على حد سواء hindlimbs نحو البطن لأكثر من 50 ٪ من الوقت مع وقف التنفيذ ، فإنه يحصل على النتيجة من 2. إذا تراجعوا بالكامل hindlimbs ولمس البطن لأكثر من 50 ٪ من الوقت مع وقف التنفيذ ، فإنه يتلقى على 3 نقاط. </li><li style=";text-align:right;direction:rtl"> مكان الماوس مرة أخرى في قفص وسجلها hindlimb درجة الشبك. </li></ol> مشية مشية هو قدر من التنسيق وظيفة العضلات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة الماوس من قفصه ووضعه على سطح مستو مع رئيسها تواجه بعيدا عن التحقيق. </li><li style=";text-align:right;direction:rtl"> مراقبة الماوس من وراء لأنه يمشي. إذا كان الماوس يتحرك بشكل طبيعي ، بكل ثقلها الجسم معتمدة على جميع الأطراف ، مع البطن على عدم ملامسة للأرض ، والمشاركة مع كل من hindlimbs بالتساوي ، فإنه يحصل على درجة من 0. إذا كان يظهر الهزة أو يبدو يعرج أثناء المشي ، فإنه يحصل على درجة من 1. إذا كان يظهر هزة شديدة ، عرج شديد ، خفضت الحوض ، أو القدمين نقطة بعيدا عن الجسم أثناء تحرك (&quot;قدم البطة&quot;) ، فإنه يحصل على النتيجة من 2. إذا كان الماوس لديه صعوبة في التحرك إلى الأمام والبطن تستمر على طول الأرض ، فإنه يحصل على درجة 3. </li><li style=";text-align:right;direction:rtl"> مكان الماوس مرة أخرى في قفص وسجل رصيده المشية. </li></ol> حداب حداب هو انحناء المميزة الظهرية من العمود الفقري الذي هو مظهر شائع من مرض الاعصاب في نماذج الماوس [2]. وهو ناتج عن فقدان قوة العضلات في العضلات الشوكي الثانوية لتنكس عصبي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة الماوس من قفصه ووضعه على سطح مستو. </li><li style=";text-align:right;direction:rtl"> كما نلاحظ أنه يمشي. إذا كان الماوس قادرة على تقويم العمود الفقري لها بسهولة لأنه يمشي ، وليس لديها حداب الثابتة ، فإنه يحصل على درجة من 0. إذا كان الماوس المعارض حداب معتدل ولكن قادرة على تقويم العمود الفقري ، فإنه يحصل على درجة من 1. إذا كان غير قادر على تقويم العمود الفقري لها تماما ويحافظ على حداب الثابتة ولكنه معتدل ، فإنه يحصل على النتيجة من 2. إذا كان الماوس يحتفظ حداب وضوحا كما يمشي أو يجلس في حين أنه ، يتم تعيينه على 3 نقاط. </li><li style=";text-align:right;direction:rtl"> مكان الماوس مرة أخرى في قفص وسجل رصيده حداب. </li></ol> ممثل النتائج مع وجود عدد كاف من الحيوانات ، وهذا الإجراء هو قادر على الكشف عن الاختلافات بين سلالات النمط الظاهري ، وضمن نفس السلالة على مر الزمن. لتحليل البيانات ، وحساب النقاط لكل تدبير من خلال اتخاذيعني من القياسات الثلاثة في كل فرد. ويمكن تحليل تدابير منفردة أو مجتمعة في مركب على درجة النمط الظاهري لكل فرد. رسم البيانات وتطبيق الأساليب الإحصائية المناسبة لتحديد الأهمية. الشكل 1 يبين نتائج التقييم من النمط الظاهري مركب نموذج الفئران المعدلة وراثيا SCA7. في هذا النموذج ، floxed SCA7 - 92Q المعدلة وراثيا ، ويحيط الإنسان ataxin - 7 الجين مع 92 من المواقع يكرر CAG loxP وأعرب من كروموسوم اصطناعية البكتيرية. النتيجة النمط الظاهري مركب يشمل الشبك ، والمشي الحافة ، والتقييمات حداب مشية ليسجل أكبر قدر ممكن من 12. ويتجلى النمط الظاهري SCA7 التدريجي في الفئران floxed - SCA7 - 92Q المعدلة وراثيا عن طريق زيادة درجة النمط الظاهري مركب ، وهو ما يتسق مع طبيعة تقدمية من الأمراض التي تصيب البشر. <img src="/files/ftp_upload/1787/1787fig1.jpg" alt="الشكل 1" /> . الشكل 1 - Floxed SCA7 - 92Q الفئران يحمل التدريجي SCA7 النمط الظاهري الذي يختلف كثيرا عن غير المعدلة وراثيا في بداية تتزاحم 20 أسبوعا (2 - طريقة ANOVA : Bonferroni بعد المخصصة ؛ *** P &lt;0.001). قيمت الفئران على نطاق و0-3 لكل اختبار الحافة ، الشبك ، مشية ، والحدب. تم احتساب درجة عن كل مركب متوسط ​​التركيب الوراثي في ​​كل سن. تمثل الأشرطة SEM.

<ol>
	<li>Chou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyglutamine-expanded+ataxin-3+causes+cerebellar+dysfunction+of+SCA3+transgenic+mice+by+inducing+transcriptional+dysregulation.">Polyglutamine-expanded ataxin-3 causes cerebellar dysfunction of SCA3 transgenic mice by inducing transcriptional dysregulation.</a> Neurobiol Dis. 31 (1), 89-101 (2008).</li><li>Thomas, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+endogenous+androgen+receptor+protein+accelerates+motor+neuron+degeneration+and+accentuates+androgen+insensitivity+in+a+mouse+model+of+X-linked+spinal+and+bulbar+muscular+atrophy.">Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy.</a> Hum Mol Genet. 15 (14), 2225-2238 (2006).</li><li>Ditzler, S., Stoeck, J., LeBlanc, M., Kooperberg, C., Hansen, S., Coppin, L., Olson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+Neurobehavioral+Assessment+Reveals+that+FK506+Delays+Symptom+Onset+in+R6/2+Huntington's+Disease+Mice.">A Rapid Neurobehavioral Assessment Reveals that FK506 Delays Symptom Onset in R6/2 Huntington's Disease Mice.</a> Preclinica Research Articles. 1 (3), 115-126 (2003).</li></ol>

تمت الموافقة على هذه الدراسات من قبل جامعة واشنطن لرعاية الحيوان واللجنة المؤسسية الاستخدام.

ويهدف هذا البروتوكول أن يكون التقييم الحساسة وأداؤها بسهولة من شدة المرض في نماذج من الماوس رنح مخيخي. سوف المكونات الفردية للنظام التهديف يكون أكثر أو أقل فعالية في نماذج مختلفة من الماوس تنكس عصبي. وقد عناصر هذا النظام التهديف ...

a simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia

وصفنا بروتوكول لالكمي السريع والحساسة من شدة المرض في نماذج الماوس من ترنح مخيخات. وهو مستمد من التقييمات النمط الظاهري المنشورة سابقا في نماذج عدة أمراض ، بما في ذلك الترنحات السبيل النخاعي ، ومرض هنتنغتون ليالي وضمور العضلات نخاعي بصلي. وتشمل التدابير أطرافهم هند الشبك ، الحافة اختبار ، والمشية والحدب. يتم تسجيل كل قياس على مقياس من 0-3 ، مع ما مجموعه 0-12 لجميع التدابير الأربعة. نتائج تميز بين الأفراد بشكل فعال المتضررة وغير المتضررة ، بينما أيضا قياس التقدم الزمني للالظواهر مرض الاعصاب. ويمكن تحليل تدابير منفردة أو مجتمعة إلى درجة النمط الظاهري المركبة لقوة أكبر الإحصائية. سيكون مزيجا مثاليا من التدابير الأربعة المذكورة تعتمد على الفوضى في السؤال. نقدم مثالا على بروتوكول المستخدمة لتقييم شدة المرض في نموذج الفأر المعدلة وراثيا من نوع رنح نخاعي مخيخي 7 (SCA7). ساهم البرت R. لا سبادا وGwenn الحديقة ألف لهذه المخطوطة على حد سواء.

Watch this Scientific Journal Video about A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia at JoVE.com

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

وصفنا بروتوكول لالكمي السريع والحساسة من شدة المرض في نماذج من الماوس رنح مخيخي. وتشمل التدابير أطرافهم هند الشبك ، الحافة اختبار ، والمشية والحدب. يميز هذا البروتوكول على نحو فعال بين الأفراد المتضررة وغير المتضررة ، ويكشف تطور الأفراد المتضررين على مر الزمن.

سجل النمط الظاهري بسيط مركب نظام لتقييم نماذج الماوس من رنح مخيخي

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

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JoVE Journal

Neuroscience

35.2K Views.  University of Washington. وصفنا بروتوكول لالكمي السريع والحساسة من شدة المرض في نماذج من الماوس رنح مخيخي. وتشمل التدابير أطرافهم هند الشبك ، الحافة اختبار ، والمشية والحدب. يميز هذا البروتوكول على نحو فعال بين الأفراد المتضررة وغير المتضررة ، ويكشف تطور الأفراد المتضررين على مر الزمن.

Video: A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

نماذج من الماوس تلين بيضاء المحيطة بالبطين

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

متعددة الماوس تشريحي عصبي التصوير بالرنين المغناطيسي

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

مخيخي الثقافات عضوي النمط : التحديات والكشف عن أفكارك

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

تحليل كمي متشابك الحويصلة التجديد حمام سباحة في الخلايا العصبية مثقف الحبيبية للدماغ باستخدام الأصباغ FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

الليزر Nanosurgery من مخيخي محاور عصبية<em&gt; في فيفو</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

فحوصات حاسة الشم لنماذج من الماوس مرض الاعصاب

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

وضع نماذج لموت الخلايا العصبية وضمور في الخلايا العصبية الأولية الحبيبية للدماغ الماوس

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

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 ...

Cerebellar Regional Dissection for Molecular Analysis

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies indicate that distinct cerebellar regions contribute to these different functions. Molecular studies examining regional cerebellar differences are lagging as they are mostly done on whole cerebellar extracts thereby masking any distinctions across specific cerebellar regions. Here we describe a technique to reproducibly and quickly dissect four different cerebellar regions: the deep cerebellar nuclei (DCN), anterior and posterior vermal cerebellar cortex, and the cerebellar cortex of the hemispheres. Dissecting out these distinct regions allows for the exploration of molecular mechanisms that may underlie their unique contributions to balance, movement, affect and cognition. This technique may also be used to explore differences in pathological susceptibility of these specific regions across various mouse disease models.

Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene expression.&#160;

تشريح المخيخ الإقليمي للتحليل الجزيئي

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.