이 작품은 NIH 개그 부여 NS052535과 EY014061 &amp; EY014997 ARL을뿐만 아니라 UL1DE019583 - 02 컨소시엄 수상에 의해 지원되었다.

편견을 방지하기 위해 평가를 수행하는 실험자이 동물의 유전자형에 대한 지식이없는 것입니다. 개별 대책이 가장 심각한 징후를 나타내는 관련 표현형 3의 부재를 나타내는 0, 0-3의 규모 득점 있습니다. 각 테스트는 점수가 재현성 있는지 확인하기 위해 여러 번 수행됩니다. 비만은 설명 모든 조치의 해석을 복잡하게합니다. 조사는 결과에 adiposity의 가능한 역할을 평가하기 위해 표현형 점수 다음 마우스의 무게를하실 수 있습니다. 레지 테스트 레지 시험은 소뇌 ataxias 및 기타 여러 neurodegenerative 장애에 장애인입니다 조정의 직접적인 측정입니다. 이 법안은 소뇌 운동 실조증 인간의 증상에 가장 직접적으로 비교됩니다. <ol><li> 케이지에서 마우스를 채취해서 케이지의 난간에 놓으십시오. 마우스는 일반적으로 난간을 따라 산책과 새장에 다시 하강을 시도합니다. </li><li> 그것이 우리를 레지 따라 산책과 새장에 스스로를 절감으로 마우스를 관찰합니다. 야생 타입 마우스 일반적으로 균형을 잃지 않고 난간을 따라 산책하며, 정상적으로 새장에 자신을 다시 낮은 것입니다, 그 갈퀴 손을 사용합니다. 이것은 0 점수를 할당됩니다. 마우스가 난간을 따라 걷는 동안의 발판을 잃고 있지만, 별도로 조율​​된 나타나면 1 점수를 받게됩니다. 케이지에 내림차순 때 효과적으로 머리보다는 그 발톱을 뒷다리, 또는 토지를 사용하지 않는 경우, 그것은 2 점수를 받게됩니다. 경우는 난간에서 떨어지는, 또는 거의 있도록 도보 또는 자체를 낮게하는 동안, 또는 쉐이크과 격려에도 불구하고 전혀 이동을 거부, 3의 점수를 받게됩니다. 일부 쥐들이 그들은 새장에 난간을 따라 산책이나 하강 장려하는 부드러운 찔러 필요합니다. </li><li> 레지 시험 점수를 기록합니다. </li></ol> Hindlimb는 clasping clasping Hindlimb 특정 소뇌 ataxias [1]을 포함한 neurodegeneration의 마우스 모델의 여러 질병 진행의 마커입니다. <ol><li> 그 기지 ​​근처에 꼬리를 파악하고 모든 주변 객체의 명확한 마우스를 올려. </li><li> 10 초 동안 hindlimb 위치를 관찰. hindlimbs가 지속적으로 복부에서 떨어져 밖으로 splayed있다면, 그것은 0 점수를 할당됩니다. 한 hindlimb 정지 시간의 50 % 이상에 대한 복부쪽으로 철회하는 경우, 그것은 1 점수를 받게됩니다. 두 h​​indlimbs 부분적으로 시간이 정지의 50 % 이상에 대한 복부쪽으로 철회하는 경우, 그것은 2 점수를 받게됩니다. 그 hindlimbs가 완전히 철회하고 중지 시간의 50 % 이상에 대한 복부를 만지고있다면, 그것은 3 점수를 받게됩니다. </li><li> 의 케이지에 다시 마우스를 놓고 그 hindlimb clasping 점수를 기록합니다. </li></ol> 보조 게이트는 조정과 근육 기능을 측정하기위한 한 방법입니다. <ol><li> 그 케이지에서 마우스를 제거하고 머리가 떨어져 조사에서 직면과 평평한 표면에 놓으십시오. </li><li> 이 산책로로 뒤에서 마우스를 관찰합니다. 마우스는 체중는 복부가 땅에 안 만질과 모든 팔다리에서 지원하고, 두 hindlimbs가 골고루 참여와 함께 정상적으로 움직이면, 그것은 0 점수를 받게됩니다. 그것은 진동을 보여줍니다 또는 도보 동안 미약으로 나타나는 경우, 그것은 1 점수를 받게됩니다. 그것이 나타나면 심한 진동, 극심한 미약는 골반을 인하하거나 발이 운동 ( &quot;오리 피트&quot;) 동안 시체에서 떨어진 지점, 그것은 2 점수를 받게됩니다. 마우스 어려움이 전진을 가지고 있으며, 지상 따라 자사의 복부를 끌 경우, 3의 점수를 받게됩니다. </li><li> 의 케이지에 다시 마우스를 놓고 그 게이트 점수를 기록합니다. </li></ol> Kyphosis Kyphosis 마우스 모델 [2]에서 neurodegenerative 질병의 일반적인 징후입니다 척추의 특성 등의 곡률이다. 그것은 neurodegeneration에 보조 척추 근육의 근육 톤의 손실로 인해 발생합니다. <ol><li> 그 케이지에서 마우스를 제거하고 평평한 표면에 놓으십시오. </li><li> 그것이 산책으로 관찰. 마우스 그것이 산책하고, 영구 kyphosis가 없습니다 쉽게 자사의 척추를 제대로 할 수있다면, 그것은 0 점수를 받게됩니다. 마우스 가벼운 kyphosis을 전시하고 있지만 척추를 제대로 할 수있다면, 1의 점수를 받게됩니다. 완전히는 척추를 펴 수 있으며, 영구하지만 온화한 kyphosis을 유지한다면, 2의 점수를 받게됩니다. 그것이 산책을하거나 앉아있는 동안, 그것이 3 점수를 할당으로 마우스 발음 kyphosis을 유지하십시오. </li><li> 의 케이지에 다시 마우스를 놓고 그 kyphosis 점수를 기록합니다. </li></ol> 대표 결과 동물의 충분한 숫자로,이 절차는 종자 사이 시간이 지남에 따라 동일한 변형 이내 표현형 차이를 검출이 가능합니다. 데이터 분석의 경우를 복용하여 각 측정에 대한 점수를 계산각의 세 가지 측정의 의미합니다. 대책은 별도로 분석 또는 각위한 복합 표현형 점수에 조합하여 사용할 수 있습니다. 데이터를 차트와 중요성을 결정하기 위해 적절한 통계 방법을 적용할 수 있습니다. 그림 1은 murine 유전자 변형 SCA7 모델의 복합 표현형 평가의 결과를 보여줍니다. 이 floxed - SCA7 - 92Q 유전자 변형 모델에서 92 배속 반복과 인간 ataxin - 7 유전자는 loxP 사이트 둘러싸인이며 세균 인공 염색체의 표현. 복합 표현형 점수 12 최대 가능한 점수에 대한 clasping, 레지 산책, 보행 및 kyphosis 평가를 포함하고 있습니다. floxed - SCA7 - 92Q 유전자 변형 생쥐의 진보 SCA7 표현형은 인간 질병의 진보 자연과 일치 증가 복합 표현형 점수에 의해 증명됩니다. <img src="/files/ftp_upload/1787/1787fig1.jpg" alt="그림 1" /> . 그림 1 Floxed - SCA7 - 92Q 생쥐는 20 주 (:; ** P &lt;0.001 Bonferroni 사후 특별 2 - 웨이 ANOVA)에서 시작 비 트렌스 제닉 littermates에서 크게 다른 진보 SCA7 표현형를 나타냅니다. 생쥐, 보행, 그리고 kyphosis을 clasping, 레지 시험 0-3 규모의 각 평가했다. 각 나이에 각각의 유전자형에 관한 종합 점수가 계산되었다. 바 SEM을 나타냅니다.

<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>

이러한 연구는 워싱턴 기관 애니멀 케어 및 사용위원회의 대학에 의해 승인되었습니다.

이 프로토콜은 소뇌 운동 실조증의 마우스 모델에서 질병 심각도의 민감하고 쉽게 수행 평가하도록 설계되었습니다. 채점 시스템의 개별 구성 요소는 neurodegeneration의 다른 마우스 모델에 어느 정도 효과 것입니다. 이 채점 시스템의 요소가 효과적으로 소뇌 ataxias, 헌팅턴의 질병 및 spinobulbar 근육 위축증 [1-3]를 포함하여 인간의 neurodegenerative 질병의 마우스 모델의 다양한, 평...

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

우리는 cerebella 운동 장애의 마우스 모델에서 질병 심각도의 신속하고 민감한 부량을위한 프로토콜을 설명합니다. 이것은 spinocerebellar ataxias, 헌팅턴의 질병 및 spinobulbar 근육 위축 등 여러 가지 질병 모델 이전에 출판 표현형 평가에서 파생됩니다. 조치는 뒷다리 clasping, 레지 테스트, 보행 및 kyphosis가 포함되어 있습니다. 각 측정은 네 가지 조치에 대해 0-12의 결합 총, 0-3의 규모에 기록됩니다. 또한 neurodegenerative 질병 phenotypes의 시간적 진행을 quantifying 동안 결과는 효과적으로 영향이 아닌 영향을 개인 간의 차별. 대책은 개별적으로 분석 이상의 통계 전원 복합 표현형 점수에 조합하여 사용할 수 있습니다. 네 개의 설명 조치의 이상적인 조합은 문제의 장애에 따라 달라집니다. 우리는 spinocerebellar 운동 장애 유형 7 (SCA7)의 유전자 변형 마우스 모델에서 질병 심각도를 평가하는 데 사용되는 프로토콜의 예를 제시한다. 알버트 R. 라 스파다 및 Gwenn A. 가든 마찬가지로이 원고에 기여.

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

우리는 소뇌 운동 장애의 마우스 모델에서 질병 심각도의 신속하고 민감한 부량을위한 프로토콜을 설명합니다. 조치는 뒷다리 clasping, 레지 테스트, 보행 및 kyphosis가 포함되어 있습니다. 이 프로토콜은 효율적으로 영향 않은 영향을 개인 간의 차별, 그리고 시간이 지남에 따라 영향을받는 개인의 진행을 감지합니다.

소뇌 운동 실조증의 마우스 모델을 평가하기위한 간단한 복합 표현형 점수 시스템

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

a-simple-composite-phenotype-scoring-system-for-evaluating-mouse

Research

JoVE Journal

Neuroscience

35.1K 조회수.  University of Washington. 우리는 소뇌 운동 장애의 마우스 모델에서 질병 심각도의 신속하고 민감한 부량을위한 프로토콜을 설명합니다. 조치는 뒷다리 clasping, 레지 테스트, 보행 및 kyphosis가 포함되어 있습니다. 이 프로토콜은 효율적으로 영향 않은 영향을 개인 간의 차별, 그리고 시간이 지남에 따라 영향을받는 개인의 진행을 감지합니다.

비디오: 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.

Periventricular Leukomalacia의 마우스 모델

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

연구

신경과학

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.

Organotypic 소뇌 문화 : Apoptotic 도전과 감지

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.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

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.

소뇌 축삭의 레이저 Nanosurgery<em&gt; 생체</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.

신경 퇴행성 질환의 마우스 모델에 대한 후각 분석 실험

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.

신경 죽음 및 마우스 기본 소 뇌과 립 신경에 변성을 모델링

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.

성인 소뇌 슬라이스에 장기 우울증 유도의 평가

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;

분자 분석을 위한 소뇌 지역 해부

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

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALS&#160;models and any C. elegans strain that exhibits altered motility.

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility.

근위축성 측삭 경화증의 C. 엘레간 모델의 모터 손상 평가

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor ...

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.