Este trabalho foi financiado pelo NIH concede NS052535 de GAG ​​e EY014061 &amp; EY014997 a ARL, bem como um prêmio UL1DE019583-02 consórcio.

Para evitar viés, o experimentador realizar as avaliações não devem ter conhecimento do genótipo do animal. Medidas individuais são pontuados em uma escala de 0-3, com 0 representando uma ausência do fenótipo relevantes e 3 representa a manifestação mais grave. Cada teste é realizado várias vezes para garantir que a pontuação é reproduzível. A obesidade vai complicar a interpretação de todas as medidas descritas. O investigador pode querer pesar camundongos após marcar fenótipo para avaliar o possível papel de adiposidade nos resultados. Teste Ledge O teste de borda é uma medida direta da coordenação, que é prejudicada em ataxias cerebelar e muitas outras doenças neurodegenerativas. Esta medida é a mais diretamente comparáveis ​​aos sinais humanos de ataxia cerebelar. <ol><li> Levante o mouse da gaiola e coloque-a borda da gaiola. Camundongos normalmente caminhar ao longo da borda e tentativa de descer de volta na gaiola. </li><li> Observe o mouse como ele anda ao longo da borda da gaiola e reduz-se em sua gaiola. Um rato do tipo selvagem, normalmente a pé ao longo da borda, sem perder o seu equilíbrio, e irá reduzir-se de volta para a jaula graciosamente, usando suas patas. Este é atribuída uma pontuação de 0. Se o mouse perde o seu pé durante a caminhada ao longo da borda, mas por outro lado aparece coordenada, ele recebe uma pontuação de 1. Se não usar eficazmente as patas traseiras, ou terras em sua cabeça ao invés de suas patas ao descer na gaiola, ele recebe uma pontuação de 2. Se ela cair fora da borda, ou quase isso, durante a caminhada ou a tentativa de menores em si, ou shakes e se recusa a mover-se a todos, apesar de encorajamento, que recebe uma pontuação de 3. Alguns mouses exigirá um pequeno empurrão para incentivá-los a caminhar ao longo da borda ou descer na gaiola. </li><li> Registre o resultado do teste borda. </li></ol> Hindlimb clasping Hindlimb clasping é um marcador de progressão da doença em uma série de modelos de mouse da neurodegeneração, incluindo certas ataxias cerebelar [1]. <ol><li> Segure a cauda perto de sua base e levante o mouse clara de todos os objectos que nos rodeiam. </li><li> Observe a posição dos membros posteriores por 10 segundos. Se a membros posteriores são consistentemente splayed para fora, longe do abdômen, é atribuída uma pontuação de 0. Se um membro posterior é retraída em direção ao abdômen há mais de 50% do tempo suspenso, ele recebe uma pontuação de 1. Se ambos os membros posteriores são parcialmente retraída em direção ao abdômen há mais de 50% do tempo suspenso, ele recebe uma pontuação de 2. Se a sua hindlimbs são totalmente retraída e tocar no abdômen há mais de 50% do tempo suspenso, ele recebe uma pontuação de 3. </li><li> Posicione o mouse de volta para sua gaiola e registrar sua pontuação hindlimb apertando. </li></ol> Marcha Marcha é uma medida de coordenação e função muscular. <ol><li> Retire o mouse de sua gaiola e coloque-o sobre uma superfície plana com a cabeça virada para o investigador. </li><li> Observe o mouse por trás como ele anda. Se o mouse se move normalmente, com o seu peso corporal compatível com todos os membros, com o seu abdômen não tocar o chão, e com ambos os membros posteriores participantes uniformemente, ele recebe uma pontuação de 0. Se ele mostra um tremor ou aparece a coxear durante a caminhada, ele recebe uma pontuação de 1. Se ele mostra um tremor grave, limp grave, baixou pélvis, ou os pés ponto longe do corpo durante a locomoção (&quot;pés de pato&quot;), ele recebe uma pontuação de 2. Se o mouse tem dificuldade em avançar e arrasta o seu abdome ao longo do chão, ele recebe uma pontuação de 3. </li><li> Posicione o mouse de volta para sua gaiola e registrar sua pontuação da marcha. </li></ol> Cifose Cifose é uma curvatura característica dorsal da coluna vertebral que é uma manifestação comum de doenças neurodegenerativas em modelos de ratos [2]. É causada por uma perda de tônus ​​muscular nos músculos da coluna vertebral secundária à neurodegeneração. <ol><li> Retire o mouse de sua gaiola e coloque-o sobre uma superfície plana. </li><li> Observe como ele anda. Se o mouse é capaz de facilmente acertar a sua espinha como ele anda, e não tem cifose persistente, que recebe uma pontuação de 0. Se o mouse apresenta cifose leve, mas é capaz de acertar a sua coluna vertebral, que recebe uma pontuação de 1. Se ele é incapaz de acertar a sua espinha completamente e mantém cifose, mas persistente leve, ele recebe uma pontuação de 2. Se o mouse mantém cifose pronunciada como caminhadas ou enquanto ele se senta, ela é atribuída uma pontuação de 3. </li><li> Posicione o mouse de volta para sua gaiola e registrar sua pontuação cifose. </li></ol> Resultados representativos Com um número suficiente de animais, este procedimento é capaz de detectar diferenças entre as linhagens fenótipo e dentro da mesma estirpe ao longo do tempo. Para análise dos dados, calcular a pontuação para cada medida, tendo amédia das três medidas em cada indivíduo. Medidas podem ser analisadas separadamente ou combinadas em uma pontuação fenótipo composto para cada indivíduo. Gráfico os dados e aplicar os métodos estatísticos adequados para determinar a significância. A Figura 1 mostra os resultados de uma avaliação fenótipo composto de um modelo murino SCA7 transgênicos. Neste modelo floxed-SCA7 92Q-transgênico, o gene da ataxina-7 humana com 92 repetições CAG é ladeado por sites loxP e expressa a partir de um cromossomo bacteriano artificial. A pontuação fenótipo composto inclui a clasping, andando borda da marcha e avaliações cifose para uma pontuação máxima possível de 12. O fenótipo SCA7 progressiva nos ratos floxed-SCA7-92Q transgênicos é demonstrada por um aumento da pontuação fenótipo composto, que é consistente com a natureza progressiva da doença humana. <img src="/files/ftp_upload/1787/1787fig1.jpg" alt="Figura 1" /> . Figura 1 Floxed-SCA7-92Q ratos exibem um fenótipo SCA7 progressiva que é significativamente diferente do não-transgênico littermates início em 20 semanas (2-way ANOVA: Bonferroni post-hoc; *** P &lt;0,001). Camundongos foram avaliados em uma escala de 0-3 para cada teste borda, apertando, marcha, e cifose. Escore composto média para cada genótipo em cada idade foi calculada. Barras representam 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>

Esses estudos foram aprovados pela Universidade de Washington Animal Care Institucional e Comitê de uso.

Este protocolo é projetado para ser uma avaliação sensível e de fácil realização da gravidade da doença em ratos de ataxia cerebelar. Componentes individuais do sistema de pontuação será mais ou menos eficaz em modelos de ratos diferentes de neurodegeneração. Elementos deste sistema de pontuação foram efetivamente utilizados para avaliar uma variedade de modelos do rato da doença neurodegenerativa humanos, incluindo ataxias cerebelar, doença de Huntington s e atrofia muscular spinobulbar [1-3]. A combinação ideal dos testes dependerá do distúrbio em questão. Este protocolo foi originalmente concebido pelos autores para avaliar modelos de ratos transgênicos do tipo ataxia espinocerebelar 7 (SCA7). No modelo de SAC7 empregado para gerar os dados aqui apresentados, os animais foram sacrificados com a idade de 40-43 semanas ou mais cedo se as anormalidades comportamentais progrediu para um estágio que um animal não é mais suficientemente móvel para manter de forma independente de nutrição ou hidratação.

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

Nós descrevemos um protocolo para a quantificação rápida e sensível da gravidade da doença em ratos de cerebelos ataxia. É derivado do fenótipo avaliações anteriormente publicados em diversos modelos de doenças, incluindo ataxias espinocerebelares, a doença de Huntington s e atrofia muscular spinobulbar. Medidas incluem membro posterior segurando, borda teste de marcha, e cifose. Cada medida é registrada em uma escala de 0-3, com um total combinado de 0-12 para todas as quatro medidas. Os resultados efetivamente discriminar entre indivíduos afetados e não afetados, ao mesmo tempo, quantificar a progressão temporal de fenótipos da doença neurodegenerativa. Medidas podem ser analisados ​​individualmente ou combinadas em uma pontuação fenótipo composto de maior poder estatístico. A combinação ideal das quatro medidas descritas dependerá do distúrbio em questão. Nós apresentamos um exemplo do protocolo utilizado para avaliar a gravidade da doença em um modelo de camundongo transgênico do tipo ataxia espinocerebelar 7 (SCA7). Albert R. La Spada e Gwenn A. Jardim contribuíram para este manuscrito de forma igual.

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

Nós descrevemos um protocolo para a quantificação rápida e sensível da gravidade da doença em ratos de ataxia cerebelar. Medidas incluem membro posterior segurando, borda teste de marcha, e cifose. Este protocolo efetivamente discrimina entre os indivíduos afetados e não afetados, e detecta a progressão dos indivíduos afetados ao longo do tempo.

Um sistema simples de pontuação Composite Fenótipo para avaliar modelos de ratos de ataxia cerebelar

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

35.4K visualizações.  University of Washington. Nós descrevemos um protocolo para a quantificação rápida e sensível da gravidade da doença em ratos de ataxia cerebelar. Medidas incluem membro posterior segurando, borda teste de marcha, e cifose. Este protocolo efetivamente discrimina entre os indivíduos afetados e não afetados, e detecta a progressão dos indivíduos afetados ao longo do tempo.

Vídeo: 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.

Modelos de mouse de leucomalácia periventricular

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

Neurociência

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.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

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.

Organotípicas Culturas Cerebelar: Desafios Apoptotic e Detecção

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.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

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

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Um método geral para a avaliação de Incubação de Craving Sacarose em Ratos

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

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.

Ensaios olfativas para o Rato modelos de doenças neurodegenerativas

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.

Modelagem de morte Neuronal e a degeneração no Mouse grânulo cerebelar primária de neurônios

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

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Transplantação dos progenitors corticais Quimiogenetically projetados do interneuron em cérebros Postnatal adiantados do rato

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

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;

Dissecção Regional Cerebelr para Análise Molecular

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.

Avaliação do Comprometimento Motor em C. elegans Modelos de Esclerose Lateral Amiotrófica

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