Este trabajo fue apoyado por subvenciones del NIH NS052535 de GAG ​​y EY014061 y EY014997 a ARL, así como un premio UL1DE019583-02 del consorcio.

Para evitar sesgos, el investigador de realizar las evaluaciones no deben tener un conocimiento del genotipo de los animales. Las medidas individuales se califican en una escala de 0-3, donde 0 representa la ausencia del fenotipo relevante y 3 representan la manifestación más grave. Cada ensayo se realiza varias veces para asegurarse de que el resultado es reproducible. La obesidad se complican la interpretación de todas las medidas descritas. El investigador puede desear para pesar los ratones después de anotar fenotipo para evaluar el posible papel de la adiposidad en los resultados. Repisa de la prueba La prueba de borde es una medida directa de la coordinación, que se ve afectada en ataxias cerebelosas y muchos otros trastornos neurodegenerativos. Esta medida es la más directamente comparables a los signos humanos de ataxia cerebelosa. <ol><li> Levantar el ratón de la jaula y colocarlo en la repisa de la jaula. Los ratones normalmente caminar por la cornisa y tratar de descender de nuevo en la jaula. </li><li> Observe el ratón a medida que camina por la cornisa jaula y se reduce en su jaula. Un ratón de tipo salvaje normalmente caminar por la cornisa sin perder su equilibrio, y se baja de la espalda en la jaula con gracia, con sus patas. Esto se le asigna una puntuación de 0. Si el ratón pierde su equilibrio mientras camina por la cornisa, pero por lo demás parece coordinada, que recibe una puntuación de 1. Si no lo hace eficaz el uso de sus patas traseras, o cae de cabeza en lugar de sus patas cuando se desciende en la jaula, que recibe una puntuación de 2. Si se cae de la repisa, o casi, al caminar o tratar a sí mismo inferior, o se sacude y se niega a moverse en absoluto a pesar del estímulo, que recibe una puntuación de 3. Algunos ratones se requiere un pequeño empujoncito para animar a caminar por la cornisa o descender en la jaula. </li><li> Registrar la calificación de la prueba cornisa. </li></ol> Miembro Posterior juntando Miembro Posterior juntando es un marcador de progresión de la enfermedad en una serie de modelos de ratón de la neurodegeneración, incluyendo ciertas ataxias cerebelosas [1]. <ol><li> Sujete la cola cerca de su base y levantar el ratón clara de todos los objetos que lo rodean. </li><li> Observar la posición de miembro posterior durante 10 segundos. Si las patas traseras son siempre abiertas hacia el exterior, lejos del abdomen, se le asigna una puntuación de 0. Si un miembro posterior se retrae hacia el abdomen desde hace más de 50% del tiempo suspendido, que recibe una puntuación de 1. Si ambos miembros posteriores son parcialmente retraído hacia el abdomen desde hace más de 50% del tiempo suspendido, que recibe una puntuación de 2. Si sus extremidades posteriores son completamente retraído y tocando el abdomen en busca de más del 50% del tiempo suspendido, que recibe una puntuación de 3. </li><li> Coloque el ratón de nuevo en su jaula, y registrar su puntuación de extremidades inferiores juntando. </li></ol> Paso La marcha es una medida de la coordinación y la función muscular. <ol><li> Quitar el ratón de su jaula y lo coloca sobre una superficie plana con la cabeza de espaldas a la investigadora. </li><li> Observe el ratón desde atrás a medida que camina. Si el ratón se mueve con normalidad, con apoyo del peso corporal en todas las extremidades, con el abdomen sin tocar el suelo, y con las dos patas traseras que participan por igual, que recibe una puntuación de 0. Si se muestra un temblor o parece cojear al caminar, que recibe una puntuación de 1. Si se muestra un temblor intenso, cojera severa, disminución de la pelvis o los pies apuntan lejos del cuerpo durante la locomoción (&quot;pies de pato&quot;), que recibe una puntuación de 2. Si el ratón tiene dificultades para seguir adelante y arrastra a su abdomen por el suelo, recibe una puntuación de 3. </li><li> Coloque el ratón de nuevo en su jaula, y registrar su puntuación de la marcha. </li></ol> Cifosis La cifosis es una curvatura característica dorsal de la columna vertebral que es una manifestación frecuente de enfermedades neurodegenerativas en modelos de ratón [2]. Es causada por una pérdida de tono muscular en los músculos de la columna vertebral secundaria a la neurodegeneración. <ol><li> Quitar el ratón de su jaula y lo coloca sobre una superficie plana. </li><li> Observe que a medida que camina. Si el ratón es capaz de enderezar su columna vertebral con facilidad a medida que camina, y no tiene cifosis persistente, que recibe una puntuación de 0. Si el ratón presenta cifosis leve, pero es capaz de enderezar su columna vertebral, que recibe una puntuación de 1. Si no es capaz de enderezar su columna vertebral completamente y mantiene la cifosis persistente pero leve, que recibe una puntuación de 2. Si mantiene el ratón cifosis pronunciada a medida que camina o mientras se está sentado, se le asigna una puntuación de 3. </li><li> Coloque el ratón de nuevo en su jaula, y registrar su puntuación de la cifosis. </li></ol> Los resultados representativos Con un número suficiente de animales, este procedimiento es capaz de detectar diferencias fenotípicas entre las cepas y dentro de la misma cepa en el tiempo. Para el análisis de los datos, calcular la puntuación para cada medida mediante la adopción de lamedia de las tres mediciones en cada individuo. Las medidas se pueden analizar por separado o combinados en una puntuación compuesta fenotipo de cada individuo. Gráfico de los datos y aplicar los métodos estadísticos apropiados para determinar la significación. La figura 1 muestra los resultados de una evaluación de fenotipo compuesto de un anticuerpo murino transgénico SCA7 modelo. En este modelo transgénico floxed-SCA7-92Q, el ser humano ataxina-7 genes con 92 repeticiones de CAG está flanqueado por sitios loxP y expresa a partir de un cromosoma artificial bacteriano. La puntuación fenotipo compuesto incluye el juntando, caminar cornisa, la marcha y evaluación de la cifosis de una puntuación máxima posible de 12. La progresiva SCA7 fenotipo de los ratones transgénicos floxed-SCA7-92Q se demuestra con una puntuación de fenotipo aumento compuesto, lo cual es consistente con la naturaleza progresiva de la enfermedad humana. <img src="/files/ftp_upload/1787/1787fig1.jpg" alt="Figura 1" /> . Figura 1 floxed-SCA7-92Q ratones muestran una progresiva SCA7 fenotipo que es significativamente diferente de los no transgénicos de camada a partir de las 20 semanas (2-way ANOVA: Bonferroni post-hoc; *** P &lt;0,001). Los ratones fueron evaluados en una escala de 0-3 cada uno para la prueba de cornisa, cruzando, la marcha, y la cifosis. Promedio total para cada genotipo en cada edad se calculó. Las barras representan 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>

Estos estudios fueron aprobados por la Universidad de Washington Cuidado de Animales institucional y el empleo.

Este protocolo está diseñado para ser una evaluación sensible y realizar fácilmente de gravedad de la enfermedad en modelos de ratón de la ataxia cerebelosa. Los componentes individuales del sistema de puntuación será más o menos eficaz en modelos de ratón diferente de la neurodegeneración. Los elementos de este sistema de puntuación han sido efectivamente utilizados para evaluar una variedad de modelos de ratón de las enfermedades neurodegenerativas humanas, incluyendo las ataxi...

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

Se describe un protocolo para la cuantificación rápida y sensible de la gravedad de la enfermedad en modelos de ratón de cerebelo ataxia. Se deriva de las evaluaciones fenotipo previamente publicado en varios modelos de enfermedades, incluyendo las ataxias espinocerebelosas, la enfermedad de Huntington y la atrofia muscular s espinobulbar. Las medidas incluyen las extremidades posteriores juntando, la cornisa de la prueba, la marcha y la cifosis. Cada medida se registra en una escala de 0-3, con un total combinado de 0-12 para las cuatro medidas. Los resultados efectivamente discriminar entre los individuos afectados y no afectados, al tiempo que la cuantificación de la progresión temporal de los fenotipos de la enfermedad neurodegenerativa. Las medidas pueden ser analizados de forma individual o combinados en una puntuación fenotipo compuesto de una mayor potencia estadística. La combinación ideal de las cuatro medidas descritas dependerá de la enfermedad en cuestión. Se presenta un ejemplo del protocolo utilizado para evaluar la gravedad de la enfermedad en un modelo de ratón transgénico de la ataxia espinocerebelosa tipo 7 (SCA7). Albert R. La Spada y Gwenn A. Jardín contribuido a este manuscrito por 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

Se describe un protocolo para la cuantificación rápida y sensible de la gravedad de la enfermedad en modelos de ratón de la ataxia cerebelosa. Las medidas incluyen las extremidades posteriores juntando, la cornisa de la prueba, la marcha y la cifosis. Este protocolo efectivamente discrimina entre los individuos afectados y no afectados, y detecta la progresión de las personas afectadas con el tiempo.

A simple fenotipo compuesto el sistema de puntaje para la evaluación de modelos de ratón de Ataxia cerebelosa

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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Research

JoVE Journal

Neuroscience

35.1K vistas.  University of Washington. Se describe un protocolo para la cuantificación rápida y sensible de la gravedad de la enfermedad en modelos de ratón de la ataxia cerebelosa. Las medidas incluyen las extremidades posteriores juntando, la cornisa de la prueba, la marcha y la cifosis. Este protocolo efectivamente discrimina entre los individuos afectados y no afectados, y detecta la progresión de las personas afectadas con el tiempo.

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.

Los modelos de ratón de la Retinopatía

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

Investigación

Neurociencias

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.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

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.

Organotípicos Culturas del cerebelo: Retos y detección de apoptosis

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.

Análisis cuantitativo de Reposición Synaptic piscina vesículas en cultivos de neuronas granulares del cerebelo con tintes 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.

Laser Nanocirugía del cerebelo Los axones<em&gt; En Vivo</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.

Los ensayos olfativos para modelos de ratón de Enfermedades 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.

Modelado de la muerte Neuronal y la degeneración en las neuronas del gránulo cerebeloso primario de ratón

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.

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

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;

Disección regional cerebelosa para análisis molecular

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.