Ce travail a été soutenu par des subventions du NIH NS052535 au GAG et EY014061 &amp; EY014997 d&#39;ARL, ainsi qu&#39;une récompense UL1DE019583-02 du consortium.

Pour éviter les biais, l&#39;expérimentateur effectuer les évaluations ne doivent pas avoir une connaissance du génotype de l&#39;animal. Les mesures individuelles sont notés sur une échelle de 0-3, où 0 représente une absence de phénotype pertinent et 3 représentant la manifestation la plus sévère. Chaque test est effectué plusieurs fois pour s&#39;assurer que le score est reproductible. L&#39;obésité va compliquer l&#39;interprétation de toutes les mesures décrites. L&#39;enquêteur peut souhaiter peser les souris après notation phénotype d&#39;évaluer le rôle possible de l&#39;adiposité dans les résultats. Test de Ledge Le test de corniche est une mesure directe de la coordination, qui est altérée dans les ataxies cérébelleuses et beaucoup d&#39;autres maladies neurodégénératives. Cette mesure est la plus directement comparable à des signes d&#39;ataxie cérébelleuse humaine. <ol><li> Soulevez la souris à partir de la cage et la placer sur le rebord de la cage. Souris typiquement longez la corniche et de tenter de redescendre dans la cage. </li><li> Respecter les souris comme il marche le long de la corniche cage et s&#39;abaisse dans sa cage. Une souris de type sauvage est généralement marcher le long de la corniche, sans perdre son équilibre, et va se le bas du dos dans la cage gracieusement, en utilisant ses pattes. Ceci est attribué un score de 0. Si la souris perd pied en marchant le long de la corniche, mais apparaît autrement coordonnée, il reçoit un score de 1. S&#39;il n&#39;utilise pas efficacement ses pattes postérieures, ou des terres sur sa tête plutôt que ses pattes en descendant dans la cage, il reçoit un score de 2. Si elle tombe de la corniche, ou presque, tout en marchant ou en tentant de se baisse, ou tremble et refuse de bouger du tout en dépit des encouragements, il reçoit un score de 3. Certaines souris nécessitera un petit coup de pouce pour les encourager à marcher le long de la corniche, ou descendre dans la cage. </li><li> Notez le score au test rebord. </li></ol> Hindlimb joignant Hindlimb serrant est un marqueur de progression de la maladie dans un certain nombre de modèles de souris de la neurodégénérescence, y compris certaines ataxies cérébelleuses [1]. <ol><li> Saisir la queue près de sa base et soulevez la souris claire de tous les objets environnants. </li><li> Observer la position des membres postérieurs pendant 10 secondes. Si les membres postérieurs sont systématiquement écartés vers l&#39;extérieur, loin de l&#39;abdomen, il est attribué un score de 0. Si l&#39;un des membres postérieurs est rétracté vers l&#39;abdomen pendant plus de 50% du temps suspendu, il reçoit un score de 1. Si les deux membres postérieurs sont partiellement rétractée vers l&#39;abdomen pendant plus de 50% du temps suspendu, il reçoit un score de 2. Si ses membres postérieurs sont entièrement rétracté et toucher le ventre pour plus de 50% du temps suspendu, il reçoit un score de 3. </li><li> Placez la souris de nouveau dans sa cage et enregistrer son score en joignant des membres postérieurs. </li></ol> Démarche La démarche est une mesure de coordination et de la fonction musculaire. <ol><li> Retirez la souris de sa cage et la placer sur une surface plane avec sa tête tournée vers l&#39;enquêteur. </li><li> Observez la souris par derrière comme il marche. Si la souris se déplace normalement, avec son poids en charge sur tous les membres, avec son abdomen ne pas toucher le sol, et avec les deux membres postérieurs qui participent régulièrement, il reçoit un score de 0. Si elle montre un tremblement ou apparaît à boiter en marchant, il reçoit un score de 1. Si elle montre un tremblement sévère, boites sévères, a abaissé le bassin, ou le point de pieds loin du corps lors de la locomotion («pieds de canard»), il reçoit un score de 2. Si la souris a du mal à aller de l&#39;avant et fait glisser son abdomen sur le sol, il reçoit un score de 3. </li><li> Placez la souris de nouveau dans sa cage et enregistrer son score démarche. </li></ol> Cyphose La cyphose est une courbure caractéristique dorsale de la colonne vertébrale qui est une manifestation fréquente des maladies neurodégénératives dans des modèles murins [2]. Elle est causée par une perte de tonus musculaire dans les muscles spinaux secondaire à une neurodégénérescence. <ol><li> Retirez la souris de sa cage et la placer sur une surface plane. </li><li> Observez comme il marche. Si la souris est en mesure de facilement redresser sa colonne vertébrale comme il marche, et n&#39;a pas cyphose persistante, il reçoit un score de 0. Si la souris montre une cyphose légère, mais est capable de redresser sa colonne vertébrale, il reçoit un score de 1. Si elle est incapable de redresser sa colonne vertébrale complètement et maintient une cyphose persistante mais doux, il reçoit un score de 2. Si la souris conserve une cyphose prononcée que ça marche ou pendant qu&#39;il est assis, il est attribué un score de 3. </li><li> Placez la souris de nouveau dans sa cage et enregistrer son score cyphose. </li></ol> Les résultats représentatifs Avec un nombre suffisant d&#39;animaux, cette procédure est capable de détecter des différences entre les souches phénotype et au sein de la même souche au cours du temps. Pour l&#39;analyse des données, calculer le score pour chaque mesure en prenant lamoyenne des trois mesures dans chaque individu. Des mesures peuvent être analysés séparément ou combinés en un score composite de phénotype de chaque individu. Tableau des données et appliquer les méthodes statistiques appropriées pour déterminer la signification. La figure 1 montre les résultats d&#39;une évaluation composite d&#39;un phénotype murin transgénique SCA7 modèle. Dans ce modèle floxés-SCA7-92Q transgéniques, l&#39;humain ataxine-7 gène avec 92 répétitions CAG est flanqué par des sites loxP et exprimé à partir d&#39;un chromosome artificiel bactérien. Le score composite inclut le phénotype en joignant, en marchant corniche, la démarche et les évaluations cyphose pour un score maximum possible de 12. Les progressistes SCA7 phénotype chez les souris floxés-SCA7-92Q transgénique est démontrée par un score composite de phénotype augmente, ce qui est cohérent avec la nature progressive de la maladie humaine. <img src="/files/ftp_upload/1787/1787fig1.jpg" alt="Figure 1" /> . Figure 1 floxés-SCA7-92Q souris présentent une progressive SCA7 phénotype qui est sensiblement différent de non transgéniques de la même portée à partir de 20 semaines (2-way ANOVA: Bonferroni post-hoc; *** p &lt;0,001). Les souris ont été évaluées sur une échelle 0-3 pour chacun des tests corniche, serrant, la démarche, et la cyphose. Moyenne score composite pour chaque génotype à chaque âge a été calculée. Les barres représentent 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>

Ces études ont été approuvés par l&#39;Université de Washington, les soins des animaux et du Comité institutionnel d&#39;utilisation.

Ce protocole est conçu pour être une évaluation sensible et facilement réalisée de la gravité de la maladie chez des souris modèles de l&#39;ataxie cérébelleuse. Les composants individuels du système de notation sera plus ou moins efficaces dans des modèles murins différents de la neurodégénérescence. Les éléments de ce système de notation ont été effectivement utilisés pour évaluer une variété de modèles murins de maladies neurodégénératives humaines, y compris l...

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

Nous décrivons un protocole pour la quantification rapide et sensible de la gravité de la maladie dans des modèles murins de cervelets ataxie. Il est dérivé du phénotype des évaluations précédemment publiées dans les modèles de plusieurs maladies, y compris les ataxies spino-cérébelleuse, maladie de Huntington et de spinobulbar atrophie musculaire. Les mesures comprennent des membres postérieurs en joignant, corniche de test, la démarche et une cyphose. Chaque mesure est enregistrée sur une échelle de 0-3, avec un total combiné de 0-12 pour les quatre mesures. Les résultats effectivement une discrimination entre les individus affectés et non affectés, tout en quantifiant l&#39;évolution temporelle des phénotypes des maladies neurodégénératives. Les mesures peuvent être analysées individuellement ou combinés en un score composite pour le phénotype plus grande puissance statistique. La combinaison idéale des quatre mesures décrites dépendra de la maladie en question. Nous présentons un exemple de protocole utilisé pour évaluer la gravité des maladies dans un modèle de souris transgénique de l&#39;ataxie spinocérébelleuse de type 7 (SCA7). Albert R. La Spada et Gwenn A. Jardin contribué à ce manuscrit même.

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

Nous décrivons un protocole pour la quantification rapide et sensible de la gravité de la maladie dans des modèles murins de l&#39;ataxie cérébelleuse. Les mesures comprennent des membres postérieurs en joignant, corniche de test, la démarche et une cyphose. Ce protocole est discriminatoire efficace entre les individus affectés et non affectés, et détecte la progression des personnes touchées au cours du temps.

Un simple système de notation composite phénotype pour l&#39;évaluation des modèles murins de l&#39;ataxie cérébelleuse

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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35.1K vues.  University of Washington. Nous décrivons un protocole pour la quantification rapide et sensible de la gravité de la maladie dans des modèles murins de l'ataxie cérébelleuse. Les mesures comprennent des membres postérieurs en joignant, corniche de test, la démarche et une cyphose. Ce protocole est discriminatoire efficace entre les individus affectés et non affectés, et détecte la progression des personnes touchées au cours du temps.

Vidéo: 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.

Les modèles murins de la leucomalacie périventriculaire

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

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Neurosciences

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.

Multiple-Mouse Imaging neuroanatomiques par résonance magnétique

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.

Cultures organotypiques cérébelleuse: Défis apoptotiques et détection

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.

Analyse quantitative des vésicules reconstitution Piscine synaptique dans des cultures de neurones granulaires du cervelet utilisant des colorants 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 nanochirurgie de cervelet axones<em&gt; In 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.

Les tests olfactifs pour les modèles murins de maladies neurodégénératives

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.

Modélisation de la mort neuronale et la dégénérescence de neurones granules primaires de cervelet de souris

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.

Évaluation de l'induction à long terme de dépression dans les tranches de cérévelateur adultes

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;

Dissection régionale cérébelleuse pour l’analyse moléculaire

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.