Ambra Southwell configurare l&#39;apparato e addestrato gli autori nel suo utilizzo. Il finanziamento è stato fornito da un finanziamento della NINDS a Paul Patterson.

1. Il presente protocollo si basa su quelli di Southwell et. al. (2009) e Carter et. al. (2001). <ol><li> L&#39;apparato è costituito raggio di 1 metro con travi in ​​una superficie piatta di 12 mm o 6 mm di larghezza) a riposo 50 cm sopra il piano del tavolo su due poli. Una scatola nera è posto alla fine della trave come punto di arrivo. (Compensato e altri tipi di legno per le travi, pali, e la scatola si possono trovare in qualsiasi negozio di ferramenta). Materiale di nidificazione da gabbie di casa si trova nella scatola nera per attirare il mouse al punto di finire. Una lampada (lampadina con 60 watt di luce) è utilizzato per illuminare al di sopra del punto di inizio e funge da stimolo avversivo. Il tempo di attraversare il centro 80 centimetri è misurata da due sensori di movimento (fabbricate da Visopia): uno a 0 cm, che avvia un timer e uno a 80 cm che ferma il timer. Un amaca in nylon (che può essere ottenuto da un deposito del tessuto locale) è allungata al di sotto del fascio, ~ 7,5 cm sopra il piano del tavolo, per attutire eventuali cadute. Una videocamera è impostata su un treppiede per registrare l&#39;efficacia. L&#39;apparecchio utilizzato in questa dimostrazione è stato costruito in loco. </li><li> Una coorte di almeno 10 topi per ogni condizione è raccomandata per questo test. I topi sono alloggiati come gruppi, quando possibile, in una luce 12 ore (07:00-7:00) / ciclo buio e acclimatati alle loro gabbie nella struttura comportamentale per almeno 2 settimane prima dell&#39;inizio del test. Circa 10 minuti prima formazione / test, i topi possono essere trasportate nel locale che contiene l&#39;apparato fascio. </li><li> Nei giorni di training, ogni topo attraversa il fascio di 12 mm 3 volte e poi il 6 millimetri fascio 3 volte. Travi di una più ampia larghezza (ovvero 28 mm) può essere utilizzato per i topi che non sono in grado di attraversare le strette o per i più grandi topi. I topi sono posti ad una estremità di una trave e il tempo necessario per attraversare alla casella fuggire dall&#39;altro lato (80 cm di distanza) si misura con i rivelatori di movimento. Il timer viene avviato il naso del mouse che entrano nel centro 80 cm, e si fermò quando l&#39;animale raggiunge la fine del 80 cm. Il resto topi per 10 minuti nelle loro gabbie a casa tra le sessioni di formazione sulle due travi. Durante questo periodo tra le prove, un altro 3-4 topi possono essere addestrati a fare un uso efficiente del tempo. Topi C57BL / 6 tipicamente attraversano il fascio con una quantità minima di arresto o di interruzione. Tuttavia, quando lo fanno stallo, annusare o guardarsi intorno senza procedere in avanti, lo sperimentatore deve incoraggiare il mouse per continuare a progredire da pungolo, colpendo, o spingendo da dietro con le dita guantate. Una volta che i topi sono nella cassetta di sicurezza, essi sono autorizzati po &#39;di tempo (~ 15 sec) a riposare lì prima della prova successiva. Dopo ogni sessione di allenamento, i topi vengono restituiti direttamente a loro gabbie a casa e portato nella loro stanza alloggi. Da notare che i topi possono stallo di più se sono più addestrati e diventare troppo familiare con il compito e apparecchi. D&#39;altra parte, la formazione può essere continuato per giorni aggiuntivi se i topi continuano a non attraversare l&#39;intero fascio di successo dopo 2 giorni di formazione. Se i trattamenti sono applicati ai topi, una misura di base della performance deve essere ottenuto prima del trattamento. </li><li> Il giorno del test, i tempi di attraversare ogni beam sono registrati. Due studi di successo in cui il mouse non stallo sulla trave sono nella media. Registrazioni video possono essere utilizzati per analisi più fine di scivolare e di altri deficit motori osservabili. Un modo alternativo per valutare camminare fascio è da un 1,2 neurologico sistema di punteggio, che i tassi l&#39;impossibilità di attraversare la trave come un punteggio di &quot;1&quot; e la capacità di attraversare il fascio normalmente con entrambe le zampe sulla superficie del fascio e meno due linguette piede come un &quot;7&quot;. Una scivolata è definito come il piede venuta fuori la parte superiore della trave. In questo sistema di indicizzazione, un punteggio di &quot;3&quot; corrisponderebbe ad attraversare il fascio trascinando gli arti. Un animale che i punteggi 1 su due prove non è ripetere il test. Descrizione delle prestazioni sulla trave relativi ai punteggi si possono trovare in Feeney et al., (1982) e Carter et al., (2001). </li><li> Le travi e box vengono puliti di escrementi di topo e si asciugò con asciugamani imbevuti con il 70% di etanolo e poi l&#39;acqua prima che il fascio successivo è situato su di esso. </li></ol> 2. Rappresentante dei risultati: <img src="/files/ftp_upload/2376/2376fig1.jpg" alt="Figura 1" /> Figura 1. Tipiche volte croce di C57Bl6 maschili e femminili su travi 6mm e 12mm. Una coorte di 9 a 11 anni settimana C57BL/6-NCRL topi WT sono stati testati su entrambi i fasci dopo 2 giorni di formazione. Tempi medi per l&#39;attraversamento dei 12 fascio mm erano 4,6 ± 0,4 sec per gli uomini (n = 14) e 3,3 ± 0,3 sec per le femmine (n = 11). Volte sulla trave 6 millimetri sono stati 6,8 ± 0,7 sec per gli uomini (n = 13) e 5,9 ± 0,5 secondi per le femmine (n = 10) (gli errori sono segnalati come SEM).

<ol>
	<li>Feeney, D. M., Gonzalez, A., Law, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine,+haloperidol,+and+experience+interact+to+affect+rate+of+recovery+after+motor+cortex+injury.">Amphetamine, haloperidol, and experience interact to affect rate of recovery after motor cortex injury.</a> Science. 217, 855-857 (1982).</li><li>Carter, R. J., Morton, J., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+coordination+and+balance+in+rodents.">Motor coordination and balance in rodents.</a> Curr Protoc Neurosci. Chapter 8, (2001).</li><li>Wallace, J. E., Krauter, E. E., Campbell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+and+reflexive+behavior+in+the+aging+rat.">Motor and reflexive behavior in the aging rat.</a> J Gerontol. 35, 364-370 (1980).</li><li>Brooks, S. P., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tests+to+assess+motor+phenotype+in+mice:+a+user's+guide.">Tests to assess motor phenotype in mice: a user's guide.</a> Nat Rev Neurosci. 10, 519-529 (2009).</li><li>Fox, G. B., Fan, L., Levasseur, R. A., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+sensory/motor+and+cognitive+deficits+with+neuronal+apoptosis+following+controlled+cortical+impact+brain+injury+in+the+mouse.">Sustained sensory/motor and cognitive deficits with neuronal apoptosis following controlled cortical impact brain injury in the mouse.</a> J Neurotrauma. 15, 599-614 (1998).</li><li>Buccafusco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of behavioral analysis in neuroscience. , (2009).</li><li>Southwell, A. L., Ko, J., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrabody+gene+therapy+ameliorates+motor,+cognitive,+and+neuropathological+symptoms+in+multiple+mouse+models+of+Huntington's+disease.">Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease.</a> J Neurosci. 29, 13589-13602 (2009).</li><li>Carter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+progressive+motor+deficits+in+mice+transgenic+for+the+human+Huntington's+disease+mutation.">Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation.</a> J Neurosci. 19, 3248-3257 (1999).</li><li>Gulinello, M., Chen, F., Dobrenis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+deficits+in+motor+coordination+and+cognitive+dysfunction+in+a+mouse+model+of+the+neurodegenerative+lysosomal+storage+disorder,+Sandhoff+disease.">Early deficits in motor coordination and cognitive dysfunction in a mouse model of the neurodegenerative lysosomal storage disorder, Sandhoff disease.</a> Behav Brain Res. 193, 315-319 (2008).</li><li>Stanley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+beam+walking+assay+offers+improved+sensitivity+over+the+mouse+rotarod+in+determining+motor+coordination+deficits+induced+by+benzodiazepines.">The mouse beam walking assay offers improved sensitivity over the mouse rotarod in determining motor coordination deficits induced by benzodiazepines.</a> J Psychopharmacol. 19, 221-227 (2005).</li></ol>

Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dal Istituzionali Animal Care Caltech e del Comitato uso (IACUC).

Prestazioni sulla trave equilibrio è una misura utile di coordinazione fine e l&#39;equilibrio ed è stato convalidato dal lavoro precedente. Il test raggio in grado di rilevare i deficit motori a causa dell&#39;età, nervoso centrale syst `lesioni, e le manipolazioni genetiche e farmacologiche nei roditori giovani e meno giovani 2,3,4. I topi con lesioni corticali impatto spesso mostrano controlaterale scivolare sulla trave 5,6. Il test beam è stato utilizzato anche per differenziare le abilità...

<ul><li> Travi: 1-m strisce di legno liscio con la piazza di piccole, medie e grandi dimensioni (cioè, 6, 12, e 28 mm di larghezza) sezioni trasversali </li><li> Supporto stand per tenere la sezione avvio del fascio sollevato (~ 1,5 cm di sezione, 50 cm di altezza) </li><li> Box obiettivo (20 cm su ogni lato) montata su un cavalletto di sostegno (3 cm di sezione, 50 cm di altezza) </li><li> Amaca-~ 7,5 cm sopra il tavolo </li><li> 60-watt (W) lampada da tavolo </li><li> Videocamera, treppiede, e videocassette vuoto </li><li> Etanolo, asciugamani di carta, materiali di pulizia </li></ul>

assessment of motor balance and coordination in mice using the balance beam

Lesione cerebrale, manipolazioni genetiche e trattamenti farmacologici possono provocare alterazioni delle capacità motorie nei topi. Coordinazione motoria fine e l&#39;equilibrio può essere valutato dal saggio breve raggio. L&#39;obiettivo di questo test è per il mouse per rimanere in piedi e camminare attraverso un fascio di elevata stretto a una piattaforma sicura. Questo test si svolge nell&#39;arco di 3 giorni consecutivi: 2 giorni di allenamento e 1 giorno di test. Prestazioni sulla trave viene quantificato misurando il tempo necessario per il mouse per attraversare il raggio e il numero di polizze di zampa che si verificano nel processo. Qui riportiamo il protocollo utilizzato nel nostro laboratorio, ed i risultati rappresentante di un gruppo di topi C57BL / 6. Questo compito è particolarmente utile per la rilevazione di sottili deficit nelle abilità motorie e di equilibrio che può non essere rilevato da altri test motore, come il rotarod.

Watch this Scientific Journal Video about Assessment of Motor Balance and Coordination in Mice using the Balance Beam at JoVE.com

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Deficit di coordinazione motoria fine può essere valutata con il test trave. Prestazioni sulla trave viene quantificato la velocità con cui è attraversato il raggio e il numero di volte il mouse scivola sulla trave.

Valutazione del bilanciamento del motore e di coordinamento nei topi utilizzando la Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

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JoVE Journal

Neuroscience

46.2K Views.  California Institute of Technology. Deficit di coordinazione motoria fine può essere valutata con il test trave. Prestazioni sulla trave viene quantificato la velocità con cui è attraversato il raggio e il numero di volte il mouse scivola sulla trave.

Video: Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Misurare Coordinamento motore in Mouse

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

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Neuroscienze

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Valutazione della morfina-indotta iperalgesia e analgesico tolleranza nei topi mediante nocicettive termica e meccanica Modalità

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Assessment of Vascular rigenerazione nel SNC Utilizzo del Retina mouse

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

La giunzione neuromuscolare: Synapse Misurazione circonferenza, la frammentazione e Variazioni Synaptic Proteine ​​Densità Uso confocale microscopia a fluorescenza

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Valutazione della dendritiche arborizzazione nel giro dentato della Regione ippocampale nei topi

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

Un uomo-macchina-interfaccia Integrazione di sensori a basso costo con una stimolazione del sistema neuromuscolare elettrica per post-ictus Balance riabilitazione

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funzionale e morfologica Valutazione della membrana innervazione da Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Valutazione della complessità dendritica ippocampale nei topi invecchiati utilizzando il metodo Golgi-Cox

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Analisi Neuroanatomical retrogrado dei motoneuroni frenico in topi

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Analisi kinematica 3D per la valutazione funzionale nel modello di ratto di lesioni da schiacciamento del nervo sciatico

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...