Name: preparation of parasagittal slices for the investigation of dorsal-ventral organization of the rodent medial entorhinal cortex
Uploaded: 2012-03-28T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex at JoVE.com

Vi er takke følgende for deres støtte: Commonwealth Scholarship Kommissionen England finansiering (HP), EPSRC (HP), BBSRC (MFN) og EU-Marie Curie-aktioner (MFN).

1. Parasagittal Slice Forberedelse 1,1 Skær ud hjernehalvdele Alle dyreforsøg bør følge lokale etisk vurdering og nationale regler. I tilfælde af forsøgene beskrevet her, er i overensstemmelse arbejdet til de britiske Dyr (Scientific Procedures) Act 1986. Vi rutinemæssigt bruger cervikal dislokation uden bedøvelse for at aflive musen, før du fjerner hjernen. Alternativt musen kan være terminalt bedøvede, men i dette tilfælde kan det være nødvendigt at be...

<ol>
	<li>O'Donnell, C., Nolan, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+synaptic+responses:+an+organizing+principle+for+optimization+of+neural+circuits.">Tuning of synaptic responses: an organizing principle for optimization of neural circuits.</a> Trends Neurosci. 34, 51-60 (2011).</li><li>Garden, D. L. F., Dodson, P. D., O'Donnell, C., White, M. D., Nolan, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+synaptic+integration+in+the+medial+entorhinal+cortex+to+the+organization+of+grid+cell+firing+fields.">Tuning of synaptic integration in the medial entorhinal cortex to the organization of grid cell firing fields.</a> Neuron. 60, 875-889 (2008).</li><li>Kuba, H., Yamada, R., Fukui, I., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonotopic+specialization+of+auditory+coincidence+detection+in+nucleus+laminaris+of+the+chick.">Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick.</a> Journal of Neuroscience. 25, 1924-1934 (2005).</li><li>Hafting, T., Fyhn, M., Molden, S., Moser, M. -. B., Moser, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+of+a+spatial+map+in+the+entorhinal+cortex.">Microstructure of a spatial map in the entorhinal cortex.</a> Nature. 436, 801-806 (2005).</li><li>Sargolini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjunctive+representation+of+position,+direction,+and+velocity+in+entorhinal+cortex.">Conjunctive representation of position, direction, and velocity in entorhinal cortex.</a> Science. 312, 758-762 (2006).</li><li>Fyhn, M., Hafting, T., Witter, M. P., Moser, E. I., Moser, M. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grid+cells+in+mice.">Grid cells in mice.</a> Hippocampus. 18, 1230-1238 (2008).</li><li>Giocomo, L. M., Zilli, E. A., Frans&#233;n, E., Hasselmo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+frequency+of+subthreshold+oscillations+scales+with+entorhinal+grid+cell+field+spacing.">Temporal frequency of subthreshold oscillations scales with entorhinal grid cell field spacing.</a> Science. 315, 1719-1722 (2007).</li><li>Giocomo, L. M., Hasselmo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+constants+of+h+current+in+layer+II+stellate+cells+differ+along+the+dorsal+to+ventral+axis+of+medial+entorhinal+cortex.">Time constants of h current in layer II stellate cells differ along the dorsal to ventral axis of medial entorhinal cortex.</a> Journal of Neuroscience. 28, 9414-9425 (2008).</li><li>Burgalossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcircuits+of+functionally+identified+neurons+in+the+rat+medial+entorhinal+cortex.">Microcircuits of functionally identified neurons in the rat medial entorhinal cortex.</a> Neuron. 70, 773-786 (2011).</li><li>Dodson, P. D., Pastoll, H., Nolan, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+organization+of+theta-like+activity+intrinsic+to+entorhinal+stellate+neurons+is+mediated+by+differences+in+stochastic+current+fluctuations.">Dorsal-ventral organization of theta-like activity intrinsic to entorhinal stellate neurons is mediated by differences in stochastic current fluctuations.</a> J. Physiol. (Lond). 589, 2993-3008 (2011).</li><li>Nolan, M., Dudman, J., Dodson, P., Santoro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN1+channels+control+resting+and+active+integrative+properties+of+stellate+cells+from+layer+II+of+the+entorhinal+cortex.">HCN1 channels control resting and active integrative properties of stellate cells from layer II of the entorhinal cortex.</a> Journal of Neuroscience. 27, (2007).</li><li>Boehlen, A., Heinemann, U., Erchova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+range+of+intrinsic+frequencies+represented+by+medial+entorhinal+cortex+stellate+cells+extends+with+age.">The range of intrinsic frequencies represented by medial entorhinal cortex stellate cells extends with age.</a> Journal of Neuroscience. 30, 4585-4589 (2010).</li><li>Klink, R., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+characteristics+of+layer+II+projection+neurons+in+the+rat+medial+entorhinal+cortex.">Morphological characteristics of layer II projection neurons in the rat medial entorhinal cortex.</a> Hippocampus. 7, 571-583 (1997).</li><li>van Groen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entorhinal+cortex+of+the+mouse:+cytoarchitectonical+organization.">Entorhinal cortex of the mouse: cytoarchitectonical organization.</a> Hippocampus. 11, 397-407 (2001).</li><li>Dolorfo, C. L., Amaral, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entorhinal+cortex+of+the+rat:+organization+of+intrinsic+connections.">Entorhinal cortex of the rat: organization of intrinsic connections.</a> The Journal of Comparative Neurology. 398, 49-82 (1998).</li></ol>

For at lette undersøgelse af MEC kredsløb egenskaber, der følger en dorsal-ventral organisation, vi har beskrevet her i detaljer en fremgangsmåde til fremstilling af en parasagittal skive præparat, der bevarer den dorsale-ventrale omfang MEC. Kritiske trin Fjernelse af hjernen fra dyret. Vær særlig omhyggelig med at undgå at lægge pres på hjernen. Dette er mere vigtigt end hurtig fjernelse af hjernen. U...

<table border="1"><tbody><tr><td></td><td> Skæring ACSF (mM) </td><td> Standard ACSF (mM) </td><td> Intern opløsning (mM) </td><td> CASNumber </td><td> Leverandør Katalognummer </td></tr><tr><td> NaCl </td><td> 86 </td><td> 124 </td><td></td><td> 7647-14-5 </td><td> Sigma S9888 </td></tr><tr><td> NaH 2 PO 4 </td><td> 1,2 </td><td> 1,2 </td><td></td><td> 13472-35-0 </td><td> Sigma 71505 </td></tr><tr><td> KCl </td><td> 2,5 </td><td> 2,5 </td><td> 10 </td><td> 7447-40-7 </td><td> Sigma P3911 </td></tr><tr><td> NaHCO3 </td><td> 25 </td><td> 25 </td><td></td><td> 144-55-8 </td><td> Fischer S/4240 </td></tr><tr><td> Glucose </td><td> 25 </td><td> 20 </td><td></td><td> 50-99-7 </td><td> Sigma G5767 </td></tr><tr><td> Saccharose </td><td> 75 </td><td></td><td></td><td> 57-50-1 </td><td> Sigma S5016 </td></tr><tr><td> CaCl2 </td><td> 0,5 </td><td> 2 </td><td></td><td> 10043-52-4 </td><td> VWR 190464K </td></tr><tr><td> MgCl2 </td><td> 7 </td><td> 1 </td><td> 2 </td><td> 7786-30-3 </td><td> Sigma 63020 </td></tr><tr><td> K Glykonat </td><td></td><td></td><td> 130 </td><td> 299-27-4 </td><td> Sigma G4500 </td></tr><tr><td> HEPES </td><td></td><td></td><td> 10 </td><td> 7365-45-9 </td><td> Sigma H3375 </td></tr><tr><td> EGTA </td><td></td><td></td><td> 0,1 </td><td> 67-42-5 </td><td> Sigma E4378 </td></tr><tr><td> Na2 ATP </td><td></td><td></td><td> 2 </td><td> 34369-07-8 </td><td> Sigma A7699 </td></tr><tr><td> Na2 GTP </td><td></td><td></td><td> 0,3 </td><td> 36051-31-7 </td><td> Sigma G8877 </td></tr><tr><td> NaPhospho-Kreatin </td><td></td><td></td><td> 10 </td><td> 19333-65-4 </td><td> Sigma P7936 </td></tr><tr><td> Biocytin (valgfrit) </td><td></td><td></td><td> 2,7 </td><td> 576-19-2 </td><td&gt; Sigma B4261 </td></tr></tbody></table> Tabel 1. Skæring ACSF, standard ACSF og K-gluconat intern løsning opskrifter.

preparation of parasagittal slices for the investigation of dorsal-ventral organization of the rodent medial entorhinal cortex

Beregning i hjernen afhængig neuroner reagerer hensigtsmæssigt på deres synaptiske input. Neuroner er forskellige i deres komplement og distribution af membran ionkanaler, der bestemmer, hvordan de reagerer på synaptiske input. Imidlertid er forholdet mellem disse cellulære egenskaber og neuronal funktion i at opføre dyr ikke godt forstået. En løsning på dette problem er at undersøge topografisk organiseret neurale kredsløb, hvor placeringen af de enkelte neuroner kort på de oplysninger, de koder eller beregninger, de foretager 1. Forsøg med denne fremgangsmåde foreslår principper for tuning af synaptiske reaktioner underliggende oplysninger kodning i sensoriske og kognitive kredsløb 2,3. Den topografiske organisering af rumlige repræsentationer langs dorsal-ventral akse mediale entorhinal cortex (MEC) giver en mulighed for at etablere relationer mellem cellulære mekanismer og beregninger important for geografisk kognition. Neuroner i lag II gnaver MEC kode placering ved hjælp af grid-lignende fyre felter 4-6. For neuroner findes i dorsale positioner i MEC afstanden mellem de individuelle affyringstider felter, som danner et gitter, er af størrelsesordenen 30 cm, hvorimod neuroner ved progressivt mere ventrale positioner denne afstand øges til mere end en meter. Adskillige undersøgelser har afsløret cellulære egenskaber af neuroner i lag II MEC, der som afstanden mellem gitteret fyres områder, også variere i henhold til deres dorsal-ventral position, hvilket antyder, at disse cellulære egenskaber er vigtige for rumlig beregning 2,7-10. Her beskriver vi procedurer for forberedelse og elektrofysiologiske optagelse fra hjernen skiver, der opretholder dorsal-ventral omfanget af MEC muliggør undersøgelse af topografiske organisation biofysiske og anatomiske egenskaber MEC neuroner. Dorsal-ventrale position identificeres neurons forhold til anatomiske landmærker er vanskeligt at fastslå præcist med protokoller, som bruger vandrette skiver af MEC 7,8,11,12, da det er vanskeligt at opstille referencepunkter for den nøjagtige dorsal-ventral placering skive. De procedurer, vi beskriver muliggør nøjagtig og konsistent måling af placeringen af registrerede celler langs den dorsale-ventrale akse MEC samt visualisering af molekylære gradienter 2,10. Procedurer er blevet udviklet til anvendelse med voksne mus (&gt; 28 dage) og er med held blevet anvendt med mus op til 1,5 år. Med justeringer de kunne bruges med yngre mus eller andre gnaverarter. Et standardiseret system af forberedelse og måling vil hjælpe systematisk undersøgelse af de cellulære og mikrokredsløb egenskaber af dette område.

Watch this Scientific Journal Video about Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex at JoVE.com

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Vi beskriver procedurer for forberedelse og elektrofysiologiske optagelse fra hjernen skiver, der opretholder dorsal-ventral akse mediale entorhinal cortex (MEC). Fordi neurale kodning af placeringen følger en dorsal-ventral organisation i MEC, disse procedurer lette efterforskningen af ​​cellulære mekanismer, der er vigtige for navigation og hukommelse.

Udarbejdelse af parasagittal skiver til undersøgelse af Dorsal-ventrale Organization of the Rodent Medial entorhinal cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Research

JoVE Journal

Neuroscience

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Fremstilling af akutte Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Samtidig Elektroencefalografi, Real-time måling af laktat Koncentration og Optogenetic Manipulation af neuronale aktivitet i Rodent Cerebral Cortex

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

En omfattende protokol for manuel segmentering af Medial temporallap Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Forberedelse af akut hjernen skiver bruger en optimeret N-Methyl-D-glucamine beskyttende genoprettelsesmetode

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Udarbejdelsen af ​​Oblique Spinal Cord Skiver for ventrale Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Virkningerne af transkranial vekselstrøm Stimulation på den primære Motor Cortex af Online kombineret tilgang med transkranial magnetisk Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Kombinerede transkranial magnetisk Stimulation og electroencefalografi i den Dorsolateral præfrontale Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Forberedelse af rytmisk-aktiv i Vitro Neonatal gnaver hjernestammen-rygmarven og tynd skive

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Stochastic Noise ansøgning om vurdering af mediale vestibulære Nucleus neuron følsomhed in vitro

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed ...