Questo studio è stato supportato da nuovi FMMU trovando fondazione e in parte fondi NIH.

1. Glia colture cellulari Cellule gliali possono essere coltivate provenienti da diverse regioni del sistema nervoso centrale. L&#39;intero processo è mostrato in figura processo. <img src="/files/ftp_upload/2111/2111process.jpg" alt="Processo 1" /> Giorno 1 cultura piastra di rivestimento e coprioggetto <ol><li> Sterilizzati a secco tondo microscopio vetro coprioggetto in autoclave. </li><li> Targhetta del coprioggetto sterilizzati sterilizzato in 24 pozzetti cultura. </li><li> Rivestire il coprioggetto con poli-lisina e incubare per 2 ore a temperatura radice. </li><li> Cappotto da 6 pozzetti cultura allo stesso modo il punto 3. </li><li> Lavare i coprioggetti e 6-bene piastra di coltura per due volte con acqua distillata e aria secca nella cappa della cultura. </li><li> Aggiungere 200 microlitri di media DMEM (con 10% FBS) in ciascun pozzetto a 24-pozzetti e 2 ml di ogni bene in 6 pozzetti e metterle in incubatrice sotto 37 gradi con il 5% di CO 2 </li></ol> Giorno 2 corteccia di isolamento e coltura di cellule gliali <ol><li> Sterilizzare il positivo cofano dissezione flusso. </li><li> Accendere la luce UV per 20 minuti. </li><li> Spruzzare tutte le superfici con il 70% di etanolo e attendere 15 minuti prima dell&#39;uso. </li><li> cambiamento teso: cuccioli di ratto Anestetizzare (P2-P6) per ipotermia, e decapitare alla base della magnum framen con le forbici operativo. </li><li> Aprire il cranio lungo la sutura sagittale con un forbice iride e staccare il cranio. </li><li> Rimuovere il proencefalo e metterle in fresco L15 media, sotto stereomicroscopio, pulire accuratamente la membrana dura e pia con i vasi sanguigni, isolare la corteccia e lavare più volte con L15 media. </li><li> Tagliare la corteccia in piccoli pezzi con forbice microchirurgia. </li><li> Aggiungi 0,125% tripsina-EDTA preparati con L15 medio a pezzi corteccia e incubare a 37 gradi per 15 min. </li><li> Trasferire blocchi di tessuto in 20 mL medium DMEM con 20% FBS in 50mL tubo, aggiungere soluzione madre DNasi per 10ug/mL concentrazione finale, aspirare la soluzione di tessuto su e giù per 20 volte con lucido vetro fuoco Pasteure pipetta, raccogliere la sospensione singole cellule. </li><li> Lavare la sospensione cellulare una volta con DMEM medio, risospendere le cellule in DMEM con 10% FBS, contare le cellule al microscopio le cellule con un emocitometro, seme a 5000-10000/cm 2. Mantenere le cellule in grado incubatore 37 5%, cambiare la metà della media 2 volte alla settimana. </li></ol> 2. Gangli della radice dorsale neuroni isolamento, la cultura e purificazione Giorno 1 Preparare Cultura Materiale <ol><li> Rivestire il coprioggetto sterilizzati con poli-lisina, coprioggetto lavare due volte con distillare l&#39;acqua e l&#39;aria secca, metterle in piastre da 24 pozzetti. </li><li> Aggiungi 100μL medio Neurobasal con il 2% B27 e 2,5 s NGF (50 ng / mL), messo piastra in 37 CO grado 5% 2. </li></ol> Giorno 2 Isolare DRG da embrioni <ol><li> Sterilizzato la cappa a flusso nello stesso modo come la cultura delle cellule gliali. </li><li> Euthanize un ratto in gravidanza (E15) per overdose di CO 2, addome sterilizzati con etanolo al 70%, gli embrioni sono stati isolati da utero e messo in fresco L15 media. </li><li> Isolare i cavi collegati spinale con i gangli spinali sotto stereomicroscopio e trasferimento a piatti da 35 mm con refrigerata L15 media. </li><li> Raccogliere sospensione singola cella e lavare una volta con NBF medio (medio Neurobasal contenente il 2% B27 e 2,5 s NGF (50 ng / ml)). </li></ol> 3 ° giorno Purify neuroni DRG <ol><li> 18h-24h dopo la semina neurone, aggiungere una soluzione concentrata di mm 1 magazzino FUDR alla cultura neurone ad una concentrazione finale di 20 micron e un volume finale di 200 ul. </li><li> 72h dopo, sostituire la metà della media con media Neurobasal contenente il 2% B27 e 2,5 s NGF (50ng/mL) senza FUDR. Dopo di che, cambiare il medium a giorni alterni. </li></ol> 3. Co-coltura neuroni DRG con le cellule gliali <ol><li> Quando la cultura di cellule gliali raggiunto confluenza (circa 20 giorni dopo la semina), erano pronti per la co-coltura con i neuroni. </li><li> 24 ore prima di aggiungere i neuroni purificate, le cellule gliali media sono stati modificati e medie Neurobasal contenente il 2% B27 e 2,5 s NGF (50 ng / ml). </li><li> Neuroni purificati sono stati raccolti dai pozzi cultura, singolo sospensione le cellule sono state ottenute facendo passare meccanico attraverso pipetta Pasteure fuoco lucido. </li><li> Dopo il conteggio del numero, i neuroni sono stati seminati a 500/cm 2 sul confluenti cellule gliali nel medio Neurobasal contenente il 2% B27 e 2,5 s NGF (50ng/mL). </li><li> Adesione dei neuroni e la crescita dei neuriti delle cellule gliali potrebbero essere registrati e analizzati in diversi momenti da un software di analisi delle immagini e immunocitochimica utilizzando anticorpi tipo specifico di cellule. </li></ol> 4. Rappresentante Risultati <ol><li> Coltura delle cellule gliali: Dopo la semina, le cellule gliali diventerà confluenti circa 20 giorni. Sotto microsc contrasto di faseOPE, è stato facilmente identificato che diverse sottopopolazioni morfologica delle cellule gliali formate diverse sottostrutture modello di crescita e la astrociti GFAP positivi rappresentato più del 90%, come indicato dalla tecnica immuocytochemisty (Figura 1, Figura 2). </li><li> DRG cultura neuroni: Nel nostro metodo, dopo 72 FUDR trattamento ore, la purezza dei neuroni DRG raggiungerà più in alto del 99% entro 6 giorni, i neuroni DRG hanno mostrato la loro morfologia unica e con una crescita dei neuriti alta densità (Figura 3, Figura 4). </li><li> Co-coltura delle cellule gliali e neuroni: i neuroni DRG adesione e crescita dei neuriti verificato facilmente sulle cellule gliali entro 4 ore dopo la semina, attente osservazioni hanno mostrato che l&#39;adesione dei neuroni e la crescita dei neuriti sono stati influenzati da sottopopolazioni di cellule gliali che formano speciale sottostrutture modello di crescita, questo potrebbe essere facilmente identificati al microscopio a contrasto di fase e mediante immunocitochimica (Figura 5, Figura 6). </li></ol><img src="/files/ftp_upload/2111/2111fig1.jpg" alt="Figura 1" /> Figura 1. Morfologia delle cellule gliali confluenti. Corticale cellule gliali sono stati placcati in polilisina coprioggetto patinata e in coltura per 20 giorni, notare le differenze nello sviluppo di cellule gliali, le cellule sulla sinistra disposti in modo irradiata. <img src="/files/ftp_upload/2111/2111fig2.jpg" alt="Figura 2" /> Figura 2. Confluenti cellule gliali etichettati da proteina acidica fibrillare gliale (GFAP) anticorpale. Noti la disposizione irradiata di GFAP (rosso), le cellule positive sul lato destro. <img src="/files/ftp_upload/2111/2111fig3.jpg" alt="Figura 3" /> Figura 3. Neuroni dei gangli dorsali è cresciuto in vitro senza trattamento FUDR. Le cellule contaminanti formata DRG sfondo neuroni &#39;. <img src="/files/ftp_upload/2111/2111fig4.jpg" alt="Figura 4" /> Figura 4. Neuroni dei gangli dorsali è cresciuto in vitro dopo il trattamento FUDR. Le cellule sfondo contaminanti erano stati eliminati completamente. <img src="/files/ftp_upload/2111/2111fig5.jpg" alt="Figura 5" /> Figura 5. Neuroni dei gangli dorsali coltivati ​​su cellule gliali. Adesione neuroni e la crescita dei neuriti sono stati inibiti sulle cellule irradiate organizzato e limitato sul lato destro cellule gliali. <img src="/files/ftp_upload/2111/2111fig6.jpg" alt="Figura 6" /> Figura 6. Neuroni dei gangli dorsali crescono sulle cellule gliali etichettati da anticorpi neurofilamenti. La neurite (verde) sono stati inibiti sul irradiata celle disposte a sinistra gliali e limitato sul lato destro, tutte le cellule gliali sono stati etichettati da anticorpi GFAP (rosso).

<ol>
	<li>Rossignol, S., Schwab, M., Schwartz, M., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+time+to+move?.">Spinal cord injury: time to move?.</a> J Neurosci. 27 (44), 11782-11792 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7 (8), 617-627 (2006).</li><li>Barres, 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=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Matyash, V., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+astrocyte+morphology+and+physiology.">Heterogeneity in astrocyte morphology and physiology.</a> Brain Res Rev. , (2009).</li><li>Wanner, I. B., Deik, A., Torres, M., Rosendahl, A., Neary, J. T., Lemmon, V. P., Bixby, 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=A+new+in+vitro+model+of+the+glial+scar+inhibits+axon+growth.+Glia.">A new in vitro model of the glial scar inhibits axon growth. Glia.</a> 56 (15), 1691-1709 (2008).</li><li>Kuffler, D. P., Sosa, I. J., Reyes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+chondroitin+sulfate+proteoglycan+inhibits+dorsal+root+ganglion+neuron+neurite+outgrowth+and+substrate+specificity+via+a+soma+and+not+a+growth+cone+mechanism.">Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.</a> J Neurosci Res. 87 (13), 2863-2871 (2009).</li><li>Kleitman, N., Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+method+for+the+study+of+myelination.">Tissue culture method for the study of myelination.</a> Culturing nerve cells. , P559-P559 (1998).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo protocollo esperimento è stato progettato per raggiungere due obiettivi per studiare le cellule gliali, l&#39;influenza di eterogeneità soprattutto astrociti sulla adesione dei neuroni e la crescita dei neuriti. Il primo obiettivo era quello di mantenere l&#39;eterogeneità astrociti il ​​più possibile, in questo esperimento, la confluenza delle cellule gliali cultura astrociti arricchito è stato mixato cultura primaria senza alcun trattamento chimico e la propagazione digestione, che possono ulteriorment...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM(high glucose)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-039</td>
<td>&nbsp;</td>
<tr>
<td>L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11415-114</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437-077</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>poly-lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4832</td>
<td>culture grade</td>
</tr>
<tr>
<td>NGF(2.5S)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
<td>&nbsp;</td>
<tr>
<td>B27 supplyment</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>0.25% trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>FUDR</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>neurofilament antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab24575</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

study glial cell heterogeneity influence on axon growth using a new coculture method

Nel sistema nervoso centrale di tutti i mammiferi, gli assoni interrotto dopo l&#39;infortunio non sono in grado di rigenerare i propri obiettivi di originale e di recupero funzionale è molto povera 1. Il fallimento della rigenerazione assonale è il risultato combinato di diversi fattori tra cui l&#39;ambiente ostile di cellule gliali, inibitori delle molecole legate mielina dei neuroni e una diminuzione intrinseca capacità rigenerativa 2. Astrociti sono il tipo più predominante delle cellule gliali nel sistema nervoso centrale e svolgono un ruolo importante nelle funzioni dell&#39;assone in fisiologia e le condizioni di patologia 3. Contrasto con l&#39;omologo oligodendrociti, gli astrociti sono una popolazione cellulare eterogenea composta da diverse sottopopolazioni astrociti con morfologie diverse e l&#39;espressione genica 4. Il significato funzionale di questa eterogeneità, come ad esempio le loro influenze sulla crescita degli assoni, è in gran parte sconosciuto. Per studiare le cellule gliali, in particolare la funzione di eterogeneità astrociti nel comportamento dei neuroni, abbiamo stabilito un nuovo metodo di co-colture di neuroni alta radice purificata gangli dorsali con le cellule gliali ottenuto dalla corteccia di ratto. Con questa tecnica, siamo stati in grado di confrontare direttamente l&#39;adesione e la crescita degli assoni dei neuroni in diverse sottopopolazioni astrociti nelle stesse condizioni. In questo rapporto, diamo il protocollo dettagliato di questo metodo per l&#39;isolamento astrociti e la cultura, i neuroni dei gangli dorsali isolamento e la purificazione, e la co-coltura di neuroni DRG con astrociti. Questo metodo potrebbe essere esteso anche ad altre regioni del cervello per studiare cellulare o regionali specifici di interazione tra i neuroni e cellule gliali.

Watch this Scientific Journal Video about Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method at JoVE.com

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In questo protocollo, abbiamo descritto un nuovo metodo per studiare l&#39;influenza delle eterogeneità delle cellule gliali sulla crescita degli assoni con un<em&gt; In vitro</em&gt; Di co-cultura di sistema. Rat cellule corticali gliali sono state coltivate a confluenza e cocultured con altamente purificato topo neuroni dei gangli dorsali. Diversi influenza delle cellule gliali sull&#39;adesione neuroni e la crescita degli assoni è stato confrontato direttamente nella stessa cultura. Questo metodo fornisce un nuovo modo di studiare direttamente la eterogeneità delle cellule gliali influenza sulla adesione dei neuroni e la crescita degli assoni.

Studio gliali eterogeneità Influenza sulla crescita cellulare Axon Utilizzando un nuovo metodo co-coltura

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

study-glial-cell-heterogeneity-influence-on-axon-growth-using-new

Research

JoVE Journal

Neuroscience

17.2K Views.  Cedars Sinai Medical Center, UCLA. In questo protocollo, abbiamo descritto un nuovo metodo per studiare l'influenza delle eterogeneità delle cellule gliali sulla crescita degli assoni con un In vitro Di co-cultura di sistema. Rat cellule corticali gliali sono state coltivate a confluenza e cocultured con altamente purificato topo neuroni dei gangli dorsali. Diversi influenza delle cellule gliali sull'adesione neuroni e la crescita degli assoni è stato confrontato direttamente nella...

Video: Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Studio genetico della rigenerazione assonale con colture di neuroni adulti Root dorsali gangliari

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Ricerca

Neuroscienze

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt; Caenorhabditis elegans</em&gt; Sistema modello per Amylopathy Studio

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

Un metodo per fare una craniotomia sul ventrale Cranio di neonato Roditori

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Indurre Plasticità di astrociti recettori per manipolazione del neuronali di cottura Prezzi

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Preparazione delle colture gliali miste primarie da adulti mouse spinale tessuto del midollo

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilizzando canali lineari Agarose per studiare<em&gt; Drosophila</em&gt; Comportamento larvale Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

Nuovi metodi per studiare la codifica gustativa

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Visualizzazione di navigazione motore Axon e quantificazione di Axon Arborization negli embrioni del Mouse utilizzando la microscopia a fluorescenza foglio leggero

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Tramite tomografia ottica di coerenza e risposta Optokinetic come strutturale e funzionale sistema visivo letture in topi e ratti

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Un metodo per la ricostruzione 3D e l'analisi della realtà virtuale delle cellule gliali e neuronali

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...