Name: assessment of vascular regeneration in the cns using the mouse retina
Uploaded: 2014-06-23T18:45:00
Description: Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

PS besidder en Canada Research Chair i Retinal Cellebiologi og Alcon Research Institute New Investigator Award. Dette arbejde blev støttet af tilskud fra den canadiske Institutes of Health Research (221.478), den canadiske diabetesforening (OG-3-11-3329-PS), naturvidenskab og teknik Forskningsråd Canada (418.637) og instituttet Fighting Blindness Canada. Der blev også leveret af Reseau de Recherche en Santé de la Vision du Québec.

Etik erklæring: Alle dyreforsøg klæber retningslinjer dyrepleje oprettet af Foreningen for Forskning i Vision og Oftalmologi (ARVO) Redegørelse for anvendelsen af dyr i Ophthalmic og Vision Research og Canadian Råd Animal Care. 1.. Oxygen retinopati (OIR) <ol><li> Optag fødselsdato museunger som P0. </li><li> Registrere alle dyrs vægt ved indrejsen i O 2 for at sikre en passende vægt rækkevidde. Bemærk: C57BL / 6 mus ved P17, bør kropsvægten ligge me...

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Hvad er den mest effektive måde at stimulere væksten af ​​nye sunde skibe i nervøs iskæmisk væv? Er det terapeutisk gyldigt at blande sig og accelerere naturligt forekommende vaskulær genvækst? I neuro-iskæmiske patologier såsom iskæmiske retinopatier eller slagtilfælde, er vaskulær degeneration forbundet med nedsat neuronal funktion 35-38. Derfor at modvirke tidlig skade, genindføre regional mikro-cirkulation i den umiddelbare / tidlig segment af sygdommen kan vise sig gavnligt. I en okulær...

Gennem CNS udvikling, nerver, immunceller og blodkar etablere bemærkelsesværdigt koblede netværk for at sikre tilstrækkelig vævsperfusion og tillade overførsel af sensorisk information 1-5. Fordelingen af vaskulære systemer resulterer i utilstrækkelig iltningen af vævet og kompromitteret metabolisk forsyning og er i stigende grad anerkendt som en vigtig bidragyder til patogenesen af neurodegenerative sygdomme 6. Vaskulær frafald og forværringen af neurovaskulær enhed i hjernen, for eksempel, er forbundet med vaskulær demens, vaskulære læsioner i den hvide substans i hjernen 7 og Alzheimers sygdom med stenose af arterioler og små fartøjer 8. Desuden er nedsat vaskulær barriere funktion menes at bidrage multipel sklerose 9 og amyotrofisk lateral sklerose 10. Direkte relevans for den retinale model beskrevet i denne protokol, blændendesygdomme, såsom diabetisk retinopati 11 og præmatur retinopati 12, er 13, kendetegnet af en fase med tidlig vaskulær degeneration. Den efterfølgende iskæmisk stress på neurovascular nethinden udløser en anden fase af overdreven og patologisk neovaskularisering som sandsynligvis stammer som en kompenserende reaktion genetablere ilt og energiforsyning 14-16. En attraktiv strategi for at overvinde den iskæmiske stress, der er centralt for sygdomsprogression er at genoprette funktionelle vaskulære netværk specifikt i iskæmiske områder af neuro-nethinden (figur 2 og 3). Fremprovokere en kontrolleret angiogene respons kan komme på tværs som kontra-intuitivt for en tilstand, hvor anti-angiogenese behandlinger såsom anti-VEGFs betragtes som tilpassede behandlinger. Alligevel er bevis for gyldigheden af ​​denne tilgang montering. For eksempel, øge &quot;fysiologisk-lignende&quot; vaskulær genvækst i ischemic retinopatier er elegant demonstreret gennem indførelse af endotel præcursorceller 17, hæmning af Müller celle-udtrykte VEGF-induceret nedregulering af andre angiogene faktorer 18, injektion af myeloide stamceller 19, hæmning af NADPH oxidase induceret apoptose 20, stigende kosten ω-3 polyumættede fedtsyrer syre indtag 21 behandling med et carboxyterminalt fragment af tryptophan tRNA-syntetase 22 og direkte indgivelse af VEGF eller FGF-2 til beskyttelse af gliaceller 23. Desuden har vi påvist, at modulerende klassiske neuronale vejledning tidskoder såsom Semaphorins eller Netrins i iskæmiske retinopatier accelererer vaskulær regenerering af sunde fartøjer i nethinden og dermed reducerer patologisk angiogenese 24, 25. Af direkte klinisk relevans, flere af de førnævnte dyreforsøg bevis for, at fremme vaskulær regeneration i den tidlige fase af iskæmisk retinopati kan reducere synstruende præ-retinal neovaskularisering 19, 23, 24, 26, sandsynligvis gennem reduktion af iskæmisk byrde. Udarbejdelse af terapeutiske strategier, der stimulerer regenerering af funktionelle fartøjer fortsat en stor udfordring for vaskulære biologer. Her beskriver vi et eksperimentelt system, der beskæftiger musemodel af ilt-induceret retinopati (OIR) at undersøge strategier til at modulere vaskulære genvækst i nethinden. Udviklet af Smith et al. I 1994 27, denne model fungerer som en proxy for menneskerettigheder proliferative retinopati og består af udsætte P7 museunger til 75% O 2, indtil P12 og derefter re-introducere hvalpene til omgivende rum O 2-spænding (Figur 1). Dette paradigme løst efterligner et scenarie, hvor en tidlig spædbarn er ventileretmed O 2. Eksponeringen af museunger til hyperoxi provokerer degeneration af retina kapillærer og microvasculature, og giver en reproducerbar område af vaso-udslettelse (VO), typisk vurderes ved udrejse fra O 2 på P12, selvom maksimal VO område nås ved 48 timer (P9) efter eksponering til O 2 28. Hos mus, de avaskulære VO zoner spontant regenerere i løbet af ugen efter re-introduktion til rumluft og til sidst VO zoner er fuldstændig re-vaskulariseret (Figur 2). Reintroduktion til rumluft af mus udsat for OIR provokerer også præ-retinal neovaskularisering (NV) (maksimal ved P17), der typisk vurderes at bestemme effektiviteten af ​​anti-angiogene behandling paradigmer. I sin reneste form, det OIR model giver en meget reproducerbar og kvantificerbar redskab til at vurdere ilt-induceret vaskulær degeneration og bestemme omfanget af destruktive præ-retinal neovaskularisering 29-31. <p class = &quot;jove_content&quot;&gt; Forskellige eksplorative behandling paradigmer, der modulerer CNS vaskulær regenerering kan undersøges ved hjælp af OIR model, herunder brug af farmakologiske forbindelser, genterapi, gendeletion og meget mere. Tilbøjelighed en given tilgang til at påvirke vaskulær genvækst vurderes trinvis i vinduet mellem P12 (maksimal VO efter exit fra hyperoxi) og P17 (maksimal NV). Evaluering af behandlingsresultatet på patologisk NV kan hurtigt og nemt bestemmes parallelt og er blevet grundigt beskrevet af Stahl og kolleger 30, 31. Her giver vi en enkel trin-for-trin procedure at undersøge graduering af fysiologiske revaskularisering inden den neurale nethinde ved farmakologiske forbindelser, potentielle lægemidler, virale vektorer eller studere indflydelsen af ​​kandidatgener i transgene eller knockout-mus.

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			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Operating Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14192</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Vannas Eye Scissors</td>
			<td style="width:181px;">Harvard Apparatus</td>
			<td style="width:175px;">72-8483</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
			<td style="width:181px;">Dynasty</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:175px;">371110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
			<td style="width:175px;">16004-368</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
			<td rowspan="2" style="width:181px;">Leica</td>
			<td rowspan="2" style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">46945</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of vascular regeneration in the cns using the mouse retina

Gnavere nethinden er måske den mest tilgængelige pattedyr system til at undersøge neurovaskulær samspil inden for det centrale nervesystem (CNS). Det er i stigende grad anerkendt, at flere neurodegenerative sygdomme som Alzheimers, multipel sklerose, og amyotrofisk lateral sklerose indeholder elementer vaskulær kompromis. Desuden er de mest fremtrædende årsager til blindhed hos pædiatriske og arbejdsvilkår aldersgrupper (præmatur retinopati og diabetisk retinopati, henholdsvis) er kendetegnet ved vaskulær degeneration og svigt af fysiologisk vaskulær genvækst. Formålet med denne tekniske rapport er at give en detaljeret protokol til at studere CNS vaskulær regenerering i nethinden. Fremgangsmåden kan anvendes til at belyse molekylære mekanismer, der fører til svigt af vaskulær vækst efter iskæmisk skade. Desuden kan udforskes potentielle terapeutiske modaliteter for at fremskynde og genoprette sunde vaskulære plexus. Resultaterne obtained ved hjælp af den beskrevne fremgangsmåde kan give terapeutiske muligheder for iskæmiske retinopatier som den, diabetes eller præmaturitet og muligvis gavne andre vaskulære forstyrrelser i centralnervesystemet.

Den OIR model er almindeligt anvendt til at studere ilt-induceret vaskulær degeneration og iskæmi-induceret patologisk neovaskularisering i nethinden og har været medvirkende i udviklingen af nuværende tidspunkt er ansat anti-angiogenese behandlinger for øjensygdomme 27, 29, 30.. Resultaterne er opnået ved hjælp af denne model kan løseligt ekstrapoleres til iskæmiske retinopatier såsom proliferativ diabetisk retinopati og retinopati hos præmature 30. Her præ...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Det gnaver nethinden har længe været anerkendt som en tilgængelig vindue til hjernen. I denne tekniske papir giver vi en protokol, der anvender musemodel af oxygen-induceret retinopati at undersøge de mekanismer, der fører til svigt af vaskulær regenerering inden for det centrale nervesystem efter iskæmisk skade. Det beskrevne system kan også udnyttes til at undersøge strategier til at fremme genvækst af funktionelle blodkar i nethinden og centralnervesystemet.

Vurdering af Vascular Regeneration i CNS Brug af musen Retina

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

assessment-of-vascular-regeneration-in-the-cns-using-the-mouse-retina

Research

JoVE Journal

Neuroscience

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Hele Mount immunfluorescensfarvning af Neonatal Mouse Retina at undersøge Angiogenesis<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Hurtig Mikro-iontophoresis af glutamat og GABA: et nyttigt redskab til Undersøge Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

Hornhindens Micropocket assay: En model af angiogenese i mus Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dynamics i Cone Lysmodtageren Axon Terminals i Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Brug af elektroretinogrammet at vurdere Funktion i gnaver Retina og beskyttende virkninger af fjernbetjening Limb iskæmisk prækonditionering

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Behandlende pattedyr Axon regenerering: In Vivo elektroporation af voksen mus dorsalrods Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Kraften i Interstimulus Interval for vurdering af tidsmæssige forarbejdes i Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Vandrette hippocampale skiver af musehjernen

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...