This work was supported by the NIH Neuroscience Blueprint Core grant (P30 NS057105, BJS) to Washington University, Program Project Grant NS032636 (BJS) and by the Hope Center for Neurological Disorders.

1. Packaging of Lentiviral Vectors
Lentiviral vectors are produced by cotransfection of a lentiviral transfer vector and other plasmids required for packaging into 293T cells by calcium phosphate transfection method. We use 10 100-mm tissue culture dishes in this protocol. It can be scaled up or down depending on applications. The 293T cell line is maintained in Dulbecco's modified Eagles medium (DMEM) with high glucose (4500 mg/L), supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 &mu;g/ml streptomycin in 37 &deg;C incubator with 5% CO2.
<ol>
	<li>Seed 293T cells at 30-40% confluence to 10 100-mm tissue culture dishes (3 x 106 cells/dish) in culture medium. Return the cells to incubator.</li>
	<li>After 20-24 h culture, check the cell density. The cells should be about 80% confluence at the time of transfection.</li>
	<li>Prepare a 50-ml tube. Add 4.4 ml TE79/10 (1 mM TrisHCl, 0.1 mM EDTA, pH 7.9) minus the total volume of the following plasmid DNA. Add 100 &mu;g lentiviral transfer plasmid (Figure 1), 58 &mu;g pMDLg/pRRE, 31 &mu;g pCMV-G, 25 &mu;g pRSV-Rev, 600 &mu;l 2M CaCl2. Gently Mix.</li>
	<li>Prepare another 50-ml tube. Add 5 ml 2x HBS (0.05 M HEPES, 0.28 M NaCl, 1.5 mM Na2HPO4, pH 7.12).</li>
	<li>Take the DNA-CaCl2 mixture by 10-ml pipette and add to the tube containing 2 x HBS, dropwise while vortexing the tube.</li>
	<li>Keep the precipitation reaction at room temperature (RT) for 30 min.</li>
	<li>Remove the culture dishes from incubator. Mix the precipitation reaction well by vortexing. Add 1 ml of suspension to each 100-mm dish containing cells. The suspension must be added slowly, dropwise while gently swirling the medium in the dish. Return these dishes to the incubator and leave for 5 h.</li>
	<li>Remove the medium from the culture. Add 6 ml fresh culture medium containing 6 mM sodium butyrate to each dish. Return the cultures to incubator. After overnight culture, if there is a fluorescent reporter in the construct, check reporter gene expression under fluorescent microscope. Usually, over 80% of the cells express the reporter gene if it is driven by a ubiquitous promoter (e.g. CMV promoter).</li>
	<li>Two days (40-44 h) after transfection, collect supernatant from 10 dishes into 2 50-ml tubes (about 30 ml each tube). Freeze the supernatant in -80 &deg;C freezer or go to next step.</li>
</ol>
2. Concentration and Purification of the Vectors
<ol>
	<li>Centrifuge the freshly collected or thawed supernatant at 900 g (about 2000 rpm) for 10 min to remove any cell debris in the supernatant.</li>
	<li>Attach a 60-ml syringe to a 0.2-&mu;m SFCA syringe filter. Transfer supernatant from 50-ml tube to the syringe. Filter the supernatant into a polyallomer centrifuge tube.</li>
	<li>Take 5 ml 20% sucrose (prepared in PBS) up in a 5-ml pipette. Insert the pipette to the bottom of the centrifuge tube containing supernatant. Slowly add the sucrose solution under the vector supernatant. Repeat these steps for supernatant from another tube.</li>
	<li>Centrifuge the supernatant at 11000 rpm and 4 &deg;C for 4 h with Beckman SW28 swing rotor.</li>
	<li>Remove the supernatant. Add 150 &mu;l 4% Lactose (prepared in PBS) to each centrifuge tube. Resuspend the pellets.</li>
	<li>Transfer the concentrated vector from all centrifuge tubes to a 1.5-ml tube. Leave the tube on ice for 15 min.</li>
	<li>Mix the vector suspension by pipetting. Spin with microcentrifuge at full speed (about 16000 g) for 1 min.</li>
	<li>Transfer supernatant to a new 1.5-ml tube. Divide the final sample into 20 &mu;l aliquots and stored them in -80 &deg;C freezer.</li>
</ol>
3. Titration of the Vectors
<ol>
	<li>Seed 5 x 104/well of HT1080 cells in 12-well plate in 1 ml DMEM medium supplemented with 10% FBS.</li>
	<li>After overnight culture, count cells from one well and score the cell number.</li>
	<li>Make 5-fold serial dilution (1:5, 1: 25; 1:125; and 1:625) of the concentrated vector with culture medium. Add I &mu;l of each diluted vector to separate wells. The samples may be duplicated to increase accuracy.</li>
	<li>Add 1 &mu;l 4 mg/ml Polybrene (Hexadimethrine Bromide) in each well containing vector and into a well without vector. Mix by gently shaking the plate. Return to incubator for 48 h.</li>
	<li>Remove medium from the cell culture wells. Wash each well with PBS. Add 250 &mu;l 1x trypsine-EDTA solution to the cells. When the cells are detached (3-5 min), add 1 ml culture medium. Resuspend the cells by pipetting. Transfer cell suspension to 1.5 ml centrifuge tubes.</li>
	<li>Centrifuge at 900 g for 6 min. For vectors with a fluorescent reporter gene (e.g. GFP), go to step 3.7 for FACS analysis. For vectors without a reporter, go to step 3.8 for real time qPCR.</li>
	<li>For vectors containing a fluorescent reporter gene, remove the supernatant and resuspend the pellet with 300 &mu;l of 3.7% formaldehyde in PBS. Determine the percentage of the reporter positive cells by FACS analysis. The titer will be represented as transduction units per milliliter concentrated vector (TU/ml).</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/4031/4031eq1.jpg" />
For example, if 1 x 105 cells was transduced with 1/25 &mu;l (0.04 &mu;l) vector and 30% cells are reporter positive, the titer will be:
<img alt="Equation 2" src="/files/ftp_upload/4031/4031eq2.jpg" />
Only use the dilutions fall in a linear relationship between the percentage of positive cells and the amount of vector added to calculate titer. The final titer should be an average of the titers obtained from transductions of at least 2 different amounts of the vector.
<ol start="8">
	<li>For vectors without a fluorescent reporter gene, extract genomic DNA from HT1080 cells using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. Amplify vector sequence in genomic DNA using ABI Prism 7000 Sequence Detection System (Applied Biosystems) with primers (in HIV-1 PBS/psi region 17) 5'-CCGTTGTCAGGCAACGTG-3' and 5'-AGCTGACAGGTGGTGGCAAT-3', and TaqMan probe 5'-FAM-AGCTCTCTCGACGCAGGACTCGGC-TAMRA-3'. Albumin gene that is a single copy gene in the genome (2 copies/cell) was also amplified with primers 5'-TGAAACATACGTTCCCAAAGAGTTT-3' and 5'-CTCTCCTTCTCAGAAAGTGTGCATAT-3', and probe 5'-FAM-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3' as an internal control. Determine the copy numbers of vector and albumin by PCR in 96-well plate according to the manufacture's instruction with the following program: 50 &deg;C for 2 min, 95 &deg;C for 10 min, and 35 cycles of 95 &deg;C for 15 sec and 60 &deg;C for 2 min. Ten-fold serial dilutions of plasmids of known concentration (represented as copy number) containing the template sequences should also be amplified to create a standard curve for quantification of unknown samples. The titer will be represented as integration units per milliliter concentrated vector (IU/ml).</li>
</ol>
<img alt="Equation 3" src="/files/ftp_upload/4031/4031eq3.jpg" />
4. Transduction of Neocortical Cultures
Neocortical cultures containing both neurons and glia are prepared from mouse cortices using a two-step plating procedure as previously described 18. Neocortices obtained from fetal mice at 14-16 days gestation are plated onto a previously established glial monolayer in MEM supplemented with 10% FBS, 20 mM glucose and 2 mM glutamine in 24-well tissue culture plate.
<ol>
	<li>After 5 days in vitro, add 10 &mu;M cytosine arabinoside (Ara-C) in neocortical culture to inhibit non-neuronal cell division. Continue culture the cells for 2 days.</li>
	<li>Warm culture medium in 37 &deg;C water bath for 5-10 min. Replace Ara-C containing medium with fresh culture medium (500 &mu;l/well).</li>
	<li>Add vector with desired MOI (multiplicity of infection; the ratio of the number of vector particles to the number of target cells) to the culture. Continue culture for 24 h. We use MOI of 1-10 (usually 5) in primary cortical cultures.</li>
	<li>Replace the culture medium with fresh medium. Continue culture. If there is a reporter gene in the vector construct, check cells under fluorescent microscope 2 days after transduction. Reporter gene expression will be visible in neurons 2-7 after transduction, depending on the vector design and the dose used.</li>
</ol>
5. Representative Results
The titers of lentiviral vectors produced with this protocol range 108-1010 IU/ml, which are suitable for transduction of a variety of cell types from CNS both in vitro and in vivo. Table 1 and figure 2 show a representative result using the vectors produced by this protocol. We transduced murine neocortical cultures with lentiviral vectors expressing green fluorescent protein (GFP) controlled by synapsin (SYN) promoter or glial fibrillary acidic protein (GFAP) promoter. Seven days after transduction, we performed immunostaining to label neurons and astrocytes with anti-NeuN and anti-GFAP antibodies, respectively. As shown in table 1 and Fig. 2A, after transduction with the vector carrying the synapsin promoter, over 90% of neurons (NeuN+ cells) express GFP and no astrocytes (GFAP+ cells) express this reporter gene. When GFAP promoter is used in the vector construct (Fig. 2B), about 80% of astrocytes (GFAP+ cells) express GFP; all GFP+ cells are astrocytes as confirmed by colocalization with GFAP and the absence of GFP expression in NeuN-labeled cells. These results demonstrate that lentiviral vectors are very efficient to deliver transgenes to cells from CNS and cell-specific gene expression can be achieved when appropriate promoters are used.
<img alt="Figure 1" src="/files/ftp_upload/4031/4031fig1.jpg" /> 
Figure 1. Schematic representation of HIV-based lentiviral vectors and the packaging plasmids. The HIV-1 provirus is shown at the top. The elements for vector production are separated into four different plasmids. The lentiviral transfer plasmid contains a hybrid 5' LTR in which the U3 region is replaced with the cytomegalovirus (CMV) promoter, the packaging signal (&psi;), the RRE sequence, the central polypurine tract (cPPT), a gene of interest (e.g. a fluorescent reporter) along with a promoter of choice, and the 3' LTR in which the cis regulatory sequences are completely removed from the U3 region. pMDLg/pRRE contains the gag and pol genes and RRE sequence from HIV-1 under the control of the CMV promoter. pRSV-Rev contains the coding sequence of Rev driven by the RSV promoter. pCMV-G contains the VSV-G protein gene under the control of the CMV promoter. PA indicates the polyadenylation signal from human &beta;-globin gene.
<img alt="Figure 2" src="/files/ftp_upload/4031/4031fig2.jpg" /> 
Figure 2. Expression of reporter genes in mouse neocortical mixed culture transduced with lentiviral vectors carrying cell type-specific promoters. The cultures were transduced with LV-SYN-GFP (A) or LV-GFAP-GFP vectors (B) at a MOI of 5. Seven days after transduction, the cells were immunostained with anti-NeuN or anti-GFAP antibody. Upper panels show GFP fluorescence, middle panels show immunostaining and lower panels are merged images (GFP: green; NeuN or GFAP: red).
<table border="1">
	<tbody>
		<tr>
			<td>Vector</td>
			<td>GFP+ cells in neurons</td>
			<td>GFP+ in astrocytes</td>
		</tr>
		<tr>
			<td>LV-SYN-GFP</td>
			<td>92.2&plusmn;7.3</td>
			<td>0</td>
		</tr>
		<tr>
			<td>LV-GFAP-GFP</td>
			<td>0</td>
			<td>78.3&plusmn;11.5</td>
		</tr>
	</tbody>
</table>
Table 1. Comparison of GFP expression in murine neocortical cultures transduced with lentiviral vectors carrying different promotersa. 
aMurine neocortical cultures (5 x 105/well in 24-well plate) were transduced with LV-SYN-GFP or LV-GFAP-GFP at an MOI of 5. Seven days after transduction, cultures were fixed and immunostained for NeuN or GFAP. The number of GFP and NeuN/GFAP expressing cells were counted in images from 10 fields per experimental condition. The values represent the percentage of neurons (NeuN+ cells) or astrocytes (GFAP+ cells) that also expressed the GFP reporter gene. The values shown are means &plusmn; SD from three independent experiments.

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No conflicts of interest declared.

In this protocol, we have shown the production of lentiviral vectors and application of these vectors in neocortical cultures. We demonstrated efficient and cell type-specific transduction with the vectors produced by these methods. When the synapsin promoter is used, GFP expression is strictly neuron specific. When the GFAP promoter is used, GFP expression is exclusively in astrocytes. If no cell type-specific expression is required, a ubiquitous promoter may be used. We found both ubiqutin and phosphoglycerate k...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Sigma-Aldrich</td>
 <td>D5796</td>
 </tr>
 <tr>
 <td>MEM</td>
 <td>Invitrogen</td>
 <td>11090-081</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Hyclone</td>
 <td>SV3001403</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Mediatech</td>
 <td>21-040-CM</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>T3924</td>
 </tr>
 <tr>
 <td>Sodium butyrate</td>
 <td>Sigma-Aldrich</td>
 <td>B5887</td>
 </tr>
 <tr>
 <td>Hexadimethrine bromide (Polybrene)</td>
 <td>Sigma-Aldrich</td>
 <td>H9268</td>
 </tr>
 <tr>
 <td>293T cells</td>
 <td>ATCC</td>
 <td>CRL-11268</td>
 </tr>
 <tr>
 <td>HT1080 cells</td>
 <td>ATCC</td>
 <td>CCL-121</td>
 </tr>
 <tr>
 <td>Falcon 100 x 20 mm tissue culture dish</td>
 <td>BD Biosciences</td>
 <td>353003</td>
 </tr>
 <tr>
 <td>1 x 3 &frac12; in polyallomoer centrifuge tube</td>
 <td>Beckman-Coulter </td>
 <td>326823 </td>
 </tr>
 <tr>
 <td>0.2-micron syringe filter</td>
 <td>Corning</td>
 <td>431219</td>
 </tr>
 <tr>
 <td>QIAamp DNA Mini Kit</td>
 <td>Qiagen</td>
 <td>51304</td>
 </tr>
 </tbody>
</table>

production of lentiviral vectors for transducing cells from the central nervous system

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

production-lentiviral-vectors-for-transducing-cells-from-central

Research

JoVE Journal

Neuroscience

35.9K 次观看.  Washington University School of Medicine. 在这个协议中，我们描述了慢病毒载体的生产，提纯和滴定。我们提供了一个在原代培养的神经元和神经胶质细胞的慢病毒载体介导的基因传递的例子。我们的方法也适用于其他类型的细胞在体外和在体内。

视频: Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...