This work is supported by an RO1 grant from NIH (NINDS).

1. Preparing viruses for injection
<ol>
 <li>rAAVs were purchased from the recommended commercial vendor, but they can also be produced in one's own lab (see discussion below). The virus solution is typically produced at a titer of &tilde;1x1012 genome copies per milliliter (GC/ml) and may be used at full titer to manipulate a large number of cells. Alternatively, they may be diluted to produce the desired level of sparse labeling. The appropriate dilution must be determined by the user, but a 1:10 dilution is recommended to start.</li>
 <li>Thaw viruses on ice immediately before use. Transfer the desired pre-dilution volume of virus solution into a small (&tilde;250&mu;l) PCR tube. Dilute the virus solution using an appropriate volume of sterile DPBS in a tissue culture hood. Store diluted virus solution on ice until it is needed.</li>
</ol>
NOTE: handling of all viruses should be conducted in accordance with a biosafety protocol approved by your institution
2. Syringe loading and equipment assembly
<ol>
 <li>Connect the loading needle to the glass syringe and draw up the virus solution from the PCR tube by gently pulling the plunger. Pull the plunger until a very small bubble of air can be seen in the syringe; this indicates that all of the solution is in the syringe and is not left in the loading needle. Alternatively, a small amount of inert dye can be added to the solution to make it visible. 
 NOTE: A small PCR tube should be used as the loading needle is typically not long enough to reach the bottom of most other tubes.</li>
 <li>Remove the loading needle and place the syringe on the syringe block of a syringe pump, securing it with the syringe retainer.</li>
 <li>Gently insert one end of the connective tubing into the syringe; insert the other end into the needle holder. Insert the injection needle into the needle holder. The end of the injection needle should directly contact the end of the connective tubing inside of the needle holder.</li>
 <li>Slowly depress the plunger manually to push the solution through the connective tubing. Push until a tiny drop of solution is visible at the tip of the injection needle.</li>
 <li>Carefully position the plunger next to the pusher block and secure it in place with the pusher block bracket.</li>
 <li>Set the injection parameters: injection rate should be approximately 8&mu;l/min (&tilde;130nl/s); injection volume is typically 1-2ul; select the appropriate syringe diameter for the syringe size you are using (see the syringe pump manual for guidelines).</li>
 <li>Set the hotplate to &tilde;38&deg;C and cover with a paper towel or other thin barrier to protect the pups during the recovery phase.</li>
</ol>
3. Injection procedure and pup recovery
<ol>
 <li>Prepare P0 or P1 mouse pups for injection by subjecting them to cold anesthesia following an IACUC approved protocol. Dampen a few paper towels with water and place on ice. Place the desired number of pups (4-5) on the paper towel (keep them well separated) and gently fold the paper towels over the pups so they are covered by damp paper towel. Gently place a small amount of crushed ice on top of the paper towels and incubate the pups for approximately 5 minutes. The pups can be kept on ice for up to 15 minutes and recover fine afterwards.</li>
 <li>After the pup is sufficiently anesthetized, place it on a mounting block (we use a Styrofoam block that normally holds 15ml tubes) and position it for best access to the injection site. For the cortex it is easiest to lay the animal flat on its belly, while for the cerebellum it is helpful to position the pup's head over the edge of the block to provide better access to the rear of the skull. The pup may be secured in place with a band-aid, if desired.</li>
 <li>Hold the pup's head in place with one hand while the needle is manually inserted through the skull at the desired site with the other hand. It is helpful to pull the skin tight to prevent it from moving during injection. For the cortex it is useful to make the injections relative to a well-defined anatomical landmark, such as the lambda suture of the skull, as this will aid in future injections. The cerebellum can be seen through the skull as a thin strip of tissue lying directly caudal to the colliculus. The depth to which the needle should be inserted varies slightly between individual animals and strains and can be gauged by predetermined markers on the needle, however for cortex and cerebellum we find that a depth of &tilde;0.5 - 1mm from the surface is usually suitable. The insertion depth for other sites should be determined experimentally by the end user. 
 NOTE: The needle should easily penetrate the skull; if it does not, DO NOT push too hard as this can injure the animal. Reposition the needle and gently try again.</li>
 <li>Inject the virus solution. The injection is started by pressing the START button on the pump or depressing a foot pedal, depending on the model of pressure injector used. In the absence of a foot pedal it is recommended to have a colleague assist with pressing the button to minimize unnecessary hand movements that may affect the injection.</li>
 <li>After the selected volume has been injected, keep the needle in place for a few seconds and then gently remove the needle by sliding it out smoothly. Be sure to hold the pup's head in place to minimize extraneous movements. Since the injection equipment is cleaned and sterilized between experiments with ethanol and the wound is small, the use of antiseptic is unnecessary.</li>
 <li>Place the pup on the covered hotplate with the temperature set at &tilde;38&deg;C to warm it up for recovery, typically 5-10 minutes. During this time you may continue injecting other pups.</li>
 <li>After all of the pups have recovered (they should be pink and moving) take a portion of the home bedding and gently rub the pups with it. Return the pups to the mother as a group.</li>
</ol>
NOTE: It is very important to make sure the pups are warm and exposed to home bedding prior to returning them to the mother and to return them as a group. Failure to do so may result in the mother injuring or killing the pups.
4. Equipment cleaning
<ol>
 <li>After the injection is complete and the pups have been returned to their mother, clean the injection setup by fully loading the syringe with acetone or 70% ethanol. Flush the solution through the injection setup several times, using fresh solution each time. This will remove any remaining virus solution and precipitates. The acetone or ethanol will then evaporate. 
 NOTE: Acetone is preferred for cleaning, but if your setup uses any acetone-sensitive components, such as cyanoacrylate adhesives, then use 70% ethanol for these steps.</li>
 <li>Disinfect the working area with bleach and discard waste in an appropriate biohazard container.</li>
</ol>
5. Analysis
<ol>
 <li>Sacrifice the animals at the desired age using an IACUC approved euthanasia method. Perfusion fixation is recommended to aid in later visualization of infected neurons, particularly in adult animals.</li>
 <li>Standard immunostaining procedures may be used to amplify the fluorescent signal. Briefly, 100 &mu;m floating vibratome sections are permeabilized with PBS+1% Triton X-100 and blocked in PBS+1% Triton X-100+10% normal goat serum. Sections are incubated in blocking solution with primary antibodies overnight at 4&deg;C, followed by additional washing and blocking. Sections are then incubated in blocking solution with secondary antibodies for 2 hours at room temperature, washed thoroughly, mounted on slides and coverslipped with anti-fade mounting media. Cryosections can be also used to visualize labeled neurons, however the endogenous fluorescence might be lost.</li>
</ol>
6. Representative Results:
A successful injection procedure will produce robust infection of neurons in the region of the injection site with no observable tissue damage or other ill effects on the animal. As illustrated in Figure 1, the spread of the infection can be observed in Cre-reporter mice (ROSA26R)12 injected with rAAV8-Cre in various anatomical sites. Injections into specific regions, such as cortex and superior colliculus, typically generate local infections with minimal spread to adjacent regions (Fig. 1A-B). Use of high titer virus solution makes infection of adjacent areas, such as the hippocampus, more likely (Fig. 1A). Injection with low titer virus solution results in sparse infection, which typically remains near the injection site, as shown in the cerebellar injection of rAAV8-Cre at the midline of ROSA26R reporter mice (Figure 1C-D). We have observed recombination in ROSA26R reporter mice within 2 days after injection of rAAV8-Cre, suggesting that infection, recombination, and expression of reporter genes can all occur relatively rapidly. Additionally, we have successfully used this technique to study the effects of gene deletion at a non-ROSA locus using a floxed conditional allele, the results of which will be published elsewhere.
The number of cells infected should correlate with the titer of the injected viral solution. For example, injecting a mix of high titer solutions of two rAAV8 viruses expressing different fluorescent proteins into the cerebellum infects many Purkinje cells that are differentially labeled (Fig. 2A). Conversely, injecting a low titer virus solution can result in sparse infection to aid in visualizing single cells (Fig. 2B). Fluorescently labeled neurons can be observed directly in fresh cut, live tissue (Fig. 2B) or can be subjected to immunostaining to enhance the endogenous fluorescent signals for later image acquisition by wide-field or confocal fluorescent microscopy (Fig. 2A, Fig. 3A-C). Lastly, rAAV8 is capable of infecting a variety of neuron types in the cortex with strong labeling of fine processes (Fig. 3A-C).
<img alt="Figure 1" src="/files/ftp_upload/2823/2823fig1.jpg" /> 
Figure 1: LacZ staining in the brain of ROSA26R reporter mice that were injected with rAAV8-Cre at P0 demonstrates Cre activity and the location of infection.
<ol type="A">
 <li>Wide-field sagittal view of a P14 brain showing high titer (1x1012 GC/ml) injections into cortex and superior colliculus. The use of high titer virus makes it more likely that adjacent areas will also be infected.</li>
 <li>Sagittal section of P14 cortex injected with lower titer (1x1011 GC/ml) virus demonstrates a local infection with less spreading.</li>
 <li>A wholemount view of a P28 cerebellum injected at the midline with low titer (1x109 GC/ml) virus solution shows that the infection is sparse and typically remains near the injection site.</li>
 <li>Sagittal view of the midline of the cerebellum in C). Note that generally only Purkinje cells are infected by rAAV8.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/2823/2823fig2.jpg" /> 
Figure 2: Cerebellar labeling by injection with high and low titers of rAAV8 expressing fluorescent proteins.
<ol type="A">
 <li>A sagittal section of P21cerebellum infected with a mix of two viruses expressing EGFP (green) and DsRed (red). A high titer mix of viruses was used to infect a large number of Purkinje cells.</li>
 <li>A purkinje cell in live P14 cerebellum from very low titer viral infection imaged immediately after dissection.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/2823/2823fig3.jpg" /> 
Figure 3: Cortical labeling by rAAV8-DsRed. 
Examples of the variety of cortical neuron types in the coronal sections of a P21 cortex that was infected by rAAV8-DsRed at P0.

<ol>
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No conflicts of interest declared.

The neonatal viral injection method presented here provides a simple and rapid way to generate in vivo mosaics for the study of postnatal brain development. The method takes advantage of floxed alleles that are currently available as well as those that are being made through the High Throughput Gene Targeting project6. Compared to the use of transgenic expression of Cre, this method provides a rapid way to test gene function in various cell types, as mice carrying the floxed alleles can be used dire...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>rAAV8-Cre, -GFP, -DsRed,</td>
			<td>Vector Biolabs</td>
			<td>#7060, #7061, custom order</td>
			<td>http://www.vectorbiolabs.com</td>
		</tr>
		<tr>
			<td>Harvard Pump 11 Plus</td>
			<td>Harvard Apparatus</td>
			<td>#702208</td>
			<td>(No foot pedal port)</td>
		</tr>
		<tr>
			<td>Retinal Pigment Epithelium Injection Kit</td>
			<td>World Precision Instruments</td>
			<td>RPE-KIT</td>
			<td>Contains connective tubing, injection needles (36G), and needle holder</td>
		</tr>
		<tr>
			<td>NanoFil Syringe, 100&mu;l</td>
			<td>World Precision Instruments</td>
			<td>NANOFIL-100</td>
			<td>Includes reusable loading needle</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Invitrogen</td>
			<td>14040-117</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GFP antibody</td>
			<td>Aves</td>
			<td>GFP-1020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DsRed antibody</td>
			<td>Clontech</td>
			<td>632496</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating Block</td>
			<td>VWR</td>
			<td>97042-610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROSA26R mouse</td>
			<td>Jackson Laboratory</td>
			<td>003309</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

mosaic analysis of gene function in postnatal mouse brain development by using virus-based cre recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

Watch this Scientific Journal Video about Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination at JoVE.com

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

mosaic-analysis-gene-function-postnatal-mouse-brain-development-using

Research

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Neuroscience

11.9K vues.  Keck School of Medicine, University of Southern California. Une In vivo Méthode pour tester la fonction des gènes dans le cerveau postnatal est décrite. AAV recombinants exprimant Cre et / ou une protéine fluorescente sont injectées dans le cerveau de souris néonatales. Inactivation d'un gène mosaïque et clairsemés étiquetage neuronale sont atteints, permettant une analyse rapide de la fonction des gènes dans les processus essentiels au développement des circuits neuronaux.

Vidéo: Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...