Name: homochronic transplantation of interneuron precursors into early postnatal mouse brains
Uploaded: 2018-06-08T14:30:00
Description: Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

This research was supported by the National Institutes of Health (K99MH104595) and the NICHD intramural research program to T.J.P. We thank Gord Fishell, in whose lab this approach was originally established.

All experimental procedures were conducted in accordance with the National Institutes of Health guidelines and were approved by the NICHD Animal Care and Use Committee (ACUC). The protocol described below utilizes Nkx2.1-CreC/+;Ai9+/- pups to harvest MGE-derived interneuron precursors, but can be performed on any desired fluorescent reporter mouse line. Both male and female early postnatal mice (P0-P2) were used indiscriminately for donor and host tissue.
1. Soluti...

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One critical aspect of this protocol is maximizing the survivability of the cells. Ensuring that the tissue and cells are always in ice cold carboxygenated sACSF is necessary to promote cell survival. This requires an efficient dissection and dissociation strategy to minimize the length of time that the cells spend in various solution and outside of the brain environment. Depending on the number of brain regions being dissected and transplanted, it can be beneficial to have a partner aid in the dissection and/or transpla...

Proper cortical function requires a balance between excitatory projection neurons and inhibitory GABAergic interneurons, an extremely heterogeneous population with distinct morphologies, electrophysiological properties, connectivity and neurochemical markers. Abnormal development and function of interneurons (and specific interneuron subgroups) has been linked to the pathobiology of psychiatric disorders such as schizophrenia, autism and epilepsy1,2,3. Furthermore, many genes implicated in these brain disorders are strongly enriched in young interneurons4. Thus, a greater understanding of the mechanisms that regulate interneuron fate determination and maturation is needed to understand normal development and potential etiologies of numerous brain diseases.
Forebrain interneurons are born primarily from two transient embryonic structures, the medial and caudal ganglionic eminences (MGE and CGE, respectively). These postmitotic cells (interneuron precursors) then undergo a protracted tangential migration phase to disperse throughout the telencephalon where they integrate into a wide variety of circuits. MGE-derived interneurons consist of three largely non-overlapping, neurochemically defined subgroups: fast spiking parvalbumin (PV+) interneurons, non-fast spiking somatostatin (SST+) interneurons, and late spiking neuronal nitric oxide synthase (nNOS+) interneurons that constitute hippocampal neurogliaform and ivy cells. Numerous labs have identified several mechanisms within the MGE that regulate initial fate decisions into PV+ or SST+ interneurons, including spatial gradients of morphogens, birthdate of interneuron precursors, and the mode of neurogenic division5,6,7,8,9,10. It has been proposed that interneurons initially differentiate into &#39;cardinal classes&#39; and then progressively mature into &#39;definitive classes&#39; as they interact with their environment11. Recent evidence indicates that some mature interneuron subtypes may be genetically hardwired as these cells become postmitotic in the ganglionic eminences, indicating that early defined intrinsic genetic programs may play a larger role than previously appreciated12,13. However, the key question of how the intrinsic genetic programs interact with environmental cues to drive differentiation into distinct interneuron subtypes remains largely unexplored.
Numerous studies have transplanted embryonic MGE cells directly into a variety of brain regions, with the consensus results that grafted cells mature and release GABA to generally inhibit the local endogenous circuitry14,15,16,17,18,19. These promising observations have generated significant interest in using human induced pluripotent stem cell (hIPSC)-derived interneurons to treat a variety of brain diseases. However, very few of these studies assess if these grafted cells mature into the expected types of mature interneurons, a critical component when one thinks about translational approaches.
To address how environment influences interneuron differentiation and maturation, a strategy was devised to transplant immature interneuron precursors into new brain environments in order to examine whether grafted interneurons adopt features of the host environment or retain features from the donor environment20. MGE transplants are not suitable to address this question because the MGE contains a mixed population of interneuron and GABAergic projection cells that disperse throughout numerous brain regions21. Without knowing where these MGE cells would have migrated, one can not fully assess how these transplantations are affected by the brain environment. By harvesting interneuron precursors at early postnatal timepoints, this problem is circumvented by obtaining immature cells that have completed their migration and reached their target brain region but have minimal interaction with the environment. By focusing on specific features of interneurons that are differentially expressed between distinct brain regions, one can then determine how the host environment changes interneuron properties. The general approach outlined in this protocol should be applicable to any investigator that wants to examine how young neurons behave when challenged in a new environment.

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			<td height="21" style="height:21px;width:247px;">Sodium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7653</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium bicarbonate</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S6297</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Potassium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">P9541</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S0751</td>
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			<td height="21" style="height:21px;width:247px;">Calcium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Magnesium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glucose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">G7528</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sucrose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7903</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Brain Matrices</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">SA-2165</td>
			<td>Only needed if harvesting striatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fine point Dumont Forceps</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-4978</td>
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			<td height="21" style="height:21px;width:247px;">Microdissecting scissors</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-5940</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Razor blades</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12-640</td>
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			<td height="21" style="height:21px;width:247px;">Pasteur pipettes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">1367820C</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Nanoject III</td>
			<td style="width:203px;">Drummond</td>
			<td style="width:136px;">3-000-207</td>
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			<td height="21" style="height:21px;width:247px;">Manual Manipulator w/ stand</td>
			<td style="width:203px;">World Precision Instruments&#160;</td>
			<td style="width:136px;">M3301R/M10</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">5 ml round bottom plastic tubes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">149591A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">60 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12556001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">100 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12565100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pronase</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">10165921001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">16140063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DNase I</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">4716728001</td>
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			<td height="21" style="height:21px;width:247px;">Celltrics 50um filters</td>
			<td style="width:203px;">Sysmex</td>
			<td style="width:136px;">04-0042327</td>
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			<td height="21" style="height:21px;width:247px;">Trypan blue</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">15-250-061</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Hemocytometer</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">02-671-6</td>
			<td></td>
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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.

This protocol demonstrates how to harvest specific brain regions from early postnatal brains (Figure 1-2), collect single cell dissociations of interneuron precursors, and transplant these cells into various brain regions in naive WT postnatal pups (Figure 3). For posthoc analysis, brains that received interneuron precursor grafts were harvested between P30-35 to characterize cell morphology, neurochemical marker...

Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

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 ...

The overall goal of this procedure is to harvest interneuron precursors from distinct brain regions in postnatal mouse pups, and transplant these cells into age-matched recipients to assess how environmental changes influence interneuron fate and maturation. This method can help answer key questions in the field of developmental neuroscience about how intrinsic genetic programs and environmental signals interact to sculpt the fate of neural cells. The main advantage of this technique is that alterations in the fate and maturation of interneuron precursors can be analyzed after their adoptive transfer into a new brain environment.Visual demonstration of the cell harvesting and grafting steps is critical because small errors in the technique can lead to inefficient and unsuccessful transplant experiments. To begin, place one razor blade through the anterior frontal lobe of a postnatal day one pup brain, and additional razor blades into the slots just posterior to the first razor blade to generate two 0.5 millimeter slices. Transfer the striatal slices into a Petri dish with bubbled sucrose artificial cerebrospinal fluid or sACSF and place the remaining brain tissue into a Petri dish with sACSF on ice.Place the slices under a dissecting microscope and use forceps to pinch the striatum from both hemispheres, placing the striatal chunks in a 50-milliliter tube of sACSF on ice as they are harvested. If the brain was harvested from a fluorescent reporter mouse, use fluorescence microscopy to distinguish the striatal td-tomato signal from the stronger fluorescent signal of the globus pallidus. To remove the hippocampus, use forceps to hemisect the brain along the midline, and place one hemisphere under a dissecting microscope medial surface up.Use forceps to remove the ventral brain tissue and identify the sausage-shaped hippocampus spanning the anteroposterior access along the dorsal and ventricular side of the cortex. Insert the tips of the forceps in front of the anterior hippocampus, and advance the forceps posteriorly while pinching along the hippocampal cortical border to gently separate the hippocampus from the cortex. Place the isolated tissue in a 50-milliliter tube of sACSF on ice and repeat the hippocampus harvest for the contralateral hemisphere.Then place the hemisected cortex medial side down and use the forceps to remove the most dorsal, ventral, anterior, and posterior portions of the cortex. Transfer the remaining square of medial cortex into a 50-milliliter tube of sACSF on ice. After removing the brain, pin the brain ventral side down through the cerebellum and anterior cortex or olfactory bulbs.Using curved forceps, gently separate the posterior cortex from the underlying hippocampus and other tissue in each hemisphere. And peel the dorsal cortex forward to expose the underlying structures. Use forceps to pinch the hippocampus in one hemisphere at the midline, and remove the hippocampus by peeling it laterally away from the brain.Place the hippocampus into a 50-milliliter tube of sACSF on ice and harvest the contralateral hippocampus in the same manner. Next gently scrape around the edges of the striatum to loosen it from the surrounding tissue. Gently pinch below the striatum to harvest the striatal tissue.After harvesting the cortex from both hemispheres, as previously demonstrated, transfer the striatum under a fluorescent dissecting scope. Use forceps to remove any bright red td-tomato positive globus pallidus tissue from each striatum. When all of the tissues have been harvested, transfer tissue to a five-millimeter round bottom tube and replace the sACSF in each collection tube with two milliliters of freshly-prepared pronase-sACSF solution for a 20-minute incubation at room temperature with occasional flicking.At the end of the incubation, carefully replace the pronase-sACSF with one to two milliliters of reconstitution solution, and mechanically dissociate the tissues with fire-polished Pasteur pipes. When the tissue is cloudy and no tissue chunks are visible, strain the cell solution through a 50-micron filter into new five-milliliter round bottom tubes to remove any cell clumps before fluorescence-activated cell sorting. After receiving the cells from flow cytometry, transfer the cell suspensions into 1.5-milliliter conical tubes for centrifugation.After centrifugation, aspirate all but the last 20 microliters of supernatant from each tube, and reconstitute the pellets for counting. If necessary, dilute the cell solution with sACSF to a one to three times 10 to the 5th cells per microliter concentration. Load a fine-tipped micropipette with mineral oil.Attach the micropipette to a nanoliter injector, and secure the nanoliter injection apparatus to a manipulator attached to a magnetic base. While the first pup is being anesthetized on ice, pipette the cell solution several times to ensure a homogenous cell distribution. Eject the mineral oil and completely fill the pipette with the single cell suspension.After confirming a lack of response to toe pinch, place the pup on a Petri dish cover with the head resting on adhesive putty, so that the top of the head is relatively flat. Cover the head with a piece of lab tape with a diamond hole, pulling the tape taught until the skin is stretched so that the head is firmly secured and lambda is visible through the hole. Move the secured pup under the nanoliter injector and lower the micropipette so that the tip is directly above lambda.Observe the X, Y coordinates on the manipulator and adjust the manipulator knobs until the micropipette is positioned at the appropriate coordinates along the medial, lateral, and anteroposterior axes of the head. Lower the micropipette until the tip creates a small concavity on the skin, and turn the z-axis manipulator knob firmly but gently to drive the micropipette through the skin and skull into the brain. Retract the micropipette slightly until the tip is surrounded by a cone of skin shaped like a tent.And view the coordinates along the z-axis. Then lower the micropipette to the appropriate experimental depth and inject the cells. Wait 15 seconds after the cells have been delivered before retracting the micropipette and repeat injection at a second location if desired.Place the pup on a heating pad and monitor until full recovery is achieved before transferring back to home cage. Grafted cells migrate throughout the targeted brain regions with variable cell survival rates ranging from dozens to several thousand. The grafted cells localize within the appropriate regions with many cells displaying interneuron morphologies and well-characterized interneuron neurochemical markers.The donor cells survive and mature even when they are grafted into new environments with heterotopic transplantations. Electrophysiological analysis of the grafted cells reveals adult-like physiological properties and distinct firing patterns, representative of well-defined interneuron subtypes, suggesting that grafted interneurons are able to properly mature in the host environment. Transplanted cells also display spontaneous excitatory postsynaptic currents.Recording from pyramidal cells adjacent to transplanted interneurons expressing channelrhodopsin-2 reveals postsynaptic GABAergic currents that are evoked by blue light in these endogenous pyramidal cells. While attempting this procedure, it's important to keep the cells on ice and triturate the cells gently with a Pasteur pipette to minimize cell death. Following this procedure, other methods such as single cell sequencing can be performed on the grafted cells to more fully define how environmental changes affect a cell's transcriptome.After watching this video, you should be able to isolate interneuron precursors from different brain regions of neonate pups and transplant these cells into various brain regions of age-matched postnatal pups.

homochronic-transplantation-interneuron-precursors-into-early

Research

JoVE Journal

Neuroscience

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.

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 ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

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 ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...