This work was supported by grants from the NIH (DC007681, A.B.), CT Stem Cell grant (A.B.), Pardee foundation (A.B.), Predoctoral Ruth L. Kirschstein National Research Service Awards (NRSA) (S.Z.Y.), and an NSF Graduate Research Fellowship (B.L.). We thank the Bordey lab members for helpful comments on the manuscript. The present material is based upon work partly supported by the State of Connecticut under the Connecticut Stem Cell Research Grants Program. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Connecticut, the Department of Public Health of the State of Connecticut or CT Innovations, Incorporated.

1. Preparation of Solutions, Dissection, and Vibratome
<ol>
	<li>Solutions must be prepared at the correct osmolarity and pH (Table 1). Compared to artificial cerebrospinal fluid (ACSF), dissection solution is prepared with lower concentrations of sodium and calcium, and higher concentrations of magnesium. This is to minimize excitotoxicity effects during slicing.</li>
	<li>Both dissection and ACSF solutions must be saturated with 95% O2/5% CO2 by bubbling for at least 10 min to reach the desired pH of 7.3.</li>
	<li>Prepare an ice bath in a tray. Place a dissection plate in the ice bath and fill with dissection solution. Bubble the dissection plate.</li>
	<li>Prepare the vibratome by creating an ice bath. Place the sectioning dish in the ice bath, fill the dish with dissection solution, and bubble.</li>
	<li>Fill the slice holding chamber with ACSF and bubble to ensure that solution is adequately aerated regardless of where slices are placed.</li>
</ol>
2. Removal of Brain Tissue
Acute brain slices tend to be healthier with younger mice. The postnatal SVZ's cellular architecture in rodents is mature around postnatal day (P)201. Therefore, we try to limit our experiments to mouse ages of P20-P30 but we have performed successful experiments in mice as old as 3 months. Throughout handling of the tissue, one must be mindful to minimize mechanical movements and pressure to the brain, maintain cooled conditions, and expose the SVZ to solution as quickly as possible following sacrifice.
<ol>
	<li>After injection of pentobarbital, the mouse is rapidly decapitated. The fur is removed and incisions are made through the base of the skull. The head is then placed in a tray filled with dissection solution.</li>
	<li>While stabilizing the head with forceps, continuous cuts are made around the skull and sometimes up the midline with microscissors. Be careful to minimize contact with the tissue. Since the olfactory bulb is the final destination of newborn neurons from the SVZ, one must be mindful of preserving that region, especially during bone removal around this area. Microscissor cuts made around the skull should be on the dorsal side, to allow for easy removal of the skull.</li>
	<li>Removal of the skull overlying the brain is now separated from the bones underneath the brain. If cuts in the previous step are made correctly, this overlying bone can be removed easily with tweezers or forceps.</li>
	<li>While still holding the head with forceps, one can use a surgical blade to cleanly remove several pieces of brain tissue prior to slicing, such as the following. A coronal cut can be made at the level of lambda. Sagittal cuts can be made on both sides of the brain lateral to the SVZ. A conservative estimate would be ~2.5 mm from the midline. If desired, the brain can also be hemisected here. These cuts will both facilitate mounting of the tissue and allow the SVZ to be reached more quickly when brain slicing. It is important to remember to exert the minimal amount of mechanical forces on the brain (e.g. not pushing or pulling).</li>
</ol>
3. Acute Brain Slice Preparation
<ol>
	<li>To prepare for subsequent steps, ensure that the super glue used to mount the tissue is free of clogs and ready to apply to the vibratome plate. It is important to go as fast as possible without damaging the brain.</li>
	<li>The brain tissue can then be scooped from underneath, which separates the tissue from underlying bone while simultaneously separating nerves.</li>
	<li>The tissue is mounted and glued on a plate and placed on the vibratome. Due to the rostral-caudal anatomy of the SVZ, we have focused on making sagittal slices as it preserves the migratory pathway of neuroblasts, the glial sheath, and the vasculature that are largely aligned in this direction. One can either mount the tissue at the midline or on the flat surface created by making sagittal cuts lateral to the SVZ. An optional step is to place a 3% agarose block behind the tissue to serve as an abutment to the blade as the slices come off. We preferentially used cyanoacrylate-based super glue.</li>
	<li>Rinse the blade with ethanol to remove oils and rinse with distilled water. Place the blade on the blade holder. Mount the blade holder to the vibratome.</li>
	<li>Adjust vibratome settings to cut tissue at a thickness of 250-350 &mu;m per slice. It is important to cut using high frequency vibrations and low speed.</li>
	<li>Section progressively through the tissue. The appearance of the olfactory bulb is a good indicator that one is near the SVZ in sagittal slices as the most lateral slices will contain neither region. Other landmarks to look for include the ventricle and the thickening of the corpus callosum, beneath which the SVZ is located. The SVZ will appear first in more lateral sagittal slices. The RMS will show in medial sagittal slices.</li>
	<li>Remove slices containing the SVZ with a plastic Pasteur pipette with the tip cut off. Transferring to the slice holding chamber. We often obtain more slices than we can use in a work day. However, slices will vary on the amount of cells containing the SVZ. It is sometimes necessary to look through several slices to find one desired for imaging.</li>
</ol>
4. Preparation of Microscope and Dyes
The slices require a one hour incubation time in ACSF to recover from the dissection and slicing. Several steps can be performed during this slice recovery period.
<ol>
	<li>Preparation of pipettes for calcium dye and/or drug application
	<ol class="lalpha">
		<li>Glass pipettes need to have a tip of 2-3 &mu;m in diameter and a tip length (~2 mm) and angle (~16&deg; for the pipette holder) that prevents contact with the perfusion chamber and allows room for placement of the objective.</li>
		<li>One can typically prepare 6-8 pressure pipettes for each day of experiments.</li>
	</ol>
	</li>
	<li>Calcium dye preparation
	<ol class="lalpha">
		<li>When preparing the working solution (4-5 &mu;M by bath, 250 &mu;M by pressure application), it is best to expose the minimum amount of DMSO to cells. This can be achieved by making highly concentrated stocks. For example, for Fluo-4, one tube containing 50 &mu;g is used per day. One can make a 10 mM stock of this aliquot by adding 4.6 &mu;l of Pluronic F-127 20% solution in DMSO.</li>
	</ol>
	</li>
	<li>Preparing microscope setup (Figure 2).
	<ol class="lalpha">
		<li>Use of a peristaltic pump for solution flow has helped minimize image drift. Run the peristaltic pump prior to allow ACSF to run through the perfusion lines and ensure that it is bubble-free.</li>
		<li>Set the tube inlet on the perfusion chamber and ensure there are no leaks.</li>
		<li>Position the vacuum tip to ensure that the solution is being aspirated from the perfusion chamber. Ensure that it is at an adequate level so that the objective is immersed at the proper working distance, but not so high such that solution may spill over the chamber. A low level of solution will also minimize flow distortions. A constant aspiration can also be verified by listening for steadiness.</li>
	</ol>
	</li>
</ol>
5. Dye Loading
This method has been described in detail in another manuscript2. Please see figures therein.
<ol>
	<li>Bath loading dye
	<ol class="lalpha">
		<li>Make a 1-2 ml working solution of the dye. For Fluo-4 AM, a concentration of 4-5 &mu;M is adequate for labeling SVZ neuroblasts.</li>
		<li>Slices are first transferred to a 35 mm culture dish with a mesh bottom that is already filled with ACSF. Replace the solution by using a 3 ml plastic transfer pipette to gently remove the ACSF, while simultaneously adding the calcium dye working solution.</li>
		<li>Incubate at 37 &deg;C for 30-60 min in a 5% CO2 gas-controlled environment.</li>
		<li>Place the slice back into the ACSF-filled holding chamber to wash away dye that has not entered cells. Alternatively, one can place it directly on the microscope perfusion chamber that is running ACSF. This wash period is 30-45 min.</li>
		<li>Transfer the slice to the perfusion chamber.</li>
	</ol>
	</li>
	<li>Pressure application of dye
	<ol class="lalpha">
		<li>Transfer the slice from the holding chamber to the perfusion chamber on the microscope setup.</li>
		<li>Fill a glass pipette with a working solution that is 250 &mu;M and place on the micromanipulator.</li>
		<li>Lower the pipette to the region of interest. The pipette can be placed such that the tip is either several micrometers above the slice surface or buried several micrometers below the slice surface, but away from the recorded region. The angle of the pipette did not affect the loading efficiency. When imaging below the slice surface, we find that placing the pipette tip below the imaging plane will label our desired imaging area better.</li>
		<li>When pressure applying the dye, set the Picospritzer so that it is &lt;3 psi. We have not estimated the volume of dye diffusion, because this is subject to variability of the pipette tip diameter, pressure applied, and density of tissue where the dye is injected. We apply simply until we achieve our desired loading as assessed by eye. Minimize damage to the slice during application, especially when the tip is placed beneath the slice surface. Apply for 1-2 min. Sometimes repeated applications are necessary depending on labeling as assessed by eye. For this concentration and application duration, we find that surface application of dye preferentially labels neuroblasts while below the surface application preferentially labels astrocytes. However, &gt;3 min application at 500 &mu;M will also label neuroblasts regardless of whether the tip is above or below the slice surface (JC Platel, unpublished observation). Allow for wash and slice recovery for 30-45 min.</li>
	</ol>
	</li>
</ol>
6. Confocal Imaging of Calcium Activity
<ol>
	<li>Find the region of interest first with a lower-power objective such as 4x or 10x. Place in the center of the field before switching to a higher power objective. At this point, one can assess general slice health by noting the appearance of SVZ cells under differential interference contrast (DIC). Healthy cells appear round and plump. In addition, healthy cells will display faint Fluo-4 loading as supposed to no loading at all or bright fluorescence (a dying cell).</li>
	<li>Lower the water-immersion objective onto the region of interest, which is now dye-loaded. Either a 40x or 60x objective has been adequate for our needs, although 40x provides a wider field of view and allows for greater pipette access. When imaging below the surface, we often go at least 15 &mu;m below the slice surface where the 3-dimentional architecture and cell health are better preserved.</li>
	<li>Verify that perfusion and vacuum are working adequately.</li>
	<li>Setup the microscope so that time-lapse resolution and spatial resolution are set to desired rates. For confocal microscopy, these factors must be balanced as an increase in temporal resolution usually results in a loss of spatial resolution due to the scanning nature of the system. For calcium activity, we have imaged a frame approximately every second with a 512x512 pixel resolution. If multiple channels acquired separately are necessary, this will also affect acquisition rates. Set duration of movie (2-10 min).</li>
	<li>Initiate run. If performing pharmacological studies, wash on antagonists or non-desensitizing agonists for at least 5-10 min, and then make another movie of 5-10 min. Agonists that may desensitize receptors can be acutely applied, such as by using a pressure pipette controlled by a micromanipulator.</li>
</ol>
7. Calcium Analysis
Analysis follows standard procedures that we described in pervious publications2-4. F0 (i.e. baseline) and F are the mean fluorescence intensities measured throughout all of the regions of interest (ROIs) and in each ROI, respectively. A change in fluorescence was considered to be a Ca2+ increase if it was &gt;15% F/F0 increase. Intracellular Ca2+ changes were calculated using Calsignal5.
8. Representative Results
We have been able to obtain selective loading of SVZ cells depending on the dye loading protocols described above and elsewhere2-4,6. Bath loading or pressure application on the slice surface of Fluo-4 AM (4-5 or 250 &mu;M, respectively) labels mostly neuroblasts. While pressure application can load neuroblasts more quickly than bath loading in a single slice, bath loading allows the simultaneous loading of multiple slices. We confirm the identity of labeled cells as neuroblasts by (1) whether they display migratory behavior4,7, (2) their morphology, (3) negative staining of sulforhodamine 101, an astrocyte-specific dye3 or (4) positive staining with DCX-DsRed expression (JC Platel, unpublished observation). Neuroblasts migrate quickly (average of 60 &mu;m/hr) at physiological temperature4,7-9, but their movement can be readily observed even at room temperature. The movement of neuroblasts does not pose an issue with regards to placing regions-of-interest (ROI) to track calcium activity since our movies are typically short in duration. However, during long waiting periods, such as during drug solution exchanges, investigators must be careful to match ROIs in control and treatment periods. It is not uncommon for neuroblasts to migrate into or out of the imaging field or focal plane.
Pressure application of Fluo-4 AM (250 &mu;M) deep into the slice and for limited duration (&lt;2 min) preferentially labels SVZ astrocytes2. Astrocyte labeling has been verified by positive labeling with sulforhodamine 101 and their morphology, especially by the presence of projections and endfeet on blood vessels which we have recently described3. Using these methods, we have observed spontaneous activity in both the SVZ neuroblast and astrocyte population (Figure 1). The activity in astrocytes often takes the form of waves that engage blood vessel3. Furthermore, using calcium imaging as an assay, the application of pharmacological agonists has revealed or confirmed expression of GABAA receptors on neuroblasts and astrocytes6, AMPA and NMDA receptors on neuroblasts4.
Figures
<img alt="Figure 1" src="/files/ftp_upload/4071/4071fig1.jpg" /> 
Figure 1. Propagating calcium activity in astrocytes. (A) Representative averaged image from a time-lapse recording of calcium activity. Astrocytes in the SVZ were loaded with pressure application of Fluo-4 AM deep into the slice. Movies were acquired at 0.75 s time-steps. Regions of interest (ROI) are placed over cells exhibiting activity. (B) Traces from ROIs placed over cells from the movie depicted in (A). Traces were filtered with a moving average and normalized. The vertical scale represents 2 x &Delta;F/F0 where F is the signal intensity and F0 is the average baseline signal and &Delta;F= F-F0.
 
<img alt="Figure 2" src="/files/ftp_upload/4071/4071fig2.jpg" /> 
Figure 2. Picture of the perfusion system. One end of a tube is submerged in 95% O2/5% CO2 gas-perfused solution. The solution is then perfused through the tube and to the bath chamber by a peristaltic pump (not shown). The other end is inserted into the solution inlet of the bath chamber mounted on a platform. The aspiration tip is then connected to the solution outlet of the chamber and set at a level to determine solution height. From the outlet, the aspiration tip is connected to the vacuum line, which aspirates the solution from the chamber into a waste container. The perfusion system is shown off the microscope stage for visual clarity.

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No conflicts of interest declared.

Calcium imaging of SVZ cells has been used to study patterns in spontaneous activity in neuroblasts10, receptor channel expression in both neuroblasts and astrocytes4,6,8 and astrocytic calcium waves3. Since cells in the SVZ are either immature or have glial properties, they do not fire action potentials11,12, meaning that millisecond changes in voltage potential indicative of activity in mature networks is not applicable in this region. Therefore, capturing the slower calcium ...

<table border="1">
	<tbody>
		<tr>
			<td>Solute</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Dissection (mM)</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>Dissection: 147 mM 
			ACSF: 0 mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>Dissection: 42 mM 
			ACSF: 126 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td>Dissection: 2.5 mM 
			ACSF: 2.5 mM</td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td>Dissection: 4.33 mM 
			ACSF: 1 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>Dissection: 1.25 mM 
			ACSF: 1.25 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td>Dissection: 10 mM 
			ACSF: 10 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>Dissection: 26 mM 
			ACSF: 26 mM</td>
		</tr>
		<tr>
			<td>CaCl2.2H2O</td>
			<td>Sigma</td>
			<td>C3306</td>
			<td>Dissection: 1.33 mM 
			ACSF: 2 mM</td>
		</tr>
	</tbody>
</table>
Table 1. Chemical list and recipes of dissection solution and ACSF.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT 1000S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Surehold or 3M</td>
			<td>Surehold 3G Super Glue or 3M Vet-Bond</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection tools</td>
			<td>Roboz or Ted Pella</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluo-4 AM calcium-sensitive dye</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green BAPTA-1 AM calcium-sensitive dye</td>
			<td>Invitrogen</td>
			<td>O6807</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pluronic F-127 20% solution in DMSO</td>
			<td>Invitrogen</td>
			<td>P3000MP</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Upright confocal microscope</td>
			<td>Olympus</td>
			<td>FV300 or FV1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water-immersion objectives</td>
			<td>Olympus</td>
			<td>LUMPlanFl 40 x W/IR (NA 0.80); LUMPlanFl 60 x W/I (NA 0.90)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Sutter</td>
			<td>MPC-200/MPC-325/MPC-385</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pressure controller</td>
			<td>Parker Hannifin</td>
			<td>Picospritzer</td>
			<td>&lt;3 PSI during application</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Sutter or Narshige</td>
			<td>Sutter P-97 or Narshige PP-830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass pipettes</td>
			<td>Sutter</td>
			<td>BF150-110-10</td>
			<td>I.D.:1.10, O.D.: 1.50</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Harvard Apparatus</td>
			<td>Model 720</td>
			<td>flow rate: 1 ml/min</td>
		</tr>
		<tr>
			<td>Chamber bath</td>
			<td>Warner Instruments</td>
			<td>RC-26 GLP</td>
			<td>Low profile allows for objective clearance</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Tygon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-324B/344B</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 2. Materials/equipment list.

preparation of acute subventricular zone slices for calcium imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

preparation-of-acute-subventricular-zone-slices-for-calcium-imaging

Research

JoVE Journal

Neuroscience

13.1K Views.  Yale University School of Medicine. A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Video: Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...