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 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 the mammalian brain, the generation of new neurons (neurogenesis) occurs after birth mainly in two regions, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus1. Considerable evidence gathered in recent years supports a critical role for postnatal neurogenesis in hippocampal and olfactory bulb memory functions1-3. Importantly, postnatal neurogenesis also holds therapeutic potential because of its relationship with degenerative neurological disorders, and the ability of neuroblasts to migrate to injured sites in the brain4-6.
The subventricular zone (SVZ) has recently emerged as a crucial neurogenic niche. SVZ-derived neuroblasts migrate towards the olfactory bulb (OB) via the rostral migratory stream (RMS), making this the longest migration process in the postnatal brain1,7,8. The mammalian SVZ/RMS/OB system has become a useful model to study different steps in neurogenesis, such as proliferation, migration and differentiation1,8. Many growth factors and extracellular cues regulate SVZ neurogenesis and migration along the RMS, but the intracellular molecular mechanisms are far from being fully understood1,9. Proper migration along the RMS is crucial for the subsequent maturation of newborn neurons10. Additionally, some studies have shown that SVZ-derived neuroblasts can migrate out of the RMS to brain injury sites4-6,11-13. Thus, investigating the signalling mechanisms regulating neuroblast migration is fundamental not only to understand neurogenesis but also for potential therapeutic applications.
Here, we describe a detailed protocol to label SVZ neural progenitors by in vivo postnatal electroporation and monitor their migration along the RMS in acute brain slice cultures using time-lapse spinning disk confocal microscopy. Electroporation is widely used in developmental studies from embryonic to adult stages14-18. It is a powerful tool to target and manipulate SVZ neural progenitors and represents a cheaper and considerably faster alternative to stereotactic injection of viral vectors or generation of transgenic models1,15,19,20. It is a relatively simple procedure that does not need surgery and has high survival rates. Electroporation of shRNA or CRE recombinase-expressing plasmids in mouse genetic models employing the LoxP system can be used to target genes of interest or to achieve permanent labeling of SVZ progenitors, thus representing a useful tool for adult neurogenesis studies21,22.
Imaging RMS neuroblast migration in the intact brain is still challenging due to current technical limitations. However, this process can be monitored using confocal spinning disk time-lapse microscopy of acute brain slices, which provide a suitable system closely resembling the in vivo condition also amenable to pharmacological manipulation23,24. Coupling in vivo postnatal electroporation with time-lapse imaging will facilitate the understanding of the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.
This procedure is in accordance with the UK Home Office Regulations (Animal Scientific Procedures Act, 1986). Scientists should follow the guidelines established and approved by their institutional and national animal regulatory organizations.
1. Postnatal Electroporation
1.1. Preparation of Glass Capillaries, DNA Solution and Electroporator
1.2. Electroporation
2. Preparation of Acute Brain Slice Cultures
2.1. Preparation of Solutions and Tools
2.2. Preparation of Brain Slices
2.3. Culturing Brain Slices
3. Time-lapse Imaging of Neuroblast Migration
4. Analyzing Neuroblast Migration
Labeling of SVZ-derived migratory neuroblasts can be observed along the RMS, usually 4-8 days after a successful electroporation (Figure 1B). Longer time points can also be chosen, but less cells will be found in the RMS since most of them will have entered the OB. Neuroblasts start acquiring typical morphology and features of mature granule cells in the OB around 2-3 weeks after electroporation (not shown). After culturing for ~1 hr, brain slices from electroporated mouse pups can be reliably imaged for up to 3-4 hr. As previously reported23,26, neuroblasts can display complex migration dynamics (Figure 3 and Video 1), which can be quantitatively analyzed using cell tracking (Figure 4).
Figure 1. Postnatal in vivo electroporation. (A) Schematic drawing of in vivo electroporation of a postnatal day 2 mouse pup. A dotted line (red) connecting the eye to the craniometrical landmark lambda serves as a positional marker for capillary insertion. The injection point is indicated as a green dot. Grey oval shapes indicate electrode position as described in Boutin et al.15 (B) Sagittal mouse forebrain slice immunostained for GFP 5 days after electroporation of a GFP-expressing plasmid. SVZ-derived migrating neuroblasts are visible along the RMS and some of them have started to reach the OB. Ctx: cortex; SVZ: subventricular zone; RMS: rostral migratory stream; OB: olfactory bulb. Bar, 400 µm. Click here to view larger image.
Figure 2. Schematic steps in the preparation of acute brain slice cultures for imaging. (A) Caudal and intrahemispheric cuts (dotted lines) are performed on a freshly dissected brain. (B) The electroporated brain hemisphere is placed onto a vibratome holder. (C) Sagittal slices are obtained with a vibratome and observed under a standard fluorescent microscope. (D) Slices with the best GFP signal are cultured for at least 1 hr on a Millicell insert inside a glass-bottom dish, and (E) subsequently placed in an environmental chamber for imaging by an inverted confocal spinning disk system. Click here to view larger image.
Figure 3. Time-lapse imaging of migrating neuroblasts. (A) Spinning disk time-lapse images of neuroblasts taken from a mouse sagittal brain slice 5 days after electroporation of a GFP-expressing plasmid. Images are 1 hr apart. Each panel is a z-stack projection of 28 consecutive images 4 µm apart. Arrowheads indicate 4 representative neuroblasts migrating along the RMS towards the olfactory bulb (located out of the picture at the bottom right corner). (B) Representative migratory paths obtained from time-lapse imaging of the 4 neuroblasts highlighted in (A). (C) Graph showing the distance migrated with time by 2 representative neuroblasts. Cells display a typical saltatory motile behavior. Bar, 70 µm. Click here to view larger image.
Figure 4. Tracking analysis of migrating neuroblasts. Sequential steps used to track migrating neuroblasts using Volocity software. Please see text for detailed description. Click here to view larger image.
Video 1: Time-lapse imaging of migrating neuroblasts. The movie shows a section of the mouse RMS with GFP-labeled migrating neuroblasts obtained 5 days after electroporation of a GFP-expressing plasmid. The OB is located out of view towards the bottom right corner. Confocal z-stacks were captured on a spinning disk confocal with a 20X objective every 3 min for 3 hr over a 120 µm interval. Playing speed: 10 frames/sec.
Efficient migration of neural progenitors along the RMS ensures their subsequent maturation into functional neurons10. Prominent streams of neural progenitors directed towards the OB are visible in human infancy and are likely to play an important role in early postnatal human brain development27. Moreover, these cells are able to target sites of the brain affected by injury and neurodegeneration4,28. Being able to monitor in real time the effect of gene manipulation on neuroblast dynamics becomes essential to fully understand how neuroblast movement is guided and regulated.
Here we have described a protocol to monitor SVZ-derived neuroblast migration by coupling in vivo postnatal electroporation with time-lapse confocal spinning disk microscopy of acute brain slice cultures. Specifically, we have shown how migrating neural progenitors can be labeled by electroporating in the SVZ a plasmid encoding GFP. Depending on the purpose of the study, several types of plasmids can be used (e.g. allowing expression of other fluorescent proteins or co-expression of wild type/mutant proteins of interest, CRE recombinase, or shRNA together with fluorescent proteins). We strongly recommend using plasmids containing the chicken beta actin CAG promoter29 for in vivo expression. Imaging of brain slices obtained from electroporated animals can also be used to monitor the radial migration of neural progenitors in the OB at longer time points (7-10 days) after electroporation30.
After an initial period of practice, in vivo postnatal electroporation becomes a very reliable method, allowing robust neuroblast labeling for both time-lapse imaging and immunofluorescence analysis. Moreover, this technique offers a fundamental advantage over transgenic mice expressing fluorescent proteins under neuroblast-specific promoters, where the majority of neuroblasts are labeled. Indeed, electroporation allows sparse labeling of neuroblasts, thus allowing detailed analysis of their morphology and migration dynamics. Compared to the stereotactic delivery of viral vectors31, this technique is cheaper, faster and is very well tolerated by mouse pups. The main drawback consists in the fact that it is limited to early postnatal stages. Indeed, we strongly recommend using postnatal day 2 mouse pups, since we observed a substantial decrease in labeling efficiency at later times, when viral delivery methods become more suitable31. However, strategies like electroporation of CRE-expressing plasmids in appropriate mouse genetic models may be used to study neurogenesis in adult stages22.
Two-photon, standard confocal, spinning disk confocal, and wide-field fluorescence microscopy can all be used to visualize neuroblast migration17,23,24. Spinning disk confocal microscopy is a cheaper alternative to two-photon microscopy. It allows 3D imaging at higher speed through multiple z planes, limiting photobleaching compared to standard confocal microscopy and offering a higher resolution than wide-field fluorescence imaging31-33. The majority of migrating neuroblasts have a clearly visible soma and a highly dynamic leading process tipped with a growth cone.
As described by others17, having a perfusion chamber would allow to directly assess the effects of control and drug treatments on the same brain slice. While we consider this an important advantage, the protocol described here shows that it is not absolutely necessary for cell viability to perfuse slices with oxygenated artificial cerebrospinal fluid (aCSF) or high glucose DMEM17,33. Moreover, using an inverted microscope allows easier objective manipulation compared to upright microscopes equipped with water immersion objectives. We found that using a 20X long-distance objective is an optimal compromise, enabling tracking of a good number of cells (usually 25-40 per slice) and producing good resolution images (Video 1). Automatic tracking is also possible, however it is not always reliable. For this reason we recommend visual and, if necessary, manual verification of automatic tracking data. Higher magnification imaging of single neuroblasts can be performed using a Nikon Fluor DIC M/N2 40X/0.80W objective. The freely available ImageJ plugin MTrackJ can also be used to measure basic track statistics33, however some raw data acquisition files may be incompatible with this software and may need to be converted to suitable formats for analysis, which in certain cases can be time-consuming. The combination of image acquisition and analysis modules provided by the Volocity software allows an immediate transition from image capturing/processing to quantitative analysis of migratory parameters (speed, displacement, etc.), which can be readily visualized in a variety of graphs (e.g. showing migratory patterns/distance/directionality/speed/persistence/time spent immobile, etc.).
Neuroblasts display a saltatory movement, alternating migratory to stationary phases34,35 (Figure 3C). In light of the fact that stationary phases can be between 4-10 min32, we believe that capturing images every 3 min represents a good compromise to follow migrating neuroblasts with minimum illumination and photodamage. We rarely see cells stopping for more than 6 min, and find that, over a 3-hr period, they generally spend an average of 30 min immobile. According to previous reports, neuroblasts have an average speed of 60-100 µm/hr and an average meandering index (actual displacement over the migrated distance), slightly above 0.623,36, which is in agreement with our observations. Typically, about 60% of cells show a "migratory" behavior (e.g. following almost linear migration paths, with a meandering index between 0.6-1) and 20-25% are "exploratory" (with a meandering index between 0-0.4), while the remaining ~20% can be classified as "intermediate" (alternating migratory and exploratory phases, with a meandering index between 0.4-0.6).
We recognize that there are still limitations to this technique, since we cannot film neuroblasts for more than 3-4 hr without observing substantial alterations in their motility. Filming duration could be increased by lowering imaging frequency (however this will affect the accuracy of the migration analysis) as well as by adopting a perfusion chamber to optimize environmental conditions for imaging. However, the protocol described here including a 3-4 hr imaging period represents a suitable compromise enabling the collection of sufficient data for quantitative analysis of migration. Indeed, so far we have managed to detect the effects produced on migration by protein overexpression/downregulation37 or by pharmacological manipulation after comparison with appropriate controls (Sonego and Zhou, unpublished results).
In conclusion, the combination of in vivo postnatal electroporation with spinning disk imaging of brain slice cultures represents a powerful tool to monitor the dynamics of neuroblast migration in a system closely resembling the in vivo environment, offering the possibility to investigate in depth the molecular mechanisms controlling neuroblast motility.
The authors have nothing to disclose.
M.S. and Y.Z. are supported by KCL and KCL-China PhD studentships. M.O. was funded by a Biotechnology and Biological Sciences Research Council PhD studentship. We thank Masaru Okabe and Jun-ichi Miyazaki for the pCX-EGFP plasmid and Alain Chedotal and Athena Ypsilanti for valuable advice on electroporation.
Millicell | Millipore | PICM0RG50 | |
35 mm Glass bottom culture dish | MatTek | P35G-0-14-C | |
Gey's Balanced media | Sigma | G9779-500ML | |
Glucose, 45% | Sigma | G8769-100ML | |
HEPES | Sigma | H3375-25G | |
Pen/Strep | GIBCO | 15140-122 | |
FCS | GIBCO | 10109-163 | |
B27 supplement | Invitrogen Life Technologies | 17504044 | |
L-Glutamine | Invitrogen Life Technologies | 25030-081 | |
DMEM (phenol red-free) | GIBCO | 31053-028 | |
Fast Green | Sigma | F7252-5G | |
Glass capillaries for injection | Harvard Apparatus | 30-0057 | |
Aspirator tube | Sigma | A5177 | |
Sutter P-97 capillary puller | Sutter Instrument | P-97 | |
ECM830 Square Wave Electroporator | Harvard Apparatus | 45-0052 | |
Platinum Tweezertrodes 7 mm | Harvard Apparatus | 45-0488 | |
Footswitch Model 1250F | Harvard Apparatus | 45-0211 | |
Gel for electrodes | Cefar Compex | 6602048 | |
Isoflurane | Merial | AP/DRUGS/220/96 | |
Vibratome | Leica | VT1000S | |
Glue | Roti coll | Roti coll 1 | |
UltraViEW VoX spinning disk system | Perkin Elmer | Customized setup (multiple laser sources can be used) equipped with Hamamatsu ORCA R2 C10600-10B CCD camera | |
Volocity software | Perkin Elmer | Acquisition, Quantitation, Visualization Modules | |
Environmental chamber for microscopy | Solent Scientific | Custom-made | |
Ti-E inverted microscope | Nikon | CFI Super Plan Fluor ELWD 20X/0.45 NA objective is recommended for the application described in this paper |