Neurons in Living Color

BacMam 2.0 Technology and Other Imaging Tools for Live-Cell Studies

The study of neurons is vitally important in understanding how the brain develops and functions. The ultimate goal of many neuroscientists extends well beyond the analysis of normal brain functioning to the dissection of the cellular changes underlying the myriad of devastating neurodegenerative disorders. As highly specialized postmitotic cells, neurons sense the environment and then either store or rapidly convey this information through electrical and chemical signals that propagate along physical pathways in the brain and peripheral body, ultimately controlling behavior. Investigating these complex and dynamic processes in living neurons requires tools that can target specific neural cell functions without disrupting normal cell activities.

Imaging Applications For Neuroscience

By examining events up close with a microscope, scientists can capture imagery that reveals molecular interactions, protein expression patterns, and the interplay of signaling pathways. These images can provide important information about structure and function that span perspectives from the molecular dimensions to the subcellular and macroscopic levels. Moreover, live-cell imaging has transitioned from requiring years of experience and ultra-specialized equipment into a technology that requires minimal training to obtain dramatic and informative data. With the increasing availability of a variety of spectrally distinguishable labels for imaging-based assays, microscopic analysis of neurons and neural networks is now more accessible than ever before.

For several decades Molecular Probes® reagents have been used with fluorescence microscopy to examine neural cell components and signaling mechanisms, as well as the anatomy of neural networks and functions of the brain and nervous system. For example, Molecular Probes® calcium indicators are commonly used to monitor calcium fluxes and record neural activity [1] (Figure 1), the FM® dyes serve as reliable membrane markers for following synaptic communication [2], and fluorescent neural tracers and labeled cholera toxins have been used to dissect the anatomy of neural populations [3]. Here we describe the latest addition to our probes for neurobiology research: BacMam 2.0 gene delivery and expression technology for the transduction of primary neurons and transient expression of exogenous genes.

Figure 1. Differentiated NG108-15 neural cells loaded with fluo-4 AM. NG108-15 cells, a hybrid of mouse neuroblastoma and rat glioma cells, are often used to model primary cells from the central nervous system. Like many stem cell–derived neural cells, NG108-15 cells are cultured in a propagation mode and then switched to a differentiation medium that arrests cell division and promotes the elaboration of axons and the expression of a variety of neural-specific genes, including voltage-gated ion channels and synaptic specializations. Shown here are differentiated NG108-15 cells grown in culture and loaded with the calcium indicator dye fluo-4 acetoxymethyl (AM) ester . The image was generated as an overlay of phase-contrast and green fluorescence images collected using the Till Photonics Polychrome V excitation source.

BacMam 2.0 Technology Enables Live-Cell Imaging in Neurons

Like many terminally differentiated, postmitotic cell types, neurons are not only difficult to culture but also resistant to the delivery and expression of recombinant genes. These challenges have greatly hampered basic research and drug discovery efforts that rely on functional measurements in living cells.

BacMam gene delivery and expression technology alleviates one of the main technological hurdles for live-cell imaging; it provides a system to deliver and transiently express targeted fluorescent proteins and biosensors in mammalian cells without perturbing normal cell function. The BacMam system relies on a baculovirus strain with envelope-protein modifications that allow the virus to infect mammalian cells (so-called pseudotyped virus particles). The genetic cargo of the virus has likewise been engineered to deliver genes encoding fluorescent protein fusions under the control of mammalian expression signals. Mammalian cells ignore the polyhedron insect cell promoters in the baculoviral DNA, expressing only the mammalian promoter–driven transgenes in the engineered cassette. The BacMam gene delivery and expression system has several important features:

• Lack of cytotoxicity—even at very high ratios of virus particles to cells
• Ease of use—simply incubate cells with the virus particles in normal culture medium
• Safety—P1 safety level: the virus cannot replicate in mammalian cells
• Straightforward modulation of gene expression—higher virus/cell ratios yield higher expression levels

The new BacMam 2.0 technology employs a VSVG-pseudotyped capsid protein for more efficient cell entry, a strong mammalian promoter, and a posttranscriptional regulatory element to boost expression levels. With these modifications, the BacMam 2.0 reagents can now be used to introduce and express fluorescent proteins and biosensors in cell types that could not be transduced with the original BacMam system, including primary neurons (Figure 2), stem cells (Figure 3), and cardiomyocytes.

Figure 2. Live-cell imaging with BacMam CellLight® reagents or Premo™ Autophagy Sensor LC3B-GFP. Cultured primary hippocampal neurons are labeled with either (left) CellLight® ER-GFP, which labels the endoplasmic reticulum with green fluorescence, or (center) Premo™ Autophagy Sensor LC3B-GFP, which labels autophagosomes with punctate green-fluorescent accumulations of the LC3B protein. (right) A Schwann cell has been double-labeled with BacMam 2.0 GFP transduction control and CellularLights™ Actin-RFP. Note that the actin-RFP in this image was transduced into Schwann cells using the first-generation (BacMam 1.0) reagent, which will label glial cells and many dividing cell types but does not work well with neurons.

Live-Cell Imaging Tools for Cell Structure, Autophagy, and Calcium Flux

Scientists at Life Technologies have introduced a suite of BacMam 2.0 probes that are compatible with live-cell imaging of primary neurons, including the CellLight® reagents and the Premo™ sensors. The CellLight® reagents are prepackaged, ready-to-use DNA constructs for the expression of organelle- or cytoskeleton-targeted fluorescent proteins (Figure 2), as well as the nontargeted BacMam GFP transduction control (Figure 3). The Premo™ Autophagy Sensors provide a LC3B–fluorescent protein chimera, which serves as a general marker for autophagic membranes. These BacMam 2.0 reagents can be used to probe a variety of neural processes, from trafficking of intracellular organelles and cytoskeletal dynamics, to the induction of autophagy. While standard cell lines such as HeLa and CHO cells typically express these fluorescent protein fusions for about 5 days, expression has been demonstrated for up to two weeks in some primary cells, and in terminally differentiated neurons we have images recorded more than three weeks after transduction.

For multiplex imaging experiments with green-fluorescent probes including the BacMam 2.0 GFP reagents, we recommend the red-shifted calcium indicator rhod-3 AM (Figure 4). This calcium sensor is ideal for reporting transient changes in neural cytosolic calcium signaling in cells expressing GFP. In addition to its red-shifted excitation and emission maxima, rhod-3 dye typically displays more uniform cytosolic distribution and improved signal compared with the traditional red-fluorescent calcium indicator rhod-2, which is used primarily to visualize mitochondrial calcium dynamics in living cells.

Figure 4. Excitation/emission spectra of rhod-3 dye and time-lapse calcium imaging in dorsal root ganglion (DRG) neurons loaded with rhod-3 AM during depolarization with elevated potassium chloride (KCl). The tight absorbance and emission spectra of rhod-3 dye are ideal for measuring cytosolic calcium in cells expressing GFP. The affinity of rhod-3 for free calcium is ~570 nM, providing an ideal dissociation constant (K_d for the measurement of cytosolic calcium as it enters through voltage gated ion channels, as shown in this time lapse series. In this experiment, cells loaded with rhod-3 AM were positioned adjacent to flowpipes containing control solution or elevated KCl during acquisition, as frames were taken every 100 milliseconds. Elevated (50 mM) KCl depolarizes the cells and opens voltage-gated calcium ion channels, allowing calcium to enter the cells and bind to rhod-3, increasing fluorescence 2- to 4-fold, depending on the preparation and calcium concentrations involved.

Our Commitment to Live Neural-Cell Imaging Studies

Few cell types are as dynamic to work with as neurons and their glial counterparts. Molecular Probes® ready-to-use live-cell imaging tools help you capture dramatic evidence of a wide range of cell processes. In addition, Life Technologies offers Gibco® neural primary and stem cells, plus a complete suite of neural cell culture products—including media, supplements, substrates, and growth factors—all developed to work together for optimal performance.

References

1. Yasuda R, Nimchinsky EA, Scheuss V et al. (2004) Sci STKE 2004:p15.
2. Amaral E, Guatimosim S, Guatimosim C (2011) Methods Mol Biol 689:137–148.
3. Mentis GZ, Alvarez FJ, Shneider NA et al. (2010) Ann N Y Acad Sci 1198:220–230.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.