Delineating precise patterns of synaptic connectivity in the mammalian nervous system is essential if we are to ultimately gain mechanistic insight into normal neural development, neurological disease, or potential avenues for brain repair. Numerous experimental approaches ranging from imaging and electrophysiology, to genetic engineering and stem cell biology, have been broadly implemented towards our working knowledge in these areas. Unfortunately, progress in this field has been somewhat tempered due to the innate complexity of brain tissue. Thus, new experimental approaches and methodologies for investigating patterns of synaptic connectivity are both useful and necessary.
We have generated a novel ES cell line with all the necessary elements for transsynaptic viral tracing in nervous tissue using genetically modified RV. Through immunohistochemistry, transsynaptic fluorescent reporter analysis, and electrophysiological recordings, we have demonstrated the efficient differentiation of these cells into functional neurons in vitro,
and have shown that that they can wire up effectively with host neuronal circuits in vitro
and in vivo
. Transsynaptic tracing technology using genetically-engineered RV is a powerful tool given its general utility to genetically identify neurons that are connected via chemical synapses32–37
. In contrast to most lipophilic dye-based synaptic tracers, lectin-based conjugates, and other viral technology, RV spread occurs exclusively in a retrograde manner, is not thought to cross gap junctions, and requires the presence of intact presynaptic structures for neuronal infection and transsynaptic transfer31–36
. However, it is important to consider other mechanisms of viral labeling and spread that can occur in vitro
. Unlike the specificity of synapse formation within intact nervous tissue, in vitro
preparations facilitate numerous “unnatural” cellular interactions. For example, differentiating cells and neurons in culture can form transient synaptic connections through time. This poses the potential for non-specific viral transfer and thus labeling of cell types that do not show long-lived functional synaptic connectivity. Moreover, in vitro
culture environments do not foster the longevity of cells in vivo
, and thus cultured neuronal cells can die off earlier than in vivo
. In this scenario, infected neurons might shed RV into the culture medium upon death, and these viral particles have the capacity to be taken up by nearby neurons without synaptic connectivity. Importantly, these non-specific labeling phenomena can be minimized when in vitro
RV infection times are kept as short as possible. For this reason, we performed all tracing experiments within 3–7 days after infection. During this time, infected cells remain alive, healthy, and function as expected for in vitro
Neuronal differentiation of modified ES cells allows the unique opportunity to investigate basic molecular and cell biological mechanisms that underlie synapse and circuit formation in vitro, but also affords the ability to efficiently cross over to in vivo experimentation via cell transplantation or generation of mutant mice. A beneficial facet of our approach that complements preexisting tracing methods is that circuit integration can be studied at any stage of neuronal differentiation and in any part of the nervous system. Having a genetically targeted ES cell line that harbors the elements for transsynaptic viral tracing allows for differentiation of these cells into virtually any cell type, and facilitates the identification of the various types of presynaptic inputs in diverse regions of the nervous system, both healthy and diseased. Experimentation becomes feasible in which ROSA-tomRITVA ES cells are differentiated into specific neuronal subtypes and subsequently combined with either neurons or tissues harboring known genetic lesions to address cell non-autonomous mechanisms of synapse formation, or alternatively, introduce compound mutations or shRNA expression into ROSA-tomRITVA cells to address cell autonomous gene function. These approaches could also be extended to in vivo applications, where transplanted ES cells could interact with intact brain tissue. Alternatively, the generation of novel mouse lines from these ES cells could be powerful in delineating patterns of connectivity in the context of normal development or disease. One application that could prove to be extremely useful would be to generate mosaic mice through morula aggregation, in which select subsets of neurons could be targeted for transsynaptic tracing in a semi-stochastic manner. This could also be combined with additional genetic alterations to tease out mechanisms of synapse wiring.
Of course we must also consider the limitations. ES cell technology is in its infancy. Although we have learned a great deal about driving neural differentiation in vitro, we still have much to learn about the generation and maintenance of certain neuronal subtypes. It will be imperative for future work to more precisely characterize and describe methodology to enrich and isolate neuronal subtypes of given neurotransmitter or receptor expression properties. Additionally, current applications for ES-derived neuronal transplants remain prominent in rodents, and in particular inbred strains of mice. Teratoma formation and immune responses have stifled applications in other mammalian systems. Induced pluripotent stem (iPS) cells offer promise in this area. It is conceivable that similar genetic expression systems for transsynaptic analysis could be introduced into tailored iPS cell lines, and that these could subsequently be used to investigate synaptic mechanisms in other tissues or model systems.
Considering the widespread use of ES cells to generate mouse models of brain development and dysfunction, the broad array of available alleles expressed in the nervous system, and advancements in iPS cell technology, in vitro experimentation that directly transfers from the culture dish to in vivo application is thus becoming much more feasible. Permutations of the experimentation we performed here using the described ROSA-tomRITVA ES cells hold promise to provide future insight into synaptic wiring mechanisms at work in the mammalian brain.