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Review
. 2013 Jan 24:7:2.
doi: 10.3389/fncir.2013.00002. eCollection 2013.

Revealing the secrets of neuronal circuits with recombinant rabies virus technology

Melanie Ginger  1 , Matthias Haberl, Karl-Klaus Conzelmann, Martin K Schwarz, Andreas Frick
Affiliations
Review

Revealing the secrets of neuronal circuits with recombinant rabies virus technology

Melanie Ginger et al. Front Neural Circuits. .

Abstract

An understanding of how the brain processes information requires knowledge of the architecture of its underlying neuronal circuits, as well as insights into the relationship between architecture and physiological function. A range of sophisticated tools is needed to acquire this knowledge, and recombinant rabies virus (RABV) is becoming an increasingly important part of this essential toolbox. RABV has been recognized for years for its properties as a synapse-specific trans-neuronal tracer. A novel genetically modified variant now enables the investigation of specific monosynaptic connections. This technology, in combination with other genetic, physiological, optical, and computational tools, has enormous potential for the visualization of neuronal circuits, and for monitoring and manipulating their activity. Here we will summarize the latest developments in this fast moving field and provide a perspective for the use of this technology for the dissection of neuronal circuit structure and function in the normal and diseased brain.

Keywords: connectivity; monosynaptic; neuronal tracer; synapses; wiring diagram.

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Figures

Figure 1
Figure 1
RABV—the trans-synaptic tracing toolbox. (A) The RABV genome encodes five proteins including the envelope glycoprotein (G). (B) Deletion of G (ΔG) is sufficient to prevent trans-synaptic spreading and results in a single-cycle vector. A gene of interest can then be inserted in the place of the deleted G. This gene of interest can encode a fluorescent protein such as GFP, permitting the visualization of virally traced neurons. It can also permit the expression of an almost unlimited choice of genetically encoded tools for the manipulation/visualization of neuronal circuits, e.g., biosensors, synapse markers, activators/repressors of neuronal activity. (C) RABV ΔG can be pseudotyped, either with its native glycoprotein or an engineered surface protein (e.g., EnvA). Pseudotyping restores the normal infection capabilities or enables cell-type selective infection, respectively. (D) RABV ΔG coated with its native glycoprotein is taken up by axon terminals and can be used to trace neurons in a retrograde manner. (E) EnvA pseudotyped RABV ΔG [RABV ΔG(EnvA)] only infects neurons expressing the TVA receptor. Trans-complementation with RG enables RABV ΔG to cross one synaptic step to infect the presynaptic partners of a defined neuron or neuronal population. The virus is then trapped in these presynaptic partner cells and cannot spread further due to the deficiency of RG in these presynaptic cells, hence limiting the strategy to a mono-trans-synaptic event. Initially infected source cells are identified by the expression of an additional fluorescent marker.
Figure 2
Figure 2
Strategies to reveal the secrets of defined neural circuits. (A,B) There are several strategies to target the rabies infection to individual neurons or specific cell types. (A) Genetic strategies such as the Cre-lox system take advantage of cell-specific promoters to restrict Cre expression to a defined cell-type. Injection of a Cre-dependent helper virus expressing RG and TVA permits RABV ΔG(EnvA) infection, and subsequent mono-trans-synaptic tracing, to be limited to a specific source cell population. (B) Patch pipettes can be used to infect individual neurons with the plasmids encoding RG and specific surface proteins (e.g., TVA). These neurons are subsequently infected with pseudotyped (e.g., EnvA) RABV ΔG. This allows the tracing of the presynaptic partners of specific neurons. (C–E) RABV ΔG mediated expression of a number of genetic tools permit the dissection of structure/function of specific networks in a temporally and spatially controlled manner. (C) RABV ΔG mediated expression of recombinases (Cre/FLP) permits loss- or gain-of-function studies through conditional expression of specific genes in the infected network. (D) Population activity of specific neuronal networks can be monitored, for instance, by RABV ΔG directed expression of calcium indicators. (E) The activity of the starter cells, and their presynaptic network can be very specifically controlled by the expression of either allatostatin receptor or light-activated ion channels. Binding of allatostatin to its cognate receptor or activation of these channels by light of a specific wavelength leads to specific inhibition/activation of RABV ΔG infected neurons.
Figure 3
Figure 3
Gaining insight into brain function by the dissection of neuronal circuits. Recent advances in RABV ΔG technology provide invaluable tools for gaining a better understanding of the function and structure of neuronal circuits. (A) For instance, the re-organization of neuronal circuits resulting from plasticity or disease can be revealed by comparing the wiring diagram of experimental and control brains. (B) We can learn more about plasticity in the adult brain by studying the integration of adult-born neurons into existing neuronal networks. Similar approaches might be used to study circuit integration following stem cell therapy. (C) The analysis of the physiological properties of synaptic connections between defined neuron types (in addition to the anatomical wiring diagram) is greatly aided by this technique. Furthermore, the spatial and temporal integration of signals from a larger number of presynaptic neurons can be examined in postsynaptic neurons. (D) The population activity of a defined neuronal circuit can also be measured during active sensory stimulation or behavior and subsequently correlated with the anatomical input network of a defined starter population. (E) The receptive field properties of individual neurons can be combined with the analysis of the anatomical wiring diagram of their presynaptic network.
Figure 4
Figure 4
RABV ΔG virus technology—what is possible? Here we provide an overview of the numerous tools that could, in the future, be developed using RABV ΔG vectors. Such possibilities include the expression of genetically encoded tools to aid imaging/microscopy approaches and tools to examine loss-of-function (knockdown) or gain-of-function of a gene/protein of interest. Other possibilities involve retargeting using engineered envelope proteins or the possibility of transfection using naked ribonucleoprotein (RNP) complexes. Some approaches are not possible with RABV ΔG vectors including the integration of lox P sites, cell specific promoters or tet-regulatory sequences, or expression of shRNA. RNP, Ribonucleoprotein, core of the RABV; EM, Electron microscopy; PALM, Photo-activated localization microscopy; VSD, Voltage-sensitive dye; miRNA, micro RNA; shRNA, short hairpin RNA.
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