Neuroanatomy goes viral!

Abstract

The nervous system is complex not simply because of the enormous number of neurons it contains but by virtue of the specificity with which they are connected. Unraveling this specificity is the task of neuroanatomy. In this endeavor, neuroanatomists have traditionally exploited an impressive array of tools ranging from the Golgi method to electron microscopy. An ideal method for studying anatomy would label neurons that are interconnected, and, in addition, allow expression of foreign genes in these neurons. Fortuitously, nature has already partially developed such a method in the form of neurotropic viruses, which have evolved to deliver their genetic material between synaptically connected neurons while largely eluding glia and the immune system. While these characteristics make some of these viruses a threat to human health, simple modifications allow them to be used in controlled experimental settings, thus enabling neuroanatomists to trace multi-synaptic connections within and across brain regions. Wild-type neurotropic viruses, such as rabies and alpha-herpes virus, have already contributed greatly to our understanding of brain connectivity, and modern molecular techniques have enabled the construction of recombinant forms of these and other viruses. These newly engineered reagents are particularly useful, as they can target genetically defined populations of neurons, spread only one synapse to either inputs or outputs, and carry instructions by which the targeted neurons can be made to express exogenous proteins, such as calcium sensors or light-sensitive ion channels, that can be used to study neuronal function. In this review, we address these uniquely powerful features of the viruses already in the neuroanatomist's toolbox, as well as the aspects of their biology that currently limit their utility. Based on the latter, we consider strategies for improving viral tracing methods by reducing toxicity, improving control of transsynaptic spread, and extending the range of species that can be studied.

Keywords: expression vectors; neuroanatomy; neurotropic virus; review; transsynaptic tracing.

Figure 1

An example of a Cre-dependent tracing strategy. The first Cre-dependent tracing virus was a PRV that expressed a gene essential for virus replication in neurons, thymidine kinase (TK), in a Cre-dependent manner (DeFalco et al., 2001). A theoretical example is as follows. (1) The virus is first injected into a region of interest with cells expressing Cre. (2) Cre recombines the PRV genome so that it can express GFP and TK. The virus can then transsynaptically move from the starter cells to input neurons, such as retinal ganglion cells (RGCs) in the retina in this theoretical example. The virus can continue to transmit polysynaptically, as once Cre recombined the viral genome, it is permanently activated for transmission.

Figure 2

Design of the RABV monosynaptic technique. Expression of the avian tumor virus receptor A (TVA) dictates the types of cells that can be infected by a transsynaptic tracing virus that has a glycoprotein with the extracellular domain of a type A avian retrovirus (EnvA). A chimeric protein with the RABV-G intracellular domain fused to the EnvA extracellular domain (aka ASLV-A/RABV-G fusion, or A/G) is often used in place of the native EnvA. (1) The TVA and RABV-G genes are encoded as Cre-dependent genes in adeno-associated virus (AAV) vectors (blue virions) that are injected into an area with Cre-expressing neurons; (2) After Cre-recombination of the AAV encoded genes, a RABV pseudotyped with EnvA (EnvA shown as purple spikes on the virion surface) can infect the TVA-expressing cells; and (3) transsynaptic transmission of the RABV to synaptically-connected input neurons can occur due to the expression of the RABV-G gene (RABV-G shown as red spikes on the virion surface) in the Cre-expressing cells. This method enables RABV to transmit from an initially infected (“starter”) cell to a directly connected presynaptic cell, but no further.

Figure 3

Infection of TVA-expressing cells determines specificity of tracing. Expression of TVA can be arranged using transgenic mouse strains, by crossing a strain expressing Cre in a neuronal cell type of interest to a strain with a Cre-dependent TVA. In addition, one can include in this cross a Cre-dependent G protein (e.g., RABV-G) to enable the tracing virus to transmit, and/or a Cre-dependent fluorescent protein to label the cells with Cre history, i.e., the TVA-expressing cells. AAV vectors encoding such Cre-dependent genes can be used as an alternative to transgenic mouse strains (as shown in Figure 2). Four versions of tracing experiments using TVA are shown. (A) A transgenic mouse strain that expresses Cre, in e.g., in LGN neurons, will enable identification of the presynaptic partners of the Cre-expression neurons. In this example, the Cre strain is crossed to a strain with a Cre-dependent TVA gene (Beier et al., 2011a), (or, alternatively, an AAV is delivered in step 2 with the Cre-dependent TVA): (1) AAV encoding a Cre-dependent (FLEx) RABV-G is injected into the LGN of mice crossed to have TVA in LGN neurons that are postsynaptic to RGCs, approximately 2 weeks prior to the initiation of tracing. The AAV will uncoat, make a second DNA strand, then initiate expression of the RABV-G protein only in cells that have Cre. Depending upon the serotype of AAV, this process can take over 1 week; (2) A second injection is then made into the same area of the LGN with a tracer virus, RABV ΔG GFP (EnvA or A/G). RABV ΔG GFP will only infect cells with TVA, i.e., those with Cre; (3) When RABV ΔG GFP infects a cell that was previously infected with AAV, and which expresses Cre, RABV ΔG GFP will be transmitted to presynaptic RGCs. Infected RGCs will become GFP+, but will not be able to transmit the virus any further due to the lack of the G gene in the virus and in the RGCs. (B) GFP expression determines tracing specificity. The same strategy outlined in (A) can be used with a transgenic mouse line that expresses GFP in the LGN: (1) Infection of the LGN with two AAVs encoding Cre-DOG (Cre-dependent upon GFP) enables the GFP-expressing cells to become Cre-expressing cells (Tang et al., in press). Co-infection with an AAV with a Cre-dependent RABV-G and TVA allows the GFP-expressing cells to express RABV-G and TVA; (2) Infection with RABV ΔG mCherry (EnvA or A/G); and (3) transmission from the starter cells to presynaptic cells then proceeds as in scheme shown in (A). (C) TVA determines the starter cell type for anterograde monosynaptic tracing. The strategy outlined in A is carried out for anterograde tracing: (1) A mouse cross or AAV are used to determine the TVA-expressing starter cell type. The direction of transsynaptic transmission is set by the choice of G gene, in this case, VSV-G for anterograde tracing. If AAV is used, infection into the cortex is done 2 weeks prior to the initiation of a tracing experiment; (2) Infection of TVA-expressing cells by injection of VSV ΔG GFP (A/G) into the cortex; (3) VSV will replicate in the TVA-expressing cells and if VSV-G is present, will allow transmission to postsynaptic cells. VSV ΔG GFP will not spread from these postsynaptic cells due to the absence of a G gene in the virus and the postsynaptic cells. (D) TVA determines the specificity of retrograde infection from starter cells defined by projection site: (1) TVA expression is initiated in cells as shown in (A), but the Cre-dependent RABV-G gene is not used. Instead, one may use a Cre-dependent tdTomato allele to enable the identification of cells with a history of Cre expression (but this is optional). If AAV is used, infection into the LGN is done 2 weeks prior to the initiation of a tracing experiment; (2) A VSV encoding the A/G fusion protein in the genome, and RABV-G protein on the virion surface, is injected, here into the LGN. The RABV-G on the virion surface allows the VSV to travel retrogradely from the LGN injection site to neurons that project into the LGN, e.g., layer 6 pyramidal neurons in V1; and (3) VSV will replicate in the pyramidal neurons and transsynaptically transmit specifically to presynaptic TVA-expressing cells in the cortex. Transmission out of these TVA-expressing cells will only occur to other presynaptic cells that express TVA.

Figure 4

Viral strategies for delivering functional tools to neuronal populations defined by their connectivity. (A) Axon terminal targeting. The most successful approach to date relies on locally-infecting viruses, such as lentivirus and adeno-associated virus (AAV), which enable expression of a functional tool within neuronal cell bodies (dark green triangles) and dendrites local to the injection site (area A) and throughout their axons (green lines) that terminate both locally and in remote areas of the brain (areas B,C,D). Specific efferent pathways originating from the injection site are targeted by restricting stimulation or imaging (gray) to one particular target area (area C). (B) Cell-type specific axon terminal targeting. In some circuits, spatially-intermixed parallel pathways converge on the same target area of interest. In order to disentangle these pathways, the same locally-infecting viruses described in (A) can be engineered to target specific cell-types local to the injection site (area A). This can be accomplished with cell-type specific promoter sequences or recombinase-dependent expression sequences that restrict expression of a functional tool to a genetically-defined subpopulation (dark green triangles in area A). Specific efferent pathways originating from that particular subpopulation (green lines) and not other parallel pathways (black lines) are targeted as in (A) by restricting stimulation or imaging (gray) to one particular target area (area C). (C) Projection neuron targeting. Retrograde viruses, such as rabies, enable expression of a functional tool in specific projection neuron populations. Retrograde virus is taken up by axonal terminations (green lines) local to the injection site (area C) and travels back to cell bodies (dark green triangles) throughout the brain (areas A,B,D). Specific afferent pathways projecting to the injection site are targeted by restricting stimulation or imaging (gray) to one particular input area (area A). Retrograde viruses alone are often unable to achieve the high levels of expression needed for long-term functional studies. (D) Intersectional approaches. A combination of the strategies described in (A,C) above have enabled high levels of expression with low levels of toxicity in specific projection-neuron populations. The most successful strategy to date involves delivering small amounts of Cre-recombinase via a retrograde virus and also injecting a locally-infecting virus encoding Cre-dependent expression of a functional tool at the location where the targeted projection neurons originate. Specifically, retrograde virus is taken up by axonal terminations (green and black lines) local to the injection site (area C) and travels back to cell bodies throughout the brain (areas A,B,D). The retrograde virus expresses Cre-recombinase within these infected projection neurons. Specific afferent pathways (dark green triangles) projecting to the injection site are targeted by injecting a locally-infecting virus, such as AAV, encoding Cre-dependent expression of a functional tool in the target area of interest (area A). This strategy can functionally isolate neurons that reside in area A and project to area C, obviating the need for site-specific stimulation or imaging (gray).