Due to the complexity of the nervous system, it is extremely valuable to be able to trace connections to or from particular cell types. To some degree, neurotropic viruses make this possible simply by virtue of the fact that they can spread across multiple synaptic steps. For example, if a virus that spreads retrogradely is injected into area A, the initial infection will be restricted to cells in area A and to cell types in other areas, say area B, that project axons to area A. As the virus propagates, it might be possible to infer that subsequent spread within area B or to areas that provide input to area B, results specifically from connections to the area B cell type which projects to area A. (See, for example [31
].) Nevertheless, this situation is far from ideal and there are many caveats that require caution in making such interpretations. Most notable among these are that most brain areas are connected with multiple structures, making it increasingly difficult to determine the exact route of spread as virus traverses increasing numbers of synaptic steps [32
]. And even when the number of synaptic steps is small, the rate of viral spread depends on the strength of connections [21
]. As Ugolini notes [21
]: “The speed of transfer of rabies virus to these cell groups was strongly correlated with the strength of their input
… with rabies, as with HSV-1 [33
]…different groups of second-order neurons were labelled at different times. This generated an overlap … between the delayed labelling of the last groups of second-order neurons and the onset of labelling of higher order neurons…”.
The utility of neurotropic viruses could therefore be greatly improved by controlling what cell types are initially infected or are permissive for viral replication, and by controlling the number of synaptic steps that are crossed. Effective genetic modifications for achieving these goals have been applied to both PRV Bartha and to the SAD-B19 strain of rabies virus.
To trace connections to specific cell types, De Falco et al. [15
] developed and used a modified form of PRV Bartha, called Ba2001, which is unable to replicate unless its genome is recombined by cre-recombinase. This strategy utilizes the fact that viral thymidine kinase (TK) is required for viral replication and also takes advantage of the fact that there are mouse lines which express cre-recombinase only in specific cell types. Combining these tools allows tracing of the inputs to cre-expressing cells. In particular, the thymidine kinase (TK) gene was deleted from the PRV genome and replaced with a “floxed stop” sequence followed by coding sequences for both TK and green fluorescent protein (GFP). Thus, in Ba2001, cre-recombinase will excise the floxed stop and allow TK plus GFP expression only in Cre-expressing cells. When Ba2001 is injected into a brain area, it infects all cell types. But only the cell types expressing cre, recombine the viral genome, allowing the subsequent GFP expression to mark the recombined virus and TK expression to allow replication. Recombined viral particles can then spread transneuronally, and the infected neurons will also be marked by GFP and replicate recombined virus for further retrograde spread. The spread of the GFP-expressing virus can therefore be used to trace direct and indirect inputs to a specific cell type [15
To trace connections to specific cell types with rabies virus, it is not possible to use a strategy based on cre-recombination. This is because the RNA genome of rabies cannot be recombined with cre and there is no DNA phase in the viral life cycle. Therefore, Wickersham et al. [35
] developed a strategy in which the initial viral infection is restricted to particular cells. This was done by deleting the RG gene from the SAD-B19 rabies genome, replacing it with GFP, and then producing viral particles with a different envelope protein in their envelope (pseudotyping). The RG-deleted virus was pseudotyped with an avian virus envelope protein, called EnvA; this new virus is called EnvA-SADdG-GFP. When this virus is injected into the brain of a normal animal, it does not infect any neurons because the mammalian brain has no receptors for EnvA. But by misexpressing the EnvA receptor, TVA, in particular cells, it was possible to selectively infect those cells.
Because RG is required for formation of new viral particles and transsynaptic spread [36
], and SAD-dG-GFP has no coding sequence for RG, RG expression is also required to allow viral spread from the targeted neurons. When neurons expressed TVA and not RG, infection with EnvA-SADdG-GFP (and GFP expression from the rabies genome) was restricted to TVA cells and did not spread from those cells [35
]. When neurons expressed both TVA and RG, the initial infection was restricted to TVA cells, and RG expression in those cells allowed transsynaptic spread to, and GFP labeling of additional neurons that were directly presynaptic to the TVA cells. Continued spread beyond the directly presynaptic neurons did not occur, because the presynaptic cells do not express RG and there is no RG coding sequence carried in the viral genome. Thus, this system has the additional advantage that rabies spread is monosynaptically-restricted, eliminating any confound between strength of connections and numbers of synapses crossed; this eliminates any ambiguity about numbers of synaptic steps that have been crossed. This method can be used in combination with, for example, cre- or Tta-expressing mouse lines to obtain cell type specific expression of TVA and RG (cf. [4
]). Subsequently, the targeted cell type can be specifically infected with EnvA-SADdG-GFP and direct inputs labeled by transsynaptic spread of the virus and GFP expression.
In view of the fact that neighboring neurons of the same type often connect with different presynaptic neurons (e.g. [38
]), it is highly desirable to have methods that can identify the connections to single neurons. Experimental evidence indicates that this is feasible with the EnvA-SADdG-GFP rabies virus described above [35
]. One needs to express TVA and RG in a single neuron and then infect that neuron selectively with EnvA-SADdG-GFP. Over the course of several days, the GFP expressing virus spreads to and labels neurons that are directly presynaptic to the single parent neuron. This method is now being combined with single cell electroporation [40
] or other strategies for obtaining sparse cell infection, in order to trace connections to single neurons in vivo.
In contrast to EnvA-SADdG-GFP, the Ba2001 system does
allow continued transneuronal spread across multiple synaptic steps [15
]. Although this can be advantageous, it has the disadvantage of possible ambiguity in interpretation of results [21
] (see above). An alternative that has been tested in the author’s lab, but not published, is to use the PRV virus Ba2000, which was described and used as a control by De Falco et al. [15
]. Unlike Ba2001, Ba2000 has the TK gene deleted from its genome and cannot express TK under any circumstances. But Ba2000 does express GFP following cre-recombination, as do any viral progeny that are produced from the recombined genome. To use this virus for monosynaptically-restricted spread from cre-expressing neurons, it is necessary to simply provide a means for TK to be expressed selectively in the cre-expressing neurons. Under these circumstances, GFP-expressing virus is replicated only in cre-expressing cells and it can then spread retrogradely to neurons providing direct input to those cells. These directly presynaptic neurons can express GFP from the viral genome, thus marking them, but the virus cannot replicate and spread any further.