A retrovirus is an enveloped, single-stranded RNA virus. Its most salient feature is that it provides for stable, long-term gene expression by virtue of its ability to stably integrate its DNA genome (created from its RNA genome upon entry into a host cell) into a host cell’s genome, and express virally-encoded genes from a wide range of integration sites (Knipe and Howley, 2013). This ability to integrate distinguishes it from other commonly used vectors, such as AAV. Target cells that are dividing gradually lose viral genomes that are not integrated, due to dilution, and thus any application that requires retention of expression over a number of cell cycles must use a virus that integrates. An example of such an application for neuroscientists might be the introduction of a retrovirus encoding a viral receptor, e.g., avian tumor virus receptor A (TVA), into a progenitor zone early in development. Integration would allow expansion of the number of cells with the integrated TVA gene, so that there would be a large number of TVA-expressing cells at some later point, to facilitate targeted infection using tracing viruses that contain the cognate glycoprotein, EnvA (as discussed in “Use of Monosynaptic and Transsynaptic Tracing in vivo” Section).

The two types of retroviruses commonly used as viral vectors are the gamma-retroviruses and lentiviruses. The critical distinction between these two types of retroviruses concerns their ability to enter a host cell’s nucleus in order to integrate the viral DNA. Viral gene expression requires that the genome be integrated, so this step is essential to the use of these viruses as vectors. Gamma-retroviruses are unable to gain entry to the nucleus through an intact nuclear envelope, and thus need a cell to undergo a nuclear envelope breakdown during M phase in order to access a host cell’s genome. In contrast, lentiviral DNA can enter the nucleus without nuclear envelope breakdown, and thus can integrate in a postmitotic cell. This feature makes lentiviruses particularly useful for gene transfer into neurons (Naldini et al., 1996). Both types of retroviruses have been modified to render them unable to replicate after infection, i.e., they are replication-incompetent. Lack of replication makes these vectors safe for use in the laboratory (Schambach et al., 2013), and similarly does not lead to disease in animals, with some rare exceptions due to alterations in the host genome due to viral integration (Hacein-Bey-Abina et al., 2003).

Genome size is an important consideration when choosing a vector for a particular application. Lentiviruses and gamma-retroviruses can package a genome of approximately 10 Kb (Kumar et al., 2001), which allows for expression of more than one gene, as well as use of more than one promoter or regulatory element. In contrast, AAV can only package approximately 4.8 Kb. Retroviruses can express cDNAs from the viral long terminal repeat (LTR) promoter, or they can be engineered to use an internal, non-retroviral promoter, e.g., for tissue specificity (Montiel-Equihua et al., 2012). In the latter case, use of a viral genome rigged to lose the activity of the viral LTR appears to give a greater probability of correct regulation of an internal promoter (Miyoshi et al., 1998; Ginn et al., 2003). However, even with such crippled LTRs, internal promoters are not always properly regulated, and the rules that govern regulation are not known. Retroviral vectors can be engineered to express small-hairpin RNAs (shRNAs) from Pol III promoters (Paddison et al., 2004; Harpavat and Cepko, 2006), and can use multiple promoters in the same genome. For example, a lentivirus was designed to use the Pol III promoters, H1 and U6, to express multiple shRNAs targeting complexin-1 and complexin-2. The same virus used the Pol II promoter, derived from the human ubiquitin-C gene, to express an shRNA-resistant form of complexin-1 (Ahmad et al., 2012).

The choice of the type of retrovirus to use is typically driven by whether the target cell type is mitotic or postmitotic. Gamma-retroviruses have been used to trace lineages in the nervous system, as they will only integrate (and thus express) in mitotic cells, which is exactly what one needs for lineage tracing (Turner and Cepko, 1987). As mentioned previously, lentiviruses are used when one wants to deliver genes to neurons. Both types of retroviruses have been used for gene therapy in humans, due to their stable integration, which provides for long-term gene expression, and again, the choice of which type to use takes into consideration whether the target cell is actively dividing (Wiznerowicz and Trono, 2005; Hacein-Bey-Abina et al., 2014).

Another issue to consider in choosing a viral vector is the percentage of cells at an injection site that need to be infected for the experimental outcome. A limitation of retroviruses is that they do not grow to as high a titer (typically in the range of 10⁸–10⁹ colony forming units per mL, cfu/mL) as AAVs or adenovirus vectors, which can be several log₁₀ units higher. The expression level should also be taken into consideration. In part, due to the fact that there is usually only a single viral genome in an infected cell, the level of expression from retroviruses tends to be modest (Wickersham et al., 2007a), though a recently developed lentiviral vector with a tetracycline regulatory element and activator may overcome this problem for some applications (Cetin and Callaway, 2014). Relatively low expression is a disadvantage of using these vectors, as not all neuronal processes are well labeled.

The genome structure of retroviruses is fairly flexible, allowing for the design of vectors using different types of promoters and cargoes. In addition, retroviruses, which have an RNA genome packaged in their capsids, create a DNA copy of themselves after infection, which is integrated into the host genome. This means that the integrated viral genome can be modified by Cre- or Flp-mediated recombination (Gonçalves et al., 2010; Sommer et al., 2010), and allows the use of cellular promoters, neither of which is possible when using RNA viruses. The virion particles also can be modified to suit certain applications. Retrovirus particles have a lipid envelope, and can accept the envelope glycoprotein from other viruses, or pseudotyped with, for example, the avian EnvA glycoprotein that targets the infection to cells that express the avian TVA receptor (Bates et al., 1993; Holland et al., 1998). This approach has been used to target infection for lineage studies (Beier et al., 2011a; Hafler et al., 2012) as well as transsynaptic tracing studies using RABV (Wickersham et al., 2007b) and vesicular stomatitis virus (VSV; Beier et al., 2011b).

AAV is a non-enveloped, single-stranded, small DNA virus of the Parvovirus family (Knipe and Howley, 2013) that has proven effective for gene transfer in the CNS (for a recent review of AAV in the CNS, see Murlidharan et al., 2014). AAV can be used for long-term, stable gene expression in neurons, with little or no toxicity (Kaspar et al., 2002). Its other notable feature is that it grows to very high titers, which can allow the simultaneous infection of cells with more than one AAV vector. There are hundreds of naturally occurring “species” of AAV (Gao et al., 2004), referred to as serotypes, which can be exploited for infection of different cell types. In addition to differences in capsid proteins, the different serotypes have different sequences in their inverted terminal repeats (ITRs), sequences required for replication of the viral genome, as well as differences in the viral replicases that recognize these sequences. The ITRs from serotype 2 have been the most extensively employed in AAV vectors. The ITR serotype is the number that is listed first in the name of an AAV vector, while the capsid type is the second number, e.g., AAV2/8 has the ITRs from AAV 2 and the capsid proteins from AAV 8 (Gao et al., 2005). Serotypes vary in terms of the speed of onset of viral gene expression, with some of the serotypes being particularly slow, needing up to several weeks for the maximum number of cells to show evidence of infection (Vandenberghe et al., 2013). Some of the reasons for this remain unknown, but uncoating and the need to replicate a second strand of DNA (as the virus only packages a single DNA strand) contribute to the lag time. AAV vectors with a genome that is self-complementary, and thus are double-stranded in the capsid, are much faster in terms of onset of expression as they do not need to produce a second DNA strand before initiating gene expression (McCarty et al., 2001; Gray et al., 2011). However, the packaging size of these vectors is very small, about half that of conventional vectors.

Another feature of a serotype to consider is the target cell specificity (e.g., Burger et al., 2004; Cearley and Wolfe, 2006; Taymans et al., 2007; Zincarelli et al., 2008; Aschauer et al., 2013), whether the capsid is transported away from the inoculation site and, if transported, whether in the retrograde (Taymans et al., 2007; Hollis et al., 2008; McFarland et al., 2009; Towne et al., 2010; Masamizu et al., 2011; Aschauer et al., 2013; Castle et al., 2014) or/and anterograde (Castle et al., 2014) directions. AAVs can also access the CNS and PNS from intravascular perfusion, with different efficacies and tropisms depending upon the capsid (Zhang et al., 2011). Toxicity also can vary with the serotype (Howard et al., 2008), or even with a given virus preparation. Toxicity can be due to a high level of expression of the encoded gene, and/or acute inflammation, though this is typically quite low for AAV (McPhee et al., 2006; Zhang et al., 2011; Sondhi et al., 2012), especially relative to adenovirus (Seiler et al., 2007). T-cell mediated toxicity for AAV-infected cells has been seen in humans due to memory T cells (Mingozzi and High, 2013), but has not been reported for rodents. In addition, there may be toxic elements in a virus preparation that do not elicit toxicity through immune system interactions. For example, we have found that different preparations of the same genome in the same capsid type can vary in toxicity, and others have reported differences in toxicity among serotypes for cells cultured from different areas of the CNS (Howard et al., 2008). Acute toxicity in certain preparations may be due to the capsid itself, or to something that is tightly complexed with the capsid, as we have been unable to eliminate toxicity through multiple purification methods. However, dilution of a stock has proven an effective approach to minimizing toxicity.

In general, AAVs have proven to be very useful for long-term, relatively high-level gene expression in the mammalian CNS. In addition, as with retroviruses, their genome is double-stranded DNA after they infect and convert their single stranded packaged genome into a double strand. Their genomes can thus be modified by recombinase technology, with the tightest Cre-dependent vectors using two pairs of loxP sites, called Flip-excision (FLEx) vectors (Schnütgen et al., 2003). Multiple Cre-dependent vectors can be used to simultaneously infect the same cells, but there are some interesting caveats that must be considered when designing such vectors, due to presumed interactions between AAV-encoded loxP sites on two or more vectors. For similar reasons, there appear to be interactions between AAV-encoded loxP sites and genomic loxP sites (Saunders et al., 2012). Cre-dependent AAV vectors have been exploited for transsynaptic tracing protocols and for targeting optogenetic protein expression to specific types of neurons that express Cre (Atasoy et al., 2008; see also “Viruses Engineered to Carry Functional Genes” Section). The popularity of AAV vectors with neuroscientists also derives from the fact that they are straightforward to engineer, and there are multiple companies and core facilities that make virus constructs as well as virus preparations.

AAVs can be produced as high titer stocks [10¹²–10¹³ genome copies per mL (gc/mL) for AAV]. Note that AAV titers are usually given in gc/mL, and retroviral titers in infectious units (e.g., cfu/mL). AAV stocks using different capsid types, and different preparations of a given capsid type (Auricchio et al., 2001), can vary in their ratio of capsids to infectious capsids, also referred to as the particle to infectious particle ratio (p/pfu), or infectivity. These ratios range from 10–1000:1 (Auricchio et al., 2001; Vandenberghe, personal communication) and will affect the percentage of cells infected at an injection site. Due to the fact that it is not always trivial to measure infectious particle activity for AAVs, the p/pfu ratio is usually not measured but is one reason why stocks vary in their infectivity. The variables of toxicity and infectivity argue for pilot experiments with each new stock before investing a great deal of time in its use.

Although the naturally occurring AAVs integrate their genome into the host chromosome, at a specific locus on human chromosome 19, the deletion of the rep genes from the vector form of AAV has led to the loss of such targeted integration (McCarty et al., 2004). Some random integration occurs at a low level, but it is not clear if such integrated genomes express viral genes. Most AAV genomes exist as non-integrated episomes, either as single viral genomes or as concatamers (Yang et al., 1999; Nakai et al., 2000). The reason(s) that these genomes remain stably associated with the host cell and persist in expression has not been determined. The lack of consistent integration makes AAV vectors a poor choice if the target cells are dividing and retention of viral gene expression through several cell cycles is required. As noted above, in such cases, retroviruses are a better choice.

The natural promoters of AAV have been replaced with promoters from other viruses, e.g., cytomegalovirus (CMV; Boshart et al., 1985), or host cells, e.g., EF1α (Fitzsimons et al., 2002). Other regulatory elements that can be included are introns, to provide for splicing, polyA sites, and a regulatory element known as the woodchuck post-transcriptional regulatory element (WPRE), which increases the cytoplasmic level of virus-encoded mRNAs (Donello et al., 1998; Zufferey et al., 1999). As with all viral vectors, the exact sequences and their arrangement in the vector can have effects on the level of expression as well as the specificity of expression. As an example, we have found that different intron sequences can have a major effect on the level of expression in the retinal pigmented epithelium (Xiong et al., Unpublished observations). Due to these variables, one should assay for expression of an easily scored gene, such as GFP, using a vector that has all of the regulatory elements and the capsid serotype that one wishes to use in future experiments. In addition, such tests should be performed on animals that are of the age that will be used for future experiments, as we have found that the age of the tissue at the time of inoculation can affect the types of cells infected (Xiong and Cepko, 2014). One can obtain a series of relatively inexpensive, small aliquots of different serotypes from companies to test for the optimum vector characteristics.

The major drawback of AAV vectors is their limited packaging capacity. AAVs can only incorporate about 4.8 Kb of DNA (Dong et al., 2010), which limits the expression to one or two small genes, and also limits the use of different promoter/enhancer elements. There have been several approaches to overcome this limitation. For example, there are trans-splicing vectors where two genomes with different exons of a large gene are co-injected, and result is expression of a large protein, but only if there is a high rate of co-infection (Wu et al., 2010). However, generally if a larger genome is desired, another vector, such as an adenovirus or lentivirus, should be considered.

An adenovirus is a non-enveloped, double-stranded DNA virus, with a larger genome and capsid than AAV, but not as large as those of the HSVs (Knipe and Howley, 2013). Human adenovirus type 5 (Ad5) has been developed for use for gene therapy and most commonly used adenovirus vectors are derived from Ad5 (Gonçalves and de Vries, 2006). The newest generation of adenoviruses (helper-dependent adenovirus or HDV) have had all of their genes removed, both to make the virus less susceptible to immune system modulation and to create space for transcriptional regulatory elements and large or multiple genes (Parks et al., 1996; Ehrhardt and Kay, 2005). These HDVs have a capacity of 35–36 Kb. Adenovirus vectors are not quite as straightforward to genetically engineer or produce (Palmer and Ng, 2003) relative to retroviruses and AAVs. In addition, they remain more pro-inflammatory than retroviruses or AAV (Seiler et al., 2007). As with AAVs, they do not integrate and thus are not used for applications where the target cells are mitotic. Also, as with AAVs, they can be grown to high titer, but unlike AAVs, they express at a high level within a few days of infection (Akli et al., 1993). Adenovirus vectors have been directly compared to AAVs and lentiviruses for gene transfer in the CNS, and have not fared well in these comparisons when the time course has gone beyond a few days (e.g., Doherty et al., 2011). Still, transient and immediate expression of a gene, such as Cre, using adenovirus vectors can be useful and high titer stocks that express Cre can be purchased from several vendors.

The canine adenoviruses (CAV) are employed for their potent retrograde transport capabilities (Kremer et al., 2000; Soudais et al., 2001; Kissa et al., 2002; Peltékian et al., 2002; Hnasko et al., 2006). In addition, these vectors have been used to express Cre, and a few other genes, e.g., GFP (Kremer et al., 2000). Cre has been used to turn on the expression of floxed endogenous genes (Hnasko et al., 2006), including floxed genes used to enable monosynaptic tracing by RABV (Pivetta et al., 2014). These vectors may become increasingly useful when a DNA virus is required, as the packaging limit of AAVs often limits the experiments that one can do with them. As adenoviruses have double-stranded DNA genomes, they are amenable to Cre or Flp-mediated recombination.

The alphaviruses, semliki forest virus (SFV) and sindbis virus (SIN), are enveloped, plus-strand single-stranded RNA viruses. They can be engineered to be replication-incompetent, and typically grow to as high a titer as retroviruses (in the range of 10⁸–10⁹ cfu/mL). However, unlike retroviruses, multiple viral genes are still expressed from these vectors, resulting in rapid gene expression, but also rapid toxicity.

They have been used in cases where this rapid, high expression is desirable (Bredenbeek et al., 1993; de Hoop et al., 1994; Gwag et al., 1998; Ehrengruber et al., 1999). For example, they have been used for robust labeling of a small number of axons of neurons near the injection site in the brain (Ghosh et al., 2011; Kuramoto et al., 2015), as well as for delivering genes to neurons (including a fluorescent tag for visualization) for single-cell electrophysiological experiments (Kopec et al., 2007; Malinow et al., 2010), among others. However, rapid toxicity limits the use of these vectors. The fast expression and toxicity of these alphaviruses make them similar to VSV, though the transgene expression from VSV is even quicker, occurring within hours (van den Pol et al., 2009; Beier et al., 2011b, 2013b), as discussed further below.

HSVs are large, enveloped, double-stranded DNA viruses which are classified into three subfamilies (Davison, 2010). PRV and HSV-1 are members of the subfamily, alphaherpesviruses, and are those used for circuitry tracing. Another subfamily is the betaherpesviruses, which includes CMV, one strain of which is the source of the popular human CMV immediate early enhancer/promoter used extensively in expression vectors (Boshart et al., 1985). The herpesviruses were the first to be extensively employed to trace neuronal circuitry. Though some of the early reports of herpesviruses traveling along nerves were made in the 1920s (Goodpasture and Teague, 1923), the first applications of this type of virus for tracing did not begin until the late 1980s (Ugolini et al., 1989). In these early studies, the virus was injected into peripheral locations, and transmission to specific areas in the brain known to project to the spinal cord was observed. This permitted the mapping of brain projections onto these peripheral sites. Later studies also examined the transmission patterns of different strains of HSVs, such as PRV, in the brain (Card et al., 1990; Strack and Loewy, 1990). All of these initial studies used viruses that were replication-competent and thus could spread across many synapses. The fact that HSV’s can spread raises a question of safety for laboratory workers. As PRV does not infect primates from peripheral sites of infection, this is not a major concern, though the virus can replicate, and kill neurons, when cells of the CNS are exposed to the virus, by direct injection (Hurst, 1933) or nasal instillation (Baskerville and Lloyd, 1977). HSV-1, in contrast, is a natural pathogen of humans, giving rise to genital and oral lesions (Lafferty et al., 2000). In addition, these viruses are toxic to cells, as is true with the other viral transsynaptic tracers.

Specific strains of the virus are able to transmit only in the retrograde direction, as in the case of the PRV Bartha strain (Card et al., 1992; Levine et al., 1994; Moore et al., 1995). Other strains, such as the HSV-1 strain H129 (Zemanick et al., 1991; Sun et al., 1996), appear to transmit transsynaptically only in the anterograde direction. The retrograde specificity of the Bartha strain is likely due to a large deletion in the viral genome, which eliminates the expression of three viral genes (Lomniczi et al., 1987), while the anterograde specificity of the HSV H129 is not understood (Szpara et al., 2010). The mechanisms of axonal transport, both retrograde and anterograde, have been studied, and while there is still much to be learned, the process is better understood than for the rhabdoviruses, RABV and VSV. However, for a good review of how difficult it is to clearly define these mechanisms, see Kratchmarov et al. (2012).

HSVs are DNA viruses, and thus their genomes can be engineered using standard recombinant DNA methods. However, as these viral genomes are very large, about 150 Kb, and have many regulatory elements and genes, even minor genome alterations can cripple the virus’s ability to replicate. Since at least one cycle of replication is required for a virus to transmit across a synapse, any modification that affects viral fitness can greatly reduce the utility of a construct. Some modifications have been made to enable the use of these viruses as tracers. Fluorescent protein genes have been successfully inserted, and there are versions which include the Brainbow or rainbow cassettes of fluorescent proteins (Boldogkoi et al., 2009; Kobiler et al., 2010). In addition, there is a version that can be used as a calcium sensor, and through the use of both GFP and DsRed, there is a version which can be used to indicate the timing of arrival of the virus in a circuit, as well as the health of infected cells (Boldogkoi et al., 2009). PRV and H129 also have been rendered dependent upon Cre expression for replication, allowing one to use Cre expression patterns in transgenic mice to dictate tracing of a particular circuit (DeFalco et al., 2001; Lo and Anderson, 2011; Figure 1), or to express a variety of transgenes which can aid in circuit mapping (Boldogkoi et al., 2009; Kobiler et al., 2010). As mentioned above, PRV and HSV-1 are toxic to cells, and have been suggested to have the ability to alter neuronal connectivity (McCarthy et al., 2009). However, a variant of PRV was recently generated that lacks the master transcriptional regulator of endogenous PRV genes, and can therefore express transgenes without replicating or apparent toxicity (Oyibo et al., 2014), auguring an expanded use of these vectors. In terms of speed, PRV has been seen to label infected tissue culture cells in 6 h (Kobiler et al., 2010), and initially infected neurons in vivo in just over 1 day post-infection, while the first instances of spread in vivo were seen at around 2 days post-infection (e.g., Card et al., 1990). As with all transsynaptic tracer viruses, the kinetics of expression are slower in variants that reduce the fitness of the virus. In addition, the kinetics of labeling of circuits are determined by such variables as the distance of the injection site to the cell bodies of the initial target cells, and the distances from the initial sites of replication to the cell bodies of the secondarily infected cells. Titers of these HSV vectors are typically 10⁷–10⁹ cfu/ml, and the p/pfu is variable and can be up to 40,000 (Flint, 2000) perhaps due to the fact that the virion particle is large and complex, leading to failures in assembly and/or uncoating.

In addition to its utility as a transsynaptic tracing vector, HSV-1 has been engineered to be a replication-incompetent retrograde tracer, and to be an effective vector for other types of gene transfer into neurons (Spaete and Frenkel, 1982; Krisky et al., 1998; Samaniego et al., 1998; Lilley et al., 2001). These replication-incompetent vectors are not toxic to cells, and can be made into forms that express for different lengths of time, including several months. Given their large size, and certain aspects of vector design, they can be engineered to express at high levels, to express multiple genes, and to use large and/or multiple regulatory elements. They are somewhat more difficult to produce than some of the other vectors discussed here, but there are core facilities that can produce them.

Rhabdoviruses are enveloped, single-stranded RNA viruses, with two members, RABV and VSV, serving as transsynaptic tracers. RABV and VSV each encode five genes, with a genome of approximately 12 Kb. Their capsids are flexible enough to allow packaging of genomes both significantly smaller (Epstein et al., 1980; Timm et al., 2014), or larger (McGettigan et al., 2003), than the native genome. The genes of RABV and VSV are expressed at high levels, permitting robust gene expression and visualization shortly after infection. RABV and VSV have a simple genome organization optimized for the levels of expression of each gene, with a gradient of expression from the 3′-most gene (highest) to the 5′-most (lowest). RABV is typically engineered to encode non-viral genes at the G locus, which is in the fourth position. This means that the expression level of such a gene (Osakada et al., 2011) is less rapid and robust than that of a gene in the first (most 3′) position (van den Pol et al., 2009; Beier et al., 2011b, 2013b; Osakada et al., 2011). However, vectors can be made in which the fluorophore is placed at the 3′-most end, which results in more robust expression (Wickersham et al., 2013).

Due to fact that the genome is RNA, and rhabdoviruses do not have a DNA phase in their lifecycle, it has been challenging to produce infectious virus from engineered plasmids that represent the genomes (Whelan et al., 1995; Mebatsion et al., 1996). Nonetheless, methods have been worked out, and after a virus preparation is made from engineered plasmids, they are then straightforward to propagate by simply infecting host cells in vitro (Osakada et al., 2011). It is worth noting that the lack of a DNA phase in their replication also means that one cannot use DNA recombinases, such as Cre, to recombine their genomes. A summary of the unique features of RABV and VSV as transsynaptic tracers is given below.

RABV, like HSV, can infect neurons from the axon terminals and be retrogradely trafficked to the cell body, as well as transmit among neurons transsynaptically in the retrograde direction (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2000). Its use has been more limited than that of HSV, however, due to the biosafety concerns of using RABV, as it is often lethal upon human infection. However, it has been very useful for retrograde transsynaptic tracing in non-human primates (Nassi and Callaway, 2006; Nassi et al., 2006; Rathelot and Strick, 2006, 2009).

The first studies linking RABV to transmission from synaptic junctions occurred in the 1970s and 1980s (Iwasaki et al., 1975; Charlton and Casey, 1979; Tsiang et al., 1983). This was followed by studies in which the time-course of RABV infection was monitored in order to construct circuit diagrams. In one such early study, the tongue muscle was injected with RABV followed by survival times of 1–4 days in different animals (Ugolini, 1995). As expected, after a single day, only first-order hypoglossal motor neurons were labeled. Between 2 and 3 days post-injection, second-order neurons in various brainstem nuclei were identified, while 4-day survival times resulted in the labeling of putatively third-order neurons, including forebrain nuclei. This approach permitted the identification of a chain of connections based on the timing of transsynaptic transmission of RABV. Similar studies using transsynaptic transport of RABV (CVS-11 strain) have revealed connectivity in the monkey visual system (Kelly and Strick, 2000; Nassi and Callaway, 2006; Nassi et al., 2006).

However, the biosafety concerns inherent in using a replication-competent RABV made the use of this virus restricted to a small number of labs that were proficient in its handling and use. A major advance occurred in 2000, when RABV was engineered to become replication-conditional by removal of the G gene from the genome (Etessami et al., 2000). This virus could be used in the laboratory to robustly label neurons projecting to a defined site (Nassi and Callaway, 2007; Wickersham et al., 2007a). In addition, because the virus could not spread, it became more accessible to neurobiology labs who were less familiar with viruses.

The next major advance was the use of this replication-conditional virus for monosynaptic retrograde transmission (Wickersham et al., 2007b). This technology permitted, for the first time, unambiguous identification of synaptic inputs onto defined neuronal types. It was also the first virus whose transsynaptic specificity was physiologically verified (Wickersham et al., 2007b). This has led to the widespread use of these vectors to trace circuits in the rodent (discussed further in “Engineering Viruses to Control Transport” Section).

As with HSVs, RABV is toxic to cells. While less rapidly cytotoxic than other transsynaptic vectors such as VSV (van den Pol et al., 2009; Beier et al., 2011b), and HSV-1 and PRV (McCarthy et al., 2009), physiological analyses on RABV-infected neurons should be performed with caution. Also, while many studies have reported the neuronal specificity of RABV transsynaptic tracing, others have reported that glial cells are occasionally observed from viral infection, just as with PRV (Viney et al., 2007; Marshel et al., 2010) and VSV (Beier et al., 2011b). Therefore, viral transmission is not always exclusively limited to traditional synapses.

The activity of VSV in the CNS was not studied as early as the other transsynaptic tracers, though a few studies reported replication in the CNS (Lundh, 1990; Plakhov et al., 1995; van den Pol et al., 2002, 2009). The first study to modify the VSV genome and examine its transmission patterns showed that the virus could indeed cross synapses and label neurons (Beier et al., 2011b). Importantly, the direction of transmission was shown to be dictated by the nature of the glycoprotein. VSV with its own G showed an anterograde transsynaptic transmission pattern, while VSV encoding the RABV G showed a retrograde transsynaptic pattern (Beier et al., 2011b).

As VSV is non-pathogenic in nature, one can safely use VSV as a polysynaptic tracer, that is, in its replication-competent form. Infection of a peripheral tissue leads to a rapid shut-down of VSV replication, via the innate immune system (Junt et al., 2007). The lack of escape from this immune response renders VSV safe for laboratory use, which is one of the reasons why VSV has been well studied by virologists and cell biologists. However, direct injection of wild type VSV into the CNS can lead to rapid spread in the brain, which can kill mice very quickly, particularly young mice (Sabin and Olitsky, 1937). The recombinant forms of VSV that encode fluorescent proteins and other viral glycoproteins, such as RABV-G, are attenuated and spread in the CNS more slowly than the wild type (Roberts et al., 1998; Beier et al., 2011b). Other genome modifications have been made to create a monosynaptic tracer (Beier et al., 2011b), following the strategy used for monosynaptic RABV (Wickersham et al., 2007b). When the glycoprotein from the lymphocytic choriomeningitis virus (LCMV-G) was supplied in trans in hippocampal organotypic slices, VSV was observed to transmit between neurons in an anterograde direction. This was confirmed electrophysiologically, by optically stimulating the starter cell and recording from the post-synaptic neuron (Beier et al., 2011b). VSV is currently being developed for use in vivo, for monosynaptic and either anterograde or retrograde transmission.

One of the major advantages of the monosynaptic RABV technique is the ability to examine direct synaptic inputs to the targeted neuronal populations without a presupposition about their identity or strength. The advantages of using the monosynaptic virus for this purpose are that it does not label axons of passage, which can be problematic when using some classical tracers, and that its high level of amplification at the cell body allows it to strongly label even weakly connected inputs. For example, this approach was used to determine the inputs to AgRP-Cre expressing neurons in the ventral hypothalamus that are critical for the control of hunger (Krashes et al., 2014). The major inputs were the dorsal medial hypothalamus (DMH, 26% of inputs), and the paraventricular hypothalamus (PVH, 18% of inputs). As the monosynaptic method suggested that these are direct inputs, the investigators then used channelrhodopsin-assisted circuit mapping techniques to show that the strength of these glutamatergic afferents to the AgRP neurons was stronger from the PVH than the DMH, and that these connections subsequently were important for the control of feeding behavior. Another study used the virus to suggest a potential feedforward circuit in the PVH (Betley et al., 2013).

In addition to identifying the presence or absence of connections, RABV can be used to elucidate the topology of neuronal circuits. For example, in the olfactory circuitry in the mouse, it was known that each glomerulus in the olfactory bulb receives projections from one type of olfactory receptor neuron, but it was unknown how the olfactory map was represented in downstream regions, such as in the anterior olfactory nucleus (AON), or in the piriform cortex. RABV was injected into downstream targets of the olfactory bulb projection neurons, the mitral and tufted cells. It was found that the olfactory bulb-AON projection maintained the dorsal-ventral topography present in the olfactory bulb, that the projection to the amygdala was dorsally biased, and that the olfactory cortex appeared to get input from a random assortment of mitral/tufted cells. Thus RABV tracing was able to identify that each of the three different efferent projection sites appeared to have a different representation of the olfactory map than the one present in the olfactory bulb, a finding supported by other studies (Stettler and Axel, 2009; Choi et al., 2011; Sosulski et al., 2011).

These represent only a few examples of the many studies that have used RABV to map connectivity in different circuits throughout the central nervous system (Yonehara et al., 2011; Sun et al., 2014) as well as the spinal cord (Stepien et al., 2010; Tripodi et al., 2011; Esposito et al., 2014).

The monosynaptic RABV technique has also been used to quantitatively compare inputs to two or more different cell populations. One study used the virus to compare the inputs to the two major dopaminergic cell populations in the ventral midbrain, the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc; Watabe-Uchida et al., 2012). The authors restricted infection to dopamine neurons using a Cre mouse line (DAT-Cre) and used injections of viruses to target the two anatomical locations. They then counted all of the monosynaptic inputs to each of these regions throughout the entire brain. The first observation was that each of these regions received direct inputs from a much wider array of brain regions than previously appreciated. Many of these inputs were not detected by previous studies, as they may have escaped detection using less sensitive methods, or their relatively low abundance may have been ascribed to an error in injection targeting. It was also surprising that the VTA and SNc largely shared the same inputs, in largely the same proportions, with a few unique exceptions that include the motor cortices and subthalamic nucleus for the SNc and the lateral hypothalamus for the VTA. A similar approach has been used to examine the inputs to different groups of serotonergic neurons (Ogawa et al., 2014; Pollak Dorocic et al., 2014; Weissbourd et al., 2014), as well as the dorsal striatum (Wall et al., 2013) and hippocampus (Sun et al., 2014).

Viral tracing methods have also been used to investigate the timing of wiring and synapse formation during development. One such study investigated neurons potentially involved in rodent whisking behavior (Takatoh et al., 2013). The monosynaptic technique is particularly useful for this application, as it is able to identify when synaptic connections between neurons are formed, which would not be possible using classical tracers. In order to find such neurons, the investigators used the fact that whisking is initiated postnatally, and therefore examined inputs to the vibrissal motor neurons at different postnatal days, with the goal of finding populations of inputs that were specifically labeled after the onset of whisking. By comparing the label produced by injections at P1 and P8, the authors discovered new inputs from the rostral part of the lateral paragigantocellularis in the hindbrain that were identified during the later time window. As these neurons appear to get direct input from the motor cortex, the authors suggest that the formation of inputs from these hindbrain neurons is critical for active whisking. Another study used RABV to examine the formation of inputs onto newly generated neurons in the dentate gyrus in the hippocampus, and adult-born neurons in the olfactory bulb (Deshpande et al., 2013). In both cases, they found that at early time points post-maturation, the majority of the transsynaptically-labeled inputs were located nearby the cells from which tracing was initiated. If the researchers initiated tracing a few weeks after maturation, they observed the presence of an increasing number of inputs located at greater distances from the targeted neurons, suggesting that newly born neurons first integrate into local circuitry before they receive inputs from distant sites. The similar pattern of observations in both the hippocampus and the olfactory bulb may indicate a common mechanism for integrating newborn neurons into already-established neural networks.