A Dual Infection Pseudorabies Virus Conditional
Reporter Approach to Identify Projections to
Collateralized Neurons in Complex Neural Circuits
J. Patrick Card1*., Oren Kobiler2., Ethan B. Ludmir2, Vedant Desai1, Alan F. Sved1, Lynn W. Enquist2
1Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2Department of Molecular Biology and the Princeton
Neuroscience Institute, Princeton University, Princeton, New Jersey, United States of America
Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural
circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize
within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and
Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a
monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell
nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the
presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and
liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle
experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right
kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the
unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons
expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the
Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique
marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished
using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in
polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners.
Citation: Card JP, Kobiler O, Ludmir EB, Desai V, Sved AF, et al. (2011) A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections
to Collateralized Neurons in Complex Neural Circuits. PLoS ONE 6(6): e21141. doi:10.1371/journal.pone.0021141
Editor: Eric J. Kremer, French National Centre for Scientific Research, France
Received March 16, 2011; Accepted May 20, 2011; Published June 16, 2011
Copyright: ? 2011 Card et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by NIH grants 1RC1NS068414, 1RO1HL093134, and P40 RR018604 and NSF grant 0918867. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Neurotropic viruses represent popular and powerful tools for
defining the identity and organization of synaptically connected
neurons [1,2,3,4,5,6,7,8,9]. The method exploits the tropism of
these viruses for neurons and their tendency to replicate and
spread from neuron-to-neuron via the intimate synaptic contacts
through which neurons communicate. Pseudorabies virus (PRV), a
DNA swine alpha herpesvirus, is one of the most widely applied
viruses for polysynaptic circuit analysis in the rodent nervous
system. The extensive use of PRV in such studies is related to the
availability of strains of reduced virulence that are transported
selectively in the retrograde direction through neural circuits and
also express unique reporter proteins (e.g., fluorescent proteins).
Restricting PRV replication to defined populations of neurons
through cell-specific gene expression is a recent advance of viral
transneuronal tracing technology . In an early report, DeFalco
and colleagues exploited Cre-Lox site-specific recombination 
of the viral genome to restrict PRV replication to phenotypically
defined neurons and synaptically connected circuits . Repli-
cation of the virus constructed for that study (PRV-2001) requires
Cre-mediated recombination of the viral genome to remove a
floxed stop cassette that prevents transcription of a thymidine
kinase gene essential for viral replication in non-mitotic cells. Once
the stop cassette is removed, the virus is permanently replication
competent and passes transneuronally to infect synaptically linked
neurons. This approach has subsequently been employed to define
neural circuits synaptically linked to LHRH- [13,14,15] and
serotonin-  containing neurons.
The aforementioned conditional approach is limited by the
requirement that Cre be present in first-order neurons infected by
PRV-2001. The approach cannot be used if the goal is to define
synaptic connections specific to a population of Cre-expressing
neurons embedded within a larger circuit, while maintaining
target cell specificity of the circuit (e.g., Cre-expressing neurons
separated from the injection site by more than one synapse). The
recent development of a replication competent PRV recombinant
that changes the profile of reporter gene expression when exposed
to Cre has circumvented this problem [17,18]. The conditional
recombinant, PRV-263, carries the Brainbow 1.0L cassette
developed by Lichtman and colleagues  and expresses a red
dTomato cytoplasmic reporter unless it has been exposed to Cre.
PLoS ONE | www.plosone.org1 June 2011 | Volume 6 | Issue 6 | e21141
In the presence of Cre, the red reporter gene is removed and either
the cyan (mCerulean) or yellow (EYFP) reporter is expressed. It is
important to emphasize that each virus can express only a single
reporter (before or after Cre-recombination) but that infected
neurons can replicate more than one viral genome, resulting in a
mixed reporter phenotype of some infected neurons. We recently
documented the utility of this approach for circuit analysis by
combining PRV-263 infection with lentivirus-mediated expression
of Cre in phenotypically-defined, anatomically localized, and
projection-specific populations of neurons .
In this report we document a dual infection transneuronal
tracing approach to identify neurons synaptically linked to
collateralized neurons within complex networks. The method
takes advantage of the Cre-conditional reporter expression of
PRV-263 and a new strain of PRV (PRV-267) that expresses both
Cre and an mRFP-capsid fusion protein (VP26-mRFP). Injection
of PRV-267 and PRV-263 into separate kidneys using a well-
characterized dual infection paradigm  produced unique
markers of collateralized neurons synaptically linked to both
kidneys (nuclear mRFP and conditional fluorescent reporter
expression from the Brainbow cassette) as well as neurons infected
by replication and transneuronal passage of progeny virus from
those neurons (only Brainbow reporters). This approach expands
the utility of dual infection viral transneuronal tracing paradigms
by providing a means of distinguishing collateralized neurons
within complex networks from the neurons that are antecedent to
Materials and Methods
All experimental procedures involving animals conformed to
regulations stipulated in the NIH Guide for the Care and Use of
Laboratory Animals and were approved by the University of
Pittsburgh IACUC (protocol number: 0909666), Recombinant
DNA Committee (reference number: 112-09), and Division of
Environmental Health and Safety (protocol number: 0909666).
The in vitro experiments used to construct and characterize PRV-
267 were conducted at Princeton University and approved by the
Recombinant DNA Technology Committee (MUA # 912).
Adult male rats (Harlan Sprague-Dawley) weighing 250 to 320
grams at the time of viral injection were used for in vivo
experiments conducted in a Biosafety Level 2 certified laboratory.
Animals were single housed and lived within this facility after virus
injection. Photoperiod (12 hours light; light on at 0700) and
temperature (22–25uC) were standardized and animals had free
access to food and water.
The genomic organization of the recombinants and related
strains of PRV are illustrated in Figure 1. The preparation of
PRV-263, a PRV-Bartha recombinant carrying the Brainbow
1.0L cassette in the US4 (gG) locus (Figure 1C), has been
previously described . PRV-267, a PRV-Bartha recombinant
expressing Cre-recombinase and a red fluorescent protein-tagged
VP26 capsid protein, is a new virus constructed for this study.
Preparation of a Cre-containing plasmid (pEL2) and construction
of the virus are described below.
To construct the pEL2 plasmid, the Cre-recombinase coding
region, including an N-terminal nuclear localization signal and
133-base-pair synthetic intron was amplified by PCR from
pBecker3 (a self-recombining bacterial artificial chromosome) as
previously described . Two PCR primers were designed with a
KpnI restriction site in the sequence homologous to the 59 Cre
open reading frame (ORF) (59-GGGGTACCATGCCCAAGAA-
GAAGAGGAAG) and an XbaI site in the sequence complemen-
tary to the 39 Cre ORF (59-GCTCTAGACATATCGCCATC-
TT CCAGCAG). The eGFP ORF of pEGFP-N1 was removed
through KpnI/XbaI digestion, followed by ligation with the am-
plified Cre ORF. The resulting plasmid, pEL2, contained the Cre-
recombinase coding region under immediate-early human cyto-
megalovirus (hCMV) promoter control. Restriction fragment
analysis and nucleotide sequencing verified the structure of the
PRV-267 was constructed as follows. We first constructed PRV-
266, a PRV-Bartha strain expressing mRFP-VP26 and diffusible
eGFP, through co-infection of porcine kidney epithelial (PK-15)
cells with PRV-152 (a PRV-Bartha strain encoding a diffusible
eGFP under an immediate-early hCMV promoter from the US4
locus, ) and PRV-756 (a mRFP-VP26 fusion protein, ).
VP26 is a surface capsid protein encoded by the UL35 gene and
the fusion protein incorporating mRFP labels viral capsids
intensely, thereby providing a unique marker of neurons
replicating PRV-267. We selected viral recombinants expressing
both mRFP-VP26 and diffusible eGFP; sequential rounds of
plaque purification of this virus, PRV-266, were then performed.
We then co-transfected PRV-266 nucleocapsid DNA (purified as
previously described, ) with linearized pEL2 in PK15 cells.
Homologous recombination resulted in PRV-267. We selected
viral recombinants expressing mRFP-VP26, but not eGFP, to
distinguish PRV-267 from PRV-266 and conducted sequential
rounds of plaque purification to isolate PRV-267.
The design of the experiment is illustrated in Figure 2A. Eight
animals were included in the study. Animals were injected in pairs
on different days and fresh aliquots of virus from the same viral
stock were thawed for each pair of injections. Animals were deeply
anesthetized using isoflurane and each kidney was exposed by a
retroperitoneal approach. A total of 2 ml of PRV-263 was injected
into the parenchyma of the left kidney (4 injections of 0.5 ml per
site) using a 10 ml Hamilton syringe. An equivalent volume of
PRV-267 was injected into the right kidney using the same
procedure. We made an effort to standardize injections between
animals by injecting at four similar sites along the greater
curvature of each kidney and by marking the needle of the
Hamilton syringe so that it penetrated the kidney parenchyma
approximately 4 mm at each injection site. After injection, surgical
incisions were sutured closed and animals received a subcutaneous
injection of analgesic (Ketofen; 2 mg/kg). Upon full recovery from
anesthesia animals were returned to their home cages in the BSL 2
laboratory where they lived for the balance of the experiment. The
purified stocks of the viruses had concentrations of 3.46108pfu/
ml (PRV-263) and 56108pfu/ml (PRV-267).
Animals deeply anesthetized with sodium pentobarbital were
perfused transcardially with paraformaldehyde-lysine-periodate
fixative  four (n=2) and five (n=6) days post inoculation.
Aldehyde fixed tissues were postfixed and cryoprotected. The
brain was sectioned with a freezing microtome at 35 mm/section
through its rostrocaudal extent. The spinal cord was divided into
cervical, thoracic and lumbosacral divisions and sectioned
horizontally at 40 mm/section. Tissue was stored in cryoprotectant
 at 220uC prior to immunocytochemical analysis. Details of
all of these procedures have been published .
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The invasive profiles of the recombinants were first determined
by immunoperoxidase localization of infected neurons. Coronal
sections at a frequency of 210 mm through the brain and
horizontal sections at a frequency of 16 mm through the spinal
cord were processed from each case. Viral immunoreactivity was
detected with a rabbit polyclonal antiserum (Rb133) generated
against acetone-inactivated virus . This antiserum recognizes
epitopes on all virally encoded proteins and was used at a 1:10,000
dilution in conjunction with affinity purified, biotinylated donkey
anti-rabbit secondary antibody (1:200; Jackson ImmunoReseach
Laboratories, Inc.; West Grove, PA) and Vectastain Elite avidin-
biotin reagents (9 ml of each reagent combined 90 minutes before
tissue incubation; Vector Laboratories; Burlingame, CA). Diami-
nobenzidine (DAB) was used as a substrate for the immunoper-
oxidase reaction; tissue was incubated in the DAB solution for
10 minutes prior to addition of 35 ml of H2O2/100 ml DAB
solution to catalyze the reaction, and the reaction was terminated
3 minutes after H2O2 addition by repeated rinses in sodium
phosphate buffer. Processed sections were mounted on Superfrost
Figure 1. PRV Genome. The genomic organization of PRV-Becker, PRV-Bartha and the recombinants prepared for this study are illustrated. The
basic organization of the PRV genome is illustrated at the top of the figure (A). Viral DNA contains unique long (UL) and unique short (US) segments
flanked by internal and terminal repeat sequences. The BamH1 restriction map of PRV (B) illustrates the location of the portions of the viral genome
engineered to express transgenes in PRV-263 and PRV-267. These regions of the restriction map are expanded in C to illustrate the location of the
genes in segments 1 and 7 (C). Boxes represent individual genes, with the formal name indicated above the box and the common name, where
appropriate, indicated within each box. The recombinants prepared for this analysis are derived from the PRV-Bartha genome, which contains a large
deletion in the US segment. The genes eliminated by this deletion reduce virulence and restrict viral transport through circuits to the retrograde
direction. The Brainbow cassette and Cre were inserted into the gG (US4) locus to create PRV-263 and PRV-267, respectively. The organization of the
Brainbow 1.0L cassette is illustrated in section D of the figure. Paired loxP and lox2272 sites are positioned within the cassette such that
recombination at loxP sites eliminates the dTomato and mCerulean genes to liberate expression of EYFP and recombination at lox2272 sites
eliminates the red reporter gene to liberate expression of mCerulean. It is important to note that Cre only cuts at like pairs (e.g., loxP:loxP or
lox2272:lox2272) and that the cassette (intact or recombined) will only express one reporter. PRV-267 also carries mRFP as part of a fusion gene at the
VP26 (UL35) locus to produce a unique marker of the surface capsid protein VP26. Construction of PRV-267 is described in the Materials and Methods.
Construction of PRV-263 has been reported .
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Figure 2. Experimental Paradigm and Neuronal Phenotypes. The experimental paradigm used in this analysis (A) and the reporter
phenotypes of neurons infected with PRV-263 and PRV-267, either alone or in combination (B–I), are illustrated. Each animal received separate
injections of PRV-263 and PRV-267 into the left or right kidney (A). Prior studies have demonstrated that PRV-Bartha recombinants are transported
from the kidney to infect postganglionic neurons in the inferior mesenteric ganglion. Subsequent replication and transneuronal passage of virus
infects sympathetic preganglionic neurons in the IML and neurons of the renal preautonomic network. The preautonomic network linked to each
kidney is largely lateralized but also contains neurons that collateralize to innervate circuits linked to each kidney. Neurons infected with both
recombinants express unique reporters (cyan and/or yellow) in response to Cre-mediated recombination of the Brainbow cassette. The color-coding
of neurons defined in the upper right box of A illustrates the various phenotypes possible in this injection paradigm. Neurons only infected with PRV-
267 are marked by the VP26-mRFP reporter, a capsid surface fusion protein that is differentially concentrated in the cell nucleus (B) but produces
punctate labeling in the cytoplasm (C) as capsids migrate out of the nucleus to be incorporated into mature virions. Selective infection with PRV-263
in the absence of Cre results in default expression of the dTomato reporter and homogeneous cytoplasmic labeling (D & E). Cre-mediated
recombination of the Brainbow cassette in neurons replicating both PRV-263 and PRV-267 results in VP26-mRFP capsid labeling and cytoplasmic
labeling by the cyan and/or yellow reporters (white arrows in F–H). Neurons infected by transneuronal passage of virus containing recombined
genomes (PRV-263re) from dual infected neurons only express the cyan and/or yellow cytoplasmic reporters (I). Figure B is from IML of thoracic spinal
cord, figures C & H are from raphe pallidus, figures D–H are from VMM. Marker bars in B, C, F, and G=20 mm and those in D, E, H, and I=25 mm.
Conditional Transneuronal Tracing with PRV
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Plus microscope slides (Fisher Scientific, Pittsburgh, PA), dehy-
drated in graded alcohols, cleared in xylenes, and coverslipped
with Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI). The
details of these procedures as applied in our laboratories have been
Sections of brain and spinal cord adjacent to those used for the
immunoperoxidase analysis were analyzed using fluorescence micros-
copy. Sections were mounted on gelatin-coated slides, air dried, and
coverslipped using Vectashield Hard Set mounting medium (Vector
Laboratories, Burlingame, CA). The fluorophor profile of infected
neurons was determined using an Olympus BX51 epifluorescence
microscope equipped with filters specific for reporter proteins encoded
by the dTomato, mCerulean, and EYFP genes as described previously
. Digital micrographs of each region were captured with a
Hamamatsu camera (Hamamatsu Photonics, Hamamatsu, Japan) and
analyzed using the procedures detailed in the next section.
We first characterized the extent of viral invasion of renal
presympathetic circuits using immunoperoxidase localization of
infected neurons in brain and spinal cord. The goals of this
analysis were to determine if the invasiveness of each virus was
equivalent and conformed to the distribution documented in our
prior dual infection analysis of renal preautonomic circuitry .
To accomplish this we mapped the location of infected neurons in
coronal sections through selected coronal planes through the
neuraxis using StereoInvestigator image analysis software (version
8; Microbrightfield, Williston, VT). We selected 24 coronal
sections that thoroughly sampled the renal preautonomic network
across a 6.23 mm portion of the brain stem and five sections
through a 0.92 mm portion of diencephalon that contained the
PVN (Figure 3). Care was taken to encode the laterality of sections
(e.g., left and right) to ensure accurate recording of viral invasion of
neural circuits innervating the left (PRV-263) and right (PRV-267)
kidneys. Similarly, we matched the rostrocaudal levels of sections
to ensure an accurate comparison of viral labeling between cases.
These maps allowed a quantitative comparison of the neuroinva-
siveness of each virus from the injected kidney (Figure 3) and also
revealed the pattern of viral spread through the preautonomic
network. To illustrate these maps we faithfully transferred labels of
individual infected neurons to templates from the Brain Maps:
Structure of the Rat Brain compiled by Swanson .
With these quantitative data in hand we then documented the
fluorescence profile of infected neurons in the thoracic spinal cord,
ventromedial medulla (VMM), rostroventrolateral medulla (RVLM),
locus coeruleus (LC), and the paraventricular hypothalamic nucleus
(PVN). These regions were selected for analysis because prior
investigations demonstrated that they would contain neurons synap-
collateralize to circuits linked to both kidneys. Comparable coronal
sections through these regions in each animal were examined and
determined using Adobe Photoshop software; fluorescence emitted by
determined by examining color channels selective for each fluorophor.
In this manner it was possible to determine with certainty the
fluorophors expressed by each infected neuron.
Our experimental design takes advantage of a well character-
ized dual infection paradigm that results in predictable retrograde
transneuronal passage of PRV recombinants from the kidneys
. The pattern of infection and the distribution of collateralized
neurons observed in the present analysis recapitulated the findings
documented in that foundational study, which used PRV
recombinants expressing unique reporters (PRV-152; EGFP and
PRV-BaBlu; b galactosidase) injected into separate kidneys. The
predictable pattern of infection produced in this model system
provided a strong foundation for the proof-of-principle observa-
tions reported in the following sections.
Invasive Profiles of PRV Recombinants
There is a finite time period after initial infection of a neuron by
PRV (about 6 hours) when the cell is permissive to infection by a
second strain of PRV [29,30]. Accordingly, we first determined the
invasive profiles of PRV-263 and PRV-267 by conducting a
quantitative analysis of the spread of each recombinant through
the preautonomic network in dual injected animals. We localized
infected neurons using immunoperoxidase procedures and ob-
tained counts of neurons on each side of the brain using an image
analysis system. Immunoperoxidase localization of viral antigens
does not distinguish between the recombinants infecting individual
neurons but does provide an informed evaluation of the extent of
spread of each recombinant through the predominantly lateralized
circuitry synaptically linked to each kidney.
The temporal kinetics and pattern of invasion of renal
preautonomic circuitry for PRV-263 and PRV-267 recapitulates
that documented in our prior studies. Replication and transneu-
ronal passage of each recombinant produced infection of
sympathetic preganglionic neurons in the intermediolateral cell
column (IML) of thoracic spinal cord and subsequent retrograde
transneuronal passage through synaptically connected neurons in
the renal preautonomic network. At four days after kidney
infection, PRV immunopositive neurons were largely confined to
areas in the brain stem that give rise to direct descending
projections to the thoracic spinal cord (e.g., RVLM and A5; data
not shown). One day later, the number of infected neurons in
regions infected at 4 days increased and the infection spread
transneuronally to neurons in other regions of the medulla,
midbrain and forebrain (Figure 2A). In every case, the distribution
of infected neurons conformed to that previously documented in
prior viral transneuronal tracing studies. Importantly, the absence
of infected neurons in the dorsal motor nucleus of the vagus (a
parasympathetic cell group innervating the visceral) demonstrated
that organ-specific transport of virus was not compromised by
leakage of viral inoculum into the peritoneal cavity.
Figure 3 illustrates the number of infected neurons on the left
and right side of the brain in multiple coronal planes sampling the
renal preautonomic network in the six cases processed 5 days after
kidney injection. The number of infected neurons on the left and
right sides of the brain were remarkable consistent in each animal,
indicating that both PRV-267 and PRV-263 invaded the
preautonomic network at similar rates and extents from each
kidney. Additionally, each recombinant infected the same cell
groups (e.g., Figures 4, 5, 6, 7).
Interestingly, the extent to which the preautonomic network was
infected varied to a considerable extent between animals surviving
five days. The post inoculation survival interval for these animals
ranged from 116 to 119 hours and the animals were all injected
and perfused midway through the light phase of the circadian
cycle (between 11 am and 3 pm). All injections were made from
the same stock of virus of constant titer and a freshly thawed
aliquot of virus was used to inject each pair of animals. Similar
variations in the magnitude of infection between animals were
noted in the study by Cano et al.  employing PRV-152 (EGFP
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Figure 3. Neuroinvasive Profiles of PRV Recombinants. The number of infected neurons in 24 coronal planes sampling the neuraxis 5
days following injection of PRV 267 into right kidney (solid line) and PRV-263 into the left kidney (dashed line) is illustrated. Cases are
arranged according to the magnitude of viral invasion. Case numbers are indicated in the upper left of each graph and the post inoculation
survival interval is listed below each case number. The location of the planes of section sampled for each case is illustrated in the sagittal
schematic included in the upper right of Case 4. The X-axis of each graph indicates the position of the 24 coronal planes relative to Bregma
(b), an anatomical marker of the confluence of bone sutures on the rostral skull. The Y-axis indicates the number of infected neurons.
Although the magnitude of infection varied between cases, the number of infected neurons on the left and right side of the brain was
comparable for each coronal plane in each case. This finding is consistent with the conclusion that each recombinant invaded preautonomic
circuitry from the kidney at equivalent rates in each experimental animal. The schematic diagram is adapted from the atlas Brain Maps:
Structure of the Rat Brain .
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reporter) and PRV-BaBlu (b galactosidase reporter) in this model.
Such variations may be the result of differences in the density of
sympathetic innervation of kidney or to variations in the number
of infect cells within the kidney that become infected and amplify
virus available for uptake and retrograde transport by sympathetic
afferents, possibly due to differences in immune response to
infection. In any event, the quantitative analysis in this study
provided strong evidence that the invasion of preautonomic
circuits from each kidney was equivalent for each recombinant
within an animal, thereby optimizing the probability of achieving
dual infection of collateralized neurons.
Fluorescence profiles of infected neurons
Neurons infected by PRV-267 express a red punctate signal that
produces a dense labeling of cell nuclei early in viral replication
(Figures 2B) followed by the appearance of red puncta in the
cytoplasm later in infection (Figures 2C). This labeling is predicted
by the demonstrated sequence of capsid assembly (nuclear) and
envelopment (cytoplasmic) characteristic of viral replication and
spread . The expression profiles of fluorescent reporters
resulting from infection of neurons with PRV-263, either alone or
in the presence of Cre, have been characterized [17,18]. In the
absence of Cre, the cytoplasm of infected cells fills with the
dTomato (red) reporter expressed from the Brainbow cassette
(Figures 2D and E). However, in the presence of Cre the red
reporter gene is excised resulting in expression of either the yellow
(EYFP) or cyan (mCerulean) reporters (Figures 2F–I).
Dual infected neurons in the present study were marked by the
punctate VP26-mRFP capsid marker of PRV-267 and the
reporters of the recombined Brainbow cassette (white arrows in
Figures 2F–H). Once Cre acts, no more recombination is possible
thus rendering the expression characteristics of the resulting
progeny virus permanent. Transneuronal passage of recombined
PRV-263 will therefore produce either cytoplasmic cyan or yellow
reporters. Whether transneuronally infected neurons express
multiple cytoplasmic reporters of PRV-263 infection and also
contain VP26-mRFP appears to depend upon the mixture of
progeny virus that passes transneuronally from dual infected
neurons (see Discussion for supporting literature). However, when
the cytoplasmic reporters of the recombined Brainbow cassette are
present in cells lacking punctate VP26-mRFP labeling (Figure 2I)
the neurons should have been infected by retrograde transneuro-
nal passage of virus from one or more dual infected neurons.
Reporter gene expression in neurons replicating only one
Reporter gene expression in sympathetic preganglionic neurons
(SPNs) of thoracic spinal cord illustrated the distinctive reporters
of neuronal infection with PRV-263 or PRV-267. Infection of
SPNs in the IML revealed the largely lateralized sympathetic
outflow to the kidneys and other autonomic targets; e.g., SPNs
innervating the kidney injected with PRV-263 displayed homo-
geneous expression of the dTomato reporter throughout the
somatodendritic compartment (Figure 4A) while SPNs infected by
retrograde transport of virus from the kidney injected with PRV-
267 contained dense concentrations of mRFP puncta in cell nuclei
The largely lateralized organization of preautonomic circuitry
innervating SPN outflow to each kidney predicts that the majority
of neurons infected with only one virus will be concentrated on the
side of the brain ipsilateral to the injected kidney. Examination of
each group of infected neurons contributing to the preautonomic
circuitry on the left and right sides of the brain confirmed this
prediction. The majority of infected neurons in each node within
the preautonomic network infected with only one virus displayed
fluorescence profiles consistent with the genotype of virus injected
into the ipsilateral kidney.
Reporter gene expression in dual infected neurons
We analyzed regions of the spinal cord, medulla and dien-
cephalon previously shown to contain neurons infected through
collateralized axonal projections to neural circuits innervating
each kidney . If neurons infected with PRV-267 are producing
biologically active Cre then we should observe dual infected
neurons expressing cyan and/or yellow reporters. Similarly, the
presence or absence of the unique reporter of PRV-267 infection
(punctate VP26-mRFP) in neurons expressing cyan and yellow
fluorescence should allow us to discriminate neurons replicating
both viruses from synaptically connected neurons infected by
transneuronal passage of virus from dual infected neurons. We
selected the thoracic spinal cord, RVLM, VMM, LC and PVN for
this analysis because of the well-known collateralized connections
Figure 4. Fluorphor Expression in Spinal Cord IML. Infected SPN neurons in the thoracic IML after injection of PRV recombinants into the
kidneys are illustrated. The approximate level of thoracic spinal cord illustrated in figures B–D is designated by the red arrow on the dissection of
brain and spinal cord shown in figure A. Figure B & C illustrate infected SPNs in IML segments ipsilateral to kidneys injected with PRV-263 (B) or
PRV-267 (C) in case 4. The default dTomato reporter fills the soma and proximal dendrites of SPNs infected only with PRV-263 while capsids tagged
with the VP26-mRFP fusion protein densely label the nucleus (white arrows) of neurons only infected with PRV-267. Figure D illustrates the
fluorophor profiles of infected SPNs in an IML segment ipsilateral to the kidney injected with PRV-267 (case 1). Labeled capsids are concentrated in
the nuclei of infected SPNs but are also apparent in the cell cytoplasm (white arrow). In addition, IML neurons in this segment express cytoplasmic
reporters of the recombined Brainbow cassette, either alone or with the VP26-mRFP reporter. The yellow arrows in d9–d0 illustrate a dual infected
neuron expressing mRFP and cytoplasmic reporters of the recombined Brainbow cassette. The neuron labeled with the asterisk in d9 & d-
expresses cytoplasmic reporters of the recombined Brainbow cassette, but no mRFP labeled capsids, and was infected by retrograde transneuronal
passage of virus from dual infected neurons. Marker bars for figures B & C=50 mm; marker bars for D and d9=20 mm. The magnification is
equivalent for d9–d-.
Conditional Transneuronal Tracing with PRV
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Figure 5. Fluorphor Expression in Caudal Brainstem. The distribution of infected neurons throughout the caudal brainstem is illustrated. The
red lines described in figure A illustrate the location of the coronal planes illustrated in figures B through E. Figures B through E map the distribution
of infected neurons detected by immunoperoxidase localization of viral antigens in case 1. Each red dot indicates the position of an infected neuron
and the position of each section with respect to Bregma (b) is indicated below each map. Figures b through e illustrate the fluorescent profiles of
neurons in the RVLM (b & d), VMM (c) and raphe pallidus (Rpal; e) in sections adjacent to those mapped for immunoperoxidase localization of viral
antigens. The relative position of each field illustrated in figures b through e is illustrated in the boxed area of figures B through E. The fluorescence in
figures b through e is a composite of that revealed by the filters specific for dTomato, mCerulean and EYFP. The fluorescence signal from individual
color channels in the boxed areas of figures b through e is shown at higher magnification in adjacent photomicrographs and insets (lower case letters
marked apostrophes). White arrows mark dual infected neurons expressing VP-mRFP labeled capsids and EYFP and/or mCerulean reporters of the
recombined Brainbow cassette. Cells marked by the asterisks are expressing reporters of the recombined Brainbow cassette but do not contain VP26-
mRFP labeled capsids. The absence of labeled capsids in these cells indicates that they were infected by transneuronal passage of virus from a dual
infected neuron. The photomicrographs illustrated in b–d are from case 1 and that shown in figure e (and at higher magnification in figure 2H) is from
Conditional Transneuronal Tracing with PRV
PLoS ONE | www.plosone.org8 June 2011 | Volume 6 | Issue 6 | e21141
of neurons in these cell groups within the renal preautonomic
As noted above, we observed large numbers of infected SPNs in
the thoracic spinal cord that were marked by only PRV-267 or
PRV-263 and were concentrated in IML segments ipsilateral to
the injected kidney (Figure 4). However, as reported previously
, we also observed interneurons within IML segments that
were infected by retrograde transneuronal passage of virus from
the contralateral IML. These neurons were most prevalent in cases
with the most extensive transport through the preautonomic
network and contained punctate VP26-mRFP labeling in the
nucleus and cytoplasm as well as yellow and blue cytoplasmic
reporters of the recombined Brainbow cassette (yellow arrows in
Figures 4d9–4d0). This pattern of reporter gene expression is
consistent with dual infection of neurons by PRV-263 and PRV-
267 through collateralized axons that synapse upon SPNs
bilaterally in thoracic spinal cord.
The profile of gene expression in RVLM neurons is predicted
by the known connectivity of the RVLM, which is characterized
by a large projection to the ipsilateral IML and lesser projections
to the contralateral RVLM and IML. Data consistent with this
prediction are shown in Figures 5B and D. We observed RVLM
neurons expressing only the cytoplasmic dTomato reporter (PRV-
263 infection), neurons only expressing the punctate nuclear
VP26-mRFP reporter (PRV-267 infection), and neurons express-
ing the conditional cytoplasmic reporters (cyan and/or yellow) in
combination with the punctate VP26-mRFP labeling of nuclei
(dual infected neurons) (Figures 5b and d). The largest proportion
of neurons expressing these phenotypes was concentrated in the
rostral aspect of RVLM, which is the portion of this cell group that
gives rise to the largest portion of the reticulospinal projection to
Reporter gene expression in VMM reflected documented
descending reticulospinal projections to thoracic cord, reciprocity
of connections to other nodes within the renal preautonomic
network (e.g., RVLM), and local circuit connections within the
VMM (Figure 5C). Direct descending projections to the thoracic
cord were marked by cytoplasmic localization of the dTomato
reporter or punctate VP26-mRFP labeling in neurons replicating
only one virus, with the largest proportions of each of these groups
present on the side of the brain ipsilateral to the injected PRV
recombinant. We also observed neurons that contained both
markers and were therefore infected by collateralization of axons
to efferent pathways synaptically linked to both kidneys (white
arrows in Figure 5c). Single and dual infected neurons were
present in all subdivisions of the VMM, including raphe pallidus,
but were most prevalent in the rostral third of this cell column,
with the highest concentration occurring in the areas immediately
lateral to the pyramids (Figure 5C and E; 5c and e).
Retrograde transneuronal infection of the LC also produced a
pattern of infected neurons that conformed to that previously
documented after injection of virus into the kidney . The cases
that displayed more limited invasion of preautonomic circuits (e.g.,
animals surviving 4 days and cases 2 and 3 from the 5 day survival
group) exhibited neurons largely confined to the ventral third of
the LC. The majority of infected neurons in a single cell group
were infected by the recombinant injected into the ipsilateral
kidney, but a subset was infected by both recombinants. In cases
with the most extensive transport of virus through preautonomic
circuitry, infected neurons were observed throughout the dorso-
ventral extent of the LC bilaterally (Figure 6A). However, neurons
replicating both recombinants remained confined to the ventral
third of the LC (white arrows in Figures 6B and C). These finding
confirm and extend those reported by Cano and colleagues
case 3. The schematic diagrams are adapted from the atlas Brain Maps: Structure of the Rat Brain . NTS=nucleus of the solitary tract;
RVLM=rostroventrolateral medulla; VMM=ventromedial medulla. Marker bars for b–e and c9=50 mm; marker bars for b9, d9, and e9=10 mm.
Figure 6. Fluorphor Expression in Rostral Brainstem and LC. The distribution of infected neurons in rostral brainstem at the level of the locus
coeruleus (LC) is illustrated. The schematic sagittal section in the upper left of figure A illustrates the position of the plane sampled in the rostrocaudal
axis of the brain. The coronal schematic in figure A maps the distribution of infected neurons detected by immunoperoxidase localization of viral
antigens in case 1. Each red dot indicates the position of an infected neuron in a section 210.10 mm relative to Bregma. Figures B and C, also from
case 1, illustrate the fluorescent profiles of LC neurons in sections adjacent to that shown in A. The box in A marks the relative position of images B
and C, which illustrate reporter fluorescence revealed by the filters specific for dTomato, mCerulean and EYFP. Fluorescence signal in the boxed areas
of B & C is shown at higher magnification in b9–b- and c9–c-. White arrows in B & C indicate neurons dual infected neurons. Cells marked by the
asterisks are expressing reporters of the recombined Brainbow cassette but do not contain VP26-mRFP labeled capsids. The schematic diagram is
adapted from the atlas Brain Maps: Structure of the Rat Brain . Marker bars in B & C=50 mm; marker bars in b- and c-=20 mm.
Conditional Transneuronal Tracing with PRV
PLoS ONE | www.plosone.org9 June 2011 | Volume 6 | Issue 6 | e21141
following injection of PRV-152 (EGFP reporter) and PRV-BaBlu
(b-galactosidase reporter) into separate kidneys.
Retrograde transneuronal infection of neurons in the PVN
occurred in the parvicellular subdivisions of this diencephalic cell
group. Only scattered infected neurons were found in the PVN in
animals analyzed four days following injection of virus into the
kidneys. Five days following kidney injection we observed
numerous infected neurons in the dorsal, medial and posterior
parvicellular subfields (Figure 7A). Figure 7B illustrates the
distribution and phenotype of neurons typically observed in
animals exhibiting the most robust infection of preautonomic
circuitry (e.g., cases 1 and 4). In each case, infected neurons were
present within in both the dorsal and medial parvicellular subfields
of PVN. Dual infected neurons (e.g., white arrows in 7b) were a
subset of a larger population infected only with the recombinant
injected into the ipsilateral kidney.
Transneuronal infection from dual infected neurons
The expression of conditional reporters of the Brainbow cassette
throughout the preautonomic network, while confirming the
presence of collateralized neurons, also revealed new insights into
the synaptic organization of preautonomic synaptology. For
example, we observed infected neurons that replicated the
recombined Brainbow cassette (expressing cyan and yellow
cytoplasmic reporters), but did not express punctate VP26-mRFP
labeling. Since the presence of punctate VP26-mRFP marks cells
infected with PRV-267, neurons that exclusively express cytoplas-
mic reporters of the recombined Brainbow cassette should have
been infected by virtue of their synaptic linkage to dual infected
neurons (i.e., are presynaptic to dual infected neurons). This
synaptic relationship cannot be distinguished in dual infection
approaches that do not involve conditional reporter expression
(e.g., injection of PRV152 & PRV-BaBlu). Neurons of this
phenotype (marked by asterisks in Figures 4, 5, 6, 7) were
observed in each of the cell groups analyzed in this study and their
prevalence appeared to vary among cell groups. For example,
neurons displaying this phenotype were prevalent within RVLM,
VMM and LC but were rarely observed within raphe pallidus. A
more detailed analysis incorporating a larger sample size and
quantitative analysis is necessary to determine the relative
proportions of these neurons within individual cell groups of the
renal preautonomic network. Nevertheless, the ability to discrim-
inate these neurons from dual infected cell groups provides
another level of insight into the synaptology of neural networks
identified in dual infection paradigms.
The findings reported in this manuscript document a new viral
transneuronal tracing approach that can be used to identify
connections to neurons within a complex network whose axons
collateralize to influence separate targets. To test the utility of this
approach, we used a well documented dual infection animal model
in which PRV recombinants that express unique reporters are
injected into separate kidneys . The use of PRV-263 and
PRV-267 in this model system provides unique insights into the
synaptic organization of complex circuits that cannot be resolved
in dual infection studies employing isogenic PRV recombinants
that constitutively express unique reporters (e.g., PRV-152 & PRV-
BaBlu). Particularly important in this regard is the ability to
discriminate neurons presynaptic to dual infected neurons.
Nevertheless, it is important to emphasize that the method does
not permit a definitive identification of all neurons providing
synaptic input to dual infected collateralized neurons and thereby
provides a qualitative rather than quantitative approach for
identifying these neurons.
The method builds upon recent studies in which we reported the
construction and characterization of PRV-263  and demon-
strated the ability of lentivirus mediated Cre expression to produce
conditional reporter expression from a Brainbow cassette 
carried by PRV-263 in targeted populations of neurons . Here
wedescribethe constructionand use ofPRV-267,whichservesboth
as a transneuronal tracer and a vector for circuit related expression
Figure 7. Fluorophor Expression in Diencephalon and PVN. The distribution of infected neurons in diencephalon at the level of the
paraventricular hypothalamic nucleus (PVN) is illustrated. Figure A maps infected neurons detected by immunoperoxidase localization of viral
antigens in case 1. Each red dot indicates the position of an infected neuron and the position of the section with respect to Bregma (b) is indicated
below the schematic. Figure B illustrates the fluorescent profiles of neurons in parvicellular PVN subdivisions in a section adjacent to that shown in A.
The fluorescence in figure B is a composite of that revealed by the filters specific for dTomato, mCerulean and EYFP and the boxed area is shown at
higher magnification in the inset. Figures b–b0 show the fluorescence for individual channels in the same area as the inset in figure B. White arrows
mark dual infected neurons and asterisks mark neurons that only express reporters of the recombined Brainbow cassette. The schematic diagram is
adapted from the atlas Brain Maps: Structure of the Rat Brain . Marker bar in figure B=50 mm and the marker bar in the inset of figure B=10 mm.
Figures b–b0 are of the same magnification and the marker bar in b=20 mm.
Conditional Transneuronal Tracing with PRV
PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e21141
ofCre. Toourknowledgethis isthefirstdemonstrationoftheability
to deliver biologically active Cre in a circuit specific fashion across
multiple synapses. The fact that Cre is expressed throughout the
polysynaptic circuit infected by PRV-267 is validated both by the
pattern and kinetics of conditional reporter expression observed
withinthe CNS followingseparate injections of PRV-267 andPRV-
263 into the kidneys. Importantly, the present data confirm the
identity and organization of neurons within the preautonomic
network previously shown to collateralize to regulate both kidneys
. Considered with evidence that recombination of the Brainbow
cassette only occurs in the presence of Cre, this is an important
confirmation that PRV-267 is producing biologically active Cre in
the neurons that it infects.
Importantly, the insights derived from the use of PRV-263 and
PRV-267 in dual infection experiments are not limited to the
ability to identify neurons that collateralize to influence separate
targets. We observed neurons that expressed reporters of the
recombined Brainbow cassette but not the unique reporter of
PRV-267 infection (punctate VP26-mRFP). These neurons can
only have been infected subsequent to Cre mediated recombina-
tion and transneuronal passage of the PRV-263 genome. Using in
vitro analysis Kobiler and colleagues demonstrated that Cre-
mediated recombination occurs prior to replication of PRV-263
and that a remarkably small number of viral genomes – as few as
seven – are expressed, replicated and assembled into virions .
This interesting bottleneck may limit the population of virions that
can spread transneuronally and express their genomes. In any
case, even if PRV-263 and PRV-267 co-infect a single neuron, the
data of Kobiler and colleagues indicates that the probability of
second- and third-order neurons being infected by both recom-
binants drops after each transneuronal passage. Therefore,
neurons displaying only cytoplasmic reporters of the recombined
PRV-263 genome, and no reporters of PRV-267 infection
(punctate VP26-RFP), likely represent neurons that were infected
from the early transneuronal passage of progeny virus containing
the recombined PRV-263 genome from a dual infected neuron.
Similarly, early transneuronal passage of PRV-267, and not PRV-
263 recombinants, from dual infected neurons would produce
neurons only expressing the PRV-267 genome that are indistin-
guishable from neurons connected only to the PRV-267 infected
kidney. Thus, data derived from this approach must be interpreted
conservatively and conclusions on the synaptology of the circuit
based only upon positive unequivocal results. In this regard, the
singular expression of PRV-263 reporters of the recombined
Brainbow cassette provides an unambiguous identification of
neurons presynaptic to dual infected neurons.
As noted above, the in vitro data of Kobiler and colleagues
demonstrated that Cre-mediated recombination of the PRV-263
genome occurs prior to replication of the virus. However, there is a
chance that several incoming PRV-263 genomes will initiate
replication before recombination can occur, even in the presence
of PRV-267. This can result in neurons that were infected with
both viruses expressing the default dTomato reporter along with
the reporters liberated by Cre mediated recombination. Under
these circumstances it is possible that a single dual infected neuron
can replicate up to four different viral genomes (PRV-267, PRV-
263red, PRV-263yellow, and PRV-263blue) and transneuronal
infection of synaptically connected neurons would sample any
combination of these replicated genomes. Indeed, we often
observed neurons in vivo that expressed dTomato (a reporter of
the uncombined PRV-263 genome) along with the mCerulean
and EYFP reporters of recombination.
The functional implications of being able to identify neurons
presynaptic to collateralized neurons are apparent in our data.
Jansen and colleagues previously documented neurons within the
preautonomic network that were co-infected by retrograde
transneuronal transport of recombinant strains of PRV from the
adrenal gland and superior cervical ganglion as a means of
identifying ‘‘command’’ neurons instrumental in the initiation of
the ‘‘fight-or-flight’’ response to stressful stimuli . The neurons
identified in their investigation are among the dual infected
neurons observed in our investigation and include areas that have
been identified as important mediators of neural responses stress.
The LC is among the regions identified in our analysis that were
not included in the ‘‘command’’ neurons identified by Loewy and
colleagues. Nevertheless, the LC is prominent among the cell
groups activated by stressful stimuli and it has been postulated to
play a prominent role in orchestrating behavioral and physiolog-
ical responses to stressors [33,34,35]. Importantly, available
evidence indicates that the LC does not exert its influence upon
sympathetic outflow through direct reticulospinal projections to
SPNs in the IML . Rather, LC neurons project to components
of the preautonomic network that, in turn, project directly to SPNs
(e.g., RVLM & VMM) and also influence sympathetic outflow
indirectly through projections to regions that influence affect .
Our data suggest that the LC contains a large population of
neurons presynaptic to dual labeled neurons, an observation
consistent with a prominent role for the LC in the global activation
of sympathetic outflow that is a cardinal feature of the fight-or-
flight response. Our data are also consistent with a similar
functional role for the hypothalamic PVN, which also contained
prominent populations of neurons presynaptic to collateralized
neurons. Definitive support for these hypotheses requires quanti-
tative analysis of a larger sample size, but the possibility illustrates
the potential power of the combined use of PRV-263 and PRV-
267 in dual infection analysis of neural circuitry.
The ability to express Cre in a circuit related manner through
PRV-267 infection and transneuronal passage also has other
experimental applications. For example, PRV-267 can be used to
mediate recombination of floxed genes in transgenic mice in a
circuit-defined manner. Given the expanding list of floxed genes
that are widely available (e.g., see the list on the web site of Andras
Nagy at the Samuel Lunenfeld Research Institute at Mount Sinai
Hospital; http://www.mshri.on.ca/nagy/default.htm) this possi-
bility markedly expands the utility of PRV-267 for functional
studies in a variety of systems. Additionally, the virus can be used
to produce circuit related conditional reporter expression in the
nervous system of the Brainbow mouse .
In conclusion, we have described a new viral tracing method
based on the polysynaptic tracing properties of PRV, the ability to
express biologically active Cre from the PRV genome, and the
conditional reporter capabilities of the Brainbow cassette. The
method enables identification of neurons that collateralize within a
complex network to exert regulatory control over distant separate
targets. It provides a means of expressing Cre in a circuit specific
fashion from a replication competent PRV recombinant (PRV-
267) and relies upon Cre-dependent combinatorial expression of
fluorescent reporters from a Brainbow cassette carried by second
PRV recombinant (PRV-263). The unique reporter phenotypes
produced in dual infection studies employing these recombinants
provides unique insights into the synaptic organization and
function of polysynaptic networks and increases the diversity of
viral transneuronal tracing tools available for circuit analysis.
We thank Jeff Lichtman for the brainbow plasmids and acknowledge
members of the Enquist and Card laboratories for advice and technical
Conditional Transneuronal Tracing with PRV
PLoS ONE | www.plosone.org11 June 2011 | Volume 6 | Issue 6 | e21141
Author Contributions Download full-text
Conceived and designed the experiments: JPC OK AFS LWE. Performed
the experiments: JPC OK AFS VD EL. Analyzed the data: JPC OK AFS
VD EL LWE. Contributed reagents/materials/analysis tools: JPC OK
LWE. Wrote the paper: JPC OK AFS EL LWE.
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