Microdissection of neural networks by conditional
reporter expression from a Brainbow herpesvirus
J. Patrick Carda,1,2, Oren Kobilerb,1, Joshua McCambridgea, Sommer Ebdlahada, Zhiying Shanc, Mohan K. Raizadac,
Alan F. Sveda, and Lynn W. Enquistb
aDepartment of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260;bDepartment of Molecular Biology and the Princeton Neuroscience Institute,
Princeton University, Princeton, NJ 08544; andcPhysiology and Functional Genomics, University of Florida, Gainesville FL 32610
Edited* by Larry W. Swanson, University of Southern California, Los Angeles, CA, and approved January 12, 2011 (received for review October 8, 2010)
Transneuronal transport of neurotropic viruses is widely used
to define the organization of neural circuitry in the mature and
developing nervous system. However, interconnectivity within
complex circuits limits the ability of viral tracing to define con-
nections specifically linked to a subpopulation of neurons within
a network. Here we demonstrate a unique viral tracing technology
that highlights connections to defined populations of neurons
within a larger labeled network. This technology was accom-
plished by constructing a replication-competent strain of pseu-
dorabies virus (PRV-263) that changes the profile of fluorescent
reporter expression in the presence of Cre recombinase (Cre).
The viral genome carries a Brainbow cassette that expresses a de-
fault red reporter in infected cells. However, in the presence of
Cre, the red reporter gene is excised from the genome and expres-
sion of yellow or cyan reporters is enabled. We used PRV-263 in
combination with a unique lentivirus vector that produces Cre
expression in catecholamine neurons. Projection-specific infection
of central circuits containing these Cre-expressing catecholamine
neurons with PRV-263 resulted in Cre-mediated recombination of
the PRV-263 genome and conditional expression of cyan/yellow
reporters. Replication and transneuronal transport of recombined
virus produced conditional reporter expression in neurons synap-
tically linked to the Cre-expressing catecholamine neurons. This
unique technology highlights connections specific to phenotypi-
cally defined neurons within larger networks infected by retro-
grade transneuronal transport of virus from a defined projection
target. The availability of other technologies that restrict Cre ex-
pression to defined populations of neurons indicates that this ap-
proach can be widely applied across functionally defined systems.
autonomic|preautonomic network|sympathetic|transneuronal tracing
foundational for understanding the way in which the ner-
vous system functions, or malfunctions, in health, disease, and
injury. Experimental approaches for circuit definition have long
exploited the axonal transport capabilities of neurons (1). A vast
literature continues to define systems organization of brain cir-
cuitry through localization of “classic” tracers that are trans-
ported through axons but do not cross synapses. Such studies
define regional associations but, in the absence of transmission
electron microscopic analysis, do not define the synaptology of
the system under study. The ability of neurotropic viruses to
replicate and spread through neural circuits has brought
a polysynaptic perspective to circuit analysis (2–6). Recent ge-
netic engineering of such viruses has produced increasingly
powerful probes that have provided novel insights into the iden-
tity, organization, and activity of neural networks in a variety of
The core strengths of the viral transneuronal tracing method
lie in the ability of neurotropic viruses to cross synapses and
generate infectious progeny in each neuron of a circuit. In effect,
these viruses represent self-amplifying neural tracers that effi-
ciently label synaptically linked neurons in a time-dependent
manner. However, these desirable attributes also limit the ability
to define details of synaptic connectivity within complex net-
nowledge of the synaptic organization of neural circuitry is
works. For example, definition of the brain’s neural network,
which regulates homeostasis through autonomic outflow, has
benefited enormously from temporal analysis of the replication
and transneuronal passage of pseudorabies virus (PRV; a DNA
swine α-herpesvirus) from peripheral tissues (e.g., refs. 10–12).
Functionally distinct neurons influential in the regulation of
cardiovascular function (e.g., blood pressure versus heart rate)
are distributed throughout this network and their activity is
coordinated through local circuits connecting network nodes
(13–15). The reciprocity of connections that ensures integrated
activity among such functionally related components of preau-
tonomic circuitry also promotes efficient viral spread throughout
the network. Thus, although viral transneuronal tracing has
proven to be an effective means of defining the full extent of
preautonomic circuitry, the efficiency of viral transport through
the entire network undermines the fine-scale resolution of syn-
aptic inputs to defined populations of neurons.
Creative approaches for limiting replication of virus to tar-
geted populations of neurons have substantially improved the
ability to define the synaptology of neural circuitry. Wickersham
et al. developed a novel tracing approach in which replication of
rabies virus (an RNA virus) is restricted to targeted populations
of neurons and their first-order synaptic partners (16). This ap-
proach was achieved by deleting the rabies glycoprotein gene
from the viral genome and pseudotyping the virus so that it
differentially infects neurons engineered to express the deleted
glycoprotein. Because the rabies glycoprotein is necessary for
retrograde transneuronal passage of progeny virus, infection is
limited to the targeted neurons and their first-order synaptic
inputs. DeFalco et al. constructed a PRV recombinant (PRV-
2001), whose replication is dependent upon the presence of the
bacterial enzyme Cre recombinase (Cre), and used it to define
polysynaptic circuits in transgenic mice that express Cre in neu-
ropeptide Y neurons (17). In this approach, Cre mediates recom-
bination of the viral genome to eliminate a floxed stop cassette
that prevents transcription of thymidine kinase, a gene essential
for viral replication in nonmitotic cells. Once the stop cassette is
eliminated, thymidine kinase can be expressed and the resulting
viral genome is permanently replication competent and passes
retrogradely to infect neurons synaptically linked to the Cre-
expressing neurons. In addition to neuropeptide Y-containing
neurons (17), this approach has been used to define circuits
synaptically linked to gonadotropin- and serotonin-containing
neurons (17–20). The considerable power inherent in both of
these adaptations of the viral tracing method lies in restricting
Author contributions: J.P.C., O.K., M.K.R., A.F.S., and L.W.E. designed research; J.P.C., O.K.,
J.M., S.E., Z.S., and A.F.S., performed research; O.K., Z.S., M.K.R., and L.W.E. contributed
new reagents/analytic tools; J.P.C., O.K., J.M., S.E., A.F.S., and L.W.E. analyzed data; and
J.P.C., O.K., A.F.S., and L.W.E. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1J.P.C. and O.K. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 22, 2011
| vol. 108
| no. 8
the ability of the virus to replicate or move transneuronally
between neurons. In addition to conferring important advan-
tages to directed analysis of defined neural circuits, these ap-
proaches also place limitations upon the way in which the
method can be applied. Chief among these limitations is the
need for targeted delivery of virus to neurons permissive to viral
replication (Table S1).
In this study, we describe a viral-tracing technology based on
PRV, where the virus is replication-competent in all neurons, but
expresses unique reporters in response to Cre-mediated re-
combination of the viral genome. This technology involves in-
sertion of the Brainbow 1.0L cassette developed by Lichtman
and colleagues (21) into the genome of a PRV recombinant
(PRV-152) commonly used for viral transneuronal tracing (Fig.
1A). The resulting virus (PRV-263) is replication-competent and
reproduces the selective retrograde invasive profile of PRV-152.
In the absence of Cre, PRV-263–infected neurons express only
a red reporter. However, when Cre is present, Cre-dependent
recombination of the Brainbow cassette efficiently, and perma-
nently, removes the red reporter gene and enables expression
either of yellow (EYFP) or cyan (mCerulean) fluorescent
reporters (22). We further demonstrate that this conditional
expression approach can be used to highlight neurons synapti-
cally linked to phenotypically defined neurons embedded within
a polysynaptic network retrogradely labeled from a projection
target. In our proof-of-principle experiments, selective expres-
sion of Cre was achieved in a specific group of rat brainstem
catecholamine neurons using a lentivirus vector in which Cre
expression is controlled by a synthetic dopamine-β-hydroxylase
(DβH) promoter (23, 24). Injection of the vector into the
brainstem followed by injection of PRV-263 into the kidney
allowed us to distinguish neurons synaptically linked to Cre-
expressing catecholamine neurons (yellow and cyan fluores-
cence) from neurons of the renal preautonomic network infected
through neural pathways that did not involve those neurons (only
red fluorescence). In this fashion, microcircuit connectivity to
targeted neurons was visualized within the context of larger
complex circuits labeled by projection-specific transport of virus
from a distant target. The availability of increasingly sophisti-
cated approaches for targeted expression of Cre indicates that
this unique approach can be applied across systems to provide
novel insights into the organization and plasticity of neural
circuits of mature and developing nervous systems.
Experimental Objectives and Design
PRV that marks neurons in a circuit but changes the expression
profile of reporter genes in phenotypically defined populations of
neurons within that circuit. There are three essential elements in
to introduce the virus to a circuit in a projection-specific fashion:
kidney) rather than depending on the presence of a molecule es-
provide a genetic means to change the profile of reporter-gene ex-
pression from the virus in a controlled, permanent, and highly re-
site-specific recombination provides a well-characterized system
for achieving this goal. The third element is to provide a method
for Cre expression in a targeted population of neurons within the
circuit. Lentivirus-mediated gene delivery is well suited for this
purpose in that Cre expression can be restricted to phenotypically
cell-specific promoters. In addition, lentivirus vectors can infect
a wide variety of animals, enabling use of this PRV technology in
different species. The details of our experimental approach are
provided in SI Materials and Methods.
Results and Discussion
Characterization of PRV-263 Neuroinvasiveness. We first compared
the neuroinvasiveness of PRV-263 to that produced by PRV-152,
the parental strain of PRV-263, in animals that were not pre-
viously injected with lentivirus vector. PRV-263 was injected into
the kidney and the pattern of retrograde transneuronal infection
was analyzed 4 to 7 d later using immunoperoxidase localization
of viral antigens. Fluorescent reporter expression was analyzed
in adjacent sections from each case. The immunoperoxidase
and fluorescence analyses demonstrated that the distribution
of infected neurons recapitulated our previous findings using
PRV-152 (12). Injection of PRV-263 into the kidney produced
a predictable course of retrograde transneuronal infection that
resulted from first-order infection of sympathetic postganglionic
neurons, transneuronal infection of sympathetic preganglionic
neurons (SPGs) in the thoracic cord, and retrograde trans-
neuronal infection of the central preautonomic network (Fig. 2).
PRV-263 invasion of this circuitry was temporally defined, with
initial infection of the brain occurring in the caudal brainstem
and progressing to circumscribed populations of neurons in all
major subdivisions of the neuraxis. Importantly, the virus re-
mained confined to the preautonomic network in all cases, with
the most extensive infection and fluorescence analysis demon-
strated that all infected neurons selectively expressed the red
dTomato reporter from the Brainbow cassette.
Dual Injections of Lentivirus Vector and PRV-263. The above
experiments demonstrated that the RVLM was first infected with
PRV-263 4 d after kidney injection and infection spread trans-
cassette in the gG locus of the PRV-152
was replaced with the Brainbow 1.0L
cassette (A) developed by Lichtman and
colleagues (21), using homologous re-
combination. Cre-mediated recombina-
tion of the viral genome occurs at either
paired lox2272 or loxP sites, permanently
eliminating the red dTomato reporter
and, depending upon the site of re-
combination, liberating expression of
either mCerulean or EYFP. Images B-D
illustrate the phenotype of cultured SCG
neurons infected with PRV-263 grown in
Cre or non-Cre cells. SCG neurons infec-
ted by PRV-263 grown in non–Cre-
expressing PK15 cells only fluoresce red
(B). In contrast, infection of neurons
from virus produced by Cre-expressing
PK-15 cells revealed expression of the EYFP or mCerulean genes of the Brainbow cassette (C and D). (Scale bars, 50 μm.)
To prepare PRV-263, the EGFP
| www.pnas.org/cgi/doi/10.1073/pnas.1015033108 Card et al.
neuronally to infect all major components of the preautonomic
network by 5 d postinoculation. Thus, we chose these survival
intervals for in vivo analysis of the fluorophor profile of infected
expression from the lentivirus vector is robust by 7 d and stable
through 64 d, we injected the vector unilaterally into the left
RVLM 7 d before injection of PRV-263 into the left kidney (Fig.
2). Thus, we had two informative survival intervals to assess Cre-
mediated recombination in RVLM neurons and transneuronal
passage to afferent neurons. Our analysis of tissue from each
survival interval was done in two steps. We first conducted a de-
tailed quantitative analysis of the distribution of infected neurons
in coronal sections using immunoperoxidase localization of viral
analysis, in which the fluorophor profiles ofinfected neurons were
documented. Sections adjacent to those processed for the immu-
noperoxidase analysis were systematically examined for fluo-
rophor expression using fluorescence microscopy.
The distribution of infected neurons revealed in immunoper-
oxidase localizations of each case was mapped in 48 coronal
sections that provided a detailed sampling of the renal pre-
autonomic network (Figs. S2 and S3). In addition, we analyzed
the extent of infection in horizontal sections of the entire spinal
cord processed for immunocytochemical localization of viral
antigens. PRV-263 invaded the circuitry linked to the kidney
with kinetics and a pattern of infection that recapitulated in-
fection when PRV-263 was injected alone (detailed above). At
the early survival interval (4 d), infection in the spinal cord was
largely confined to the sympathetic preganglionic neurons of the
thoracic spinal cord and infected brainstem neurons were dif-
ferentially concentrated in cell groups that constitute the baro-
receptor reflex (RVLM, caudal ventrolateral medulla, and
nucleus of the solitary tract) and in the A5 catecholamine cell
group. It is well established that the dense descending projec-
tions of A5 and the RVLM account for the early infection of this
brainstem circuitry (12). The paraventricular nucleus (PVN) of
the hypothalamus also projects densely to SPG in the thoracic
intermediolateral cell column (IML) and a subset of animals at
the short survival interval exhibited a small number of infected
PVN neurons (Fig. S2). The extent of infection within the pre-
autonomic network was substantially greater 5 d postinocula-
tion. Prominent among the expanded circuitry infected 5 d after
injection of PRV-263 into the kidney, was the ventromedial me-
dulla (VMM) surrounding the pyramidal tracts, the locus coe-
ruleus (LC), and hypothalamic cells groups that play essential
roles in coordinating behavioral state and arousal with adaptive
changes in physiology.
Immunofluorescence analysis revealed differential expression
of the three fluorescent reporters from the Brainbow cassette
based upon the location of the neurons within the preautonomic
circuitry. Neurons in the sympathetic ganglia and the IML only
expressed the red dTomato reporter (Fig. S4). This expression
profile is consistent with selective retrograde transport of PRV-
263 from the kidney and the absence of Cre from this component
of the circuitry (Fig. 2). The left RVLM, which was injected with
the lentivirus vector, demonstrated Cre-mediated recombination
of the PRV-263 genome in all animals. It is important to note that
the overlapping wavelengths for detection of EGFP (the reporter
of the Brainbow cassette) do not permit their differential locali-
zation in the fluorescence analysis. Nevertheless, our parametric
expressed in the presence of Cre and that lentivirus-mediated
transgeneexpression isconfinedtotheC1catecholamine neurons
in the RVLM. Thus, although we cannot unambiguously distin-
guish EYFP and EGFP in the fluorescence analysis, we can assert
with confidence that mCerulean and EYFP expression in the
preautonomic network are the result of Cre-mediated recom-
bination of the PRV-263 genome in C1 neurons.
Within the left RVLM we observed neurons with mixed and
pure color profiles with a distribution consistent with the known
circuit organization of the RVLM (Fig. 3 B and C). At early
survival intervals, the majority of these neurons were concen-
trated in the rostral portion of the RVLM that is the principal
source of descending C1 projections to SPGs in the IML of the
thoracic spinal cord (27, 28). A day later, neurons throughout the
RVLM exhibited yellow, green, and cyan fluorescence, either
alone or in combination. Importantly, a population of RVLM
neurons also exhibited only red fluorescence, even in animals
with the most extensive infection of CNS circuitry (Fig. 3 B and
C). Although it is well documented that C1 and non-C1 neurons
both contribute to the descending reticulospinal projections of
the RVLM to the IML (29–31), little is known regarding the
synaptic interaction between neurons giving rise to these path-
ways within the RVLM. The fact that both of these populations
(red only and cyan/yellow) remain stable in cases with the most
extensive infection raises the possibility that projections from the
RVLM to the spinal cord are organized in parallel. Nevertheless,
a more detailed analysis incorporating longer surviving animals is
necessary to rigorously test this hypothesis.
We sought further evidence of the utility of the method for
deciphering the synaptic organization of microcircuits within
complex neural networks by examining the fluorescence profiles
of neurons known to project to the RVLM. Toward this end, we
documented the fluorophor profile of infected neurons through-
out the renal preautonomic network. That analysis revealed
neurons expressing the conditional reporters were a clear and
reproducible subset of the neurons within the network. Areas
expressing cyan/yellow reporters included the contralateral
RVLM, VMM, LC, PVN, and the suprachiasmatic nucleus
(SCN) (Fig. 3 A–J). Data derived from each of these regions
illustrated. Selective expression of Cre in C1 neurons of the RVLM was ach-
ieved using a replication defective lentivirus vector that expresses Cre under
the control of a synthetic DβH promoter. Seven days after vector injection,
renal preautonomic circuitry was infected by retrograde transneuronal
transport of PRV-263 from the kidney. The sagittal schematic of the rat brain
illustrates the major cell groups that comprise the renal preautonomic net-
work (black). Transneuronal passage of PRV-263 to the RVLM infected C1
and non-C1 neurons via their projections to the intermediolateral cell col-
umn (IML) of the thoracic spinal cord. Restricted expression of Cre in C1
neurons limited recombination of the PRV-263 genome to this catechol-
amine population, permanently removing the red dTomato gene and en-
abling the expression of either EYFP or mCerulean transgenes. Infected
neurons in the RVLM, and in parallel pathways that did not contain Cre, did
not undergo recombination of the viral genome and remained red. A5, A5
catecholamine cell group; BN, Barrington’s nucleus; DMN, dorsomedial hy-
pothalamic nucleus; IC, insular cortex; LC, locus coeruleus; LHA, lateral hy-
pothalamic nucleus; MnPO, median preoptic nucleus; MoC, motor cortex;
PAG, periaqueductal gray; PVN, paraventricular hypothalamic nucleus; R,
raphe; RVLM, rostroventrolateral medulla; VMM, ventromedial medulla.
The experimental paradigm used for proof-of-principle studies is
Card et al.PNAS
| February 22, 2011
| vol. 108
| no. 8
validated the experimental approach and also provided unique
insights into the way in which the RVLM functions within the
larger renal preautonomic network. In interpreting these find-
ings it is important to note that, although we know Cre-
expression was restricted to C1 neurons, it is likely that the
lentivirus injection only produced Cre-expression in a subset of
the C1 population. Thus, it is also likely that reticulospinal C1
neurons are among the RVLM neurons expressing only the de-
fault dTomato reporter. In any event, the presence of condi-
tional reporters in infected neurons that project to the RVLM
provides unique insights into the components of the renal pre-
autonomic network synaptically linked to the C1 cell group.
Prominent replication of the recombined genome of PRV-263
(expression of cyan/yellow reporters) following retrograde trans-
neuronal transport of virus from Cre-expressing C1 neurons was
consistently observed within distinct subdivisions of the VMM
(Fig. 3 D–G). These subdivisions included neurons within the
raphe pallidus (Fig. 3D), the parapyramidal region that contains
the raphe magnus (Fig. 3E), and a compact group of neurons in
the area of the lateral paragigantocellular cell group (Fig. 3 F
and G). The distribution of these neurons within these regions
was consistent among all of the animals in the 5-d survival group
and, importantly, constituted a subset of the total population of
infected neurons. Neurons expressing the red dTomato reporter
were observed throughout the rostrocaudal extent of the raphe
pallidus, but cyan and yellow fluorescing neurons were differ-
entially concentrated in the rostral two thirds of this serotonergic
cell group. Serotonergic neurons of the raphe magnus are a
prominent component of this region and, although we were not
able to characterize the neurotransmitter phenotype, the mor-
phology and distribution of the cyan/yellow-labeled neurons
suggest that at least a portion of these neurons express serotonin.
CFP/YFP-expressing neurons in the lateral paragigantocellular
region also formed a distinctive grouping immediately lateral to
vector into the RVLM are illustrated. The red lines in the sagittal schematic diagram A define the relative location of the regions included in the analysis. The
red boxes in the coronal schematics included in the upper left portions of B to J illustrate the location of the cell group for each photographed area. Neurons
exhibiting red, yellow, and cyan fluorescence were prominent within the RVLM injected with the Cre-expressing vector (B and C). Neurons in all major
subdivisions of the VMM exhibited neurons expressing the cyan and yellow reporters among neurons only expressing the red reporter. These included
neurons in the rostral portion of the raphe pallidus (D) and both the medial (E) and lateral (F and G) subdivisions of the VMM. Neurons in the ventral tier of
the LC were infected by retrograde transneuronal passage of PRV-263 (H). The presence of yellow and cyan reporters in essentially all of these neurons
indicates that the LC exerts its influence upon cardiovascular function through synaptic contacts with the C1 catecholamine neurons of the RVLM. Cyan and
yellow fluorophors were differentially concentrated within a distinct subfield of the medial parvicellular subdivision of the PVN (arrow in I) and within the
SCN (J) of animals with the most extensive infection of the preautonomic circuit. See text for discussion of these data. (Scale bars, 50 μm.)
Fluorophor profiles of neurons in different regions of the renal preautonomic network in animals infected with PRV-263 7 d after injection of the Cre-
| www.pnas.org/cgi/doi/10.1073/pnas.1015033108 Card et al.
the pyramids that was present bilaterally, but most extensive
ipsilateral to Cre-expressing C1 neurons (Fig. 3 F and G). The
VMM is a well-documented component of the preautonomic net-
work labeled by viral transport from a variety of peripheral tissues
(e.g., refs. 11, 12). Functional roles for VMM neurons in the
control of thermogenesis, cardiovascular function, and noci-
ception are well established (32–36). In addition, Mason (37) has
provided evidence that neurons in this region play an important
role in multimodal sensory processing, some of which is relevant
to cardiovascular homeostasis. Our data bring further clarity to
the identity of the subset of neurons within this region that in-
fluence arterial blood pressure through their connections with
the C1 catecholamine neurons.
A considerable amount of literature has demonstrated that the
LC plays an important role in coordinating behavioral state and
arousal with adaptive changes in physiology (38, 39). Activity in
the LC mirrors behavioral state, with increased arousal and task-
related behavior correlating with an increased firing rate of LC
neurons (40). Our data are consistent with the prior demon-
strations that neurons in the ventral tier of the LC project upon
the RVLM and other nodes within the preautonomic network
(Fig. 3H). The data further demonstrate that the vast majority of
neurons infected by retrograde transneuronal transport of PRV-
263 from the kidney expressed yellow and cyan reporters (Fig.
3H). This finding demonstrates that ventral tier LC neurons
exert their influence upon the RVLM through synaptic con-
nections with C1 catecholamine neurons.
The PVN exerts prominent influences over endocrine and
autonomic regulation, particularly as it relates to cardiovascular
homeostasis and responses to stress (41, 42). Retrograde trans-
neuronal passage of virus from peripheral tissues infects PVN
neurons in all of the preautonomic subdivisions of this complex
cell group, but the precise pathways through which the PVN
controls autonomic outflow in an organ-specific fashion are not
known. Retrograde transneuronal infection of the PVN by in-
jection of PRV-263 into the kidney recapitulated the findings of
prior viral-tracing studies. We observed infected neurons in the
dorsal and medial parvicellular PVN subdivisions, but also
documented a differential pattern of reporter-gene expression in
the medial and dorsal parvicellular subfields of the PVN (Fig.
3I). Whereas neurons exhibiting red reporter-gene expression
were present in both of these subfields, neurons expressing cyan
and yellow reporters were largely confined to a distinct region
within the medial parvicellular PVN (arrow in Fig. 3I). These
data indicate that the influence of the PVN upon RVLM C1
neurons arises from neurons in the medial parvicellular PVN,
and that dorsal parvicellular PVN neurons are acting through
a pathway independent of the C1 population.
We also observed neurons in the SCN in the animals with the
most extensive viral transport (e.g., see cases 10 and 11 in Fig.
S2). These neurons were present in both the dorsomedial and
ventrolateral subdivisions of the SCN and were of mixed phe-
notypes (Fig. 3J). The majority of the neurons expressed the red
fluorophor but a subset expressed the cyan and yellow fluo-
rophors. It is well known that cardiovascular homeostasis
exhibits a circadian rhythm that is subject to the control of the
SCN (43, 44). In addition, a recent study demonstrated that
a mutation in the Per2 clock protein, an integral component of
the molecular machinery in SCN neurons responsible for the
generation of rhythmicity, interferes with the circadian expres-
sion of rhythms of heart rate and blood pressure (45). Prior
studies have demonstrated retrograde transneuronal infection of
SCN neurons after injection of PRV into the adrenal gland (46),
pineal gland (47), liver (48), and autonomic ganglia (49). On the
basis of these data it has been postulated that the SCN orches-
trates the rhythmic functional activity of autonomic targets.
Furthermore, dual infection studies with viruses expressing
unique reporters have provided evidence that there is functional
parcellation of the SCN with respect to the peripheral organ
systems that the clock regulates (46). Our data are in accord with
this conclusion in that the neurons expressing cyan and yellow
reporters are a subset of the total population of infected neurons
in the SCN. The fact that these neurons are only present in the
cases with the most extensive infection further supports the
conclusion that the influence of the SCN upon the RVLM C1
population occurs through a relay. Further directed analysis of
this circuitry provides a means of defining this relay.
Summary and Conclusions
Our experimental approach using PRV Brainbow recombinants
provides a powerful beginning to defining the identity and orga-
nization of circuits synaptically linked to phenotypically identified
design, the power of the PRV Brainbow virus derives from local-
ized expression of Cre within components of a circuit and reveals
unique insights into the distributed circuit of neurons synaptically
linked to reticulospinal C1 neurons in the renal preautonomic
network. In preliminary studies presented in Fig. S5, we demon-
a universal promoter can be effectively applied to define routes of
viral transport through a node within a circuit independent of the
neurotransmitter phenotype of the resident neurons. This ability
andtheincreasing availability oftransgenic animalsengineered to
express Cre in phenotypically defined neurons substantially ex-
pand the experimental scope of the approach.
from in vitro studies with PRV-263 that demonstrated a limited
number of genomes are expressed in infected cells (22). We also
used this cassette to optimize the possibility that a recombination
of lox sites for Cre-mediated recombination. This feature may
have contributed to the remarkably bright and diverse hues
produced by combinatorial expression of different fluorescent
proteins observed in our analysis. This feature permitted un-
ambiguous identification of neurons replicating recombined
genomes, and also offers the possibility of circuit analysis at the
single-cell level. However, it should be noted that Cre-mediated
expression of a single novel reporter (e.g., cyan) would further
expand the utility of the conditional approach by allowing phe-
notypic characterization of neurons expressing the conditional
reporter in dual labeling immunofluorescence localizations.
This conditional reporter approach for viral transneuronal
circuit analysis also holds substantial promise for production of
more powerful probes of circuit organization that will increase
the versatility of the viral-tracing method. For example, construc-
tion of viruses that conditionally express membrane-tethered
fluorescent proteins can expand the power of the approach by
densely labeling the axons of identified infected neurons. Con-
sidered collectively, these unique tools have the potential to
substantially improve the resolution of viral tracing by revealing
details of synaptology of identified populations of neurons within
the context of functionally related partners in complex networks.
Materials and Methods
are provided in SI Materials and Methods. SI Materials and Methods also
describes the in vitro studies conducted to validate Cre-mediated re-
combination of the PRV-263 genome. All experimental procedures were ap-
ACKNOWLEDGMENTS. We thank Jeff Lichtman for the Brainbow plasmids
and acknowledge members of the L.W.E. and J.P.C. laboratories for advice,
encouragement, and technical assistance. O.K. is funded by the Interna-
tional Human Frontier Science Program. This research was supported
by National Institute of Health Grants 1RC1NS068414, 1R01HL093134, and
P40 RR018604, and National Science Foundation Grant 0918867.
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Card et al.PNAS
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