Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.
ABSTRACT The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.
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ABSTRACT: The proper connectivity between neurons is essential for the implementation of the algorithms used in neural computations, such as the detection of directed motion by the retina. The analysis of neuronal connectivity is possible with electron microscopy, but technological limitations have impeded the acquisition of high-resolution data on a large enough scale. Here we show, using serial block-face electron microscopy and two-photon calcium imaging, that the dendrites of mouse starburst amacrine cells make highly specific synapses with direction-selective ganglion cells depending on the ganglion cell's preferred direction. Our findings indicate that a structural (wiring) asymmetry contributes to the computation of direction selectivity. The nature of this asymmetry supports some models of direction selectivity and rules out others. It also puts constraints on the developmental mechanisms behind the formation of synaptic connections. Our study demonstrates how otherwise intractable neurobiological questions can be addressed by combining functional imaging with the analysis of neuronal connectivity using large-scale electron microscopy.Nature 03/2011; 471(7337):183-8. · 38.60 Impact Factor
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ABSTRACT: Understanding the brain will require unraveling its synaptic circuitry, but methods that can reliably identify connected neurons are often excruciatingly slow. Although light microscopy can provide much higher throughput, synapses are smaller than the diffraction limit and cannot readily be assigned to particular presynaptic and postsynaptic cells without specialized labeling methods. Here we review the ongoing development of techniques that allow direct imaging of neural networks by specifically marking connected cells or their synapses.Current opinion in neurobiology 02/2012; 22(1):121-7. · 7.21 Impact Factor
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ABSTRACT: A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.Proceedings of the National Academy of Sciences 08/2013; · 9.81 Impact Factor
Fluorescence-Based Monitoring of In Vivo Neural Activity
Using a Circuit-Tracing Pseudorabies Virus
Andrea E. Granstedt, Moriah L. Szpara, Bernd Kuhn, Samuel S.-H. Wang, Lynn W. Enquist*
Department of Molecular Biology, Neuroscience Institute, Princeton University, Princeton, New Jersey, United States of America
The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report
activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically
connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal.
Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro.
With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine
peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct
electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a
time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent
and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence
calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects
of infection with single cell resolution.
Citation: Granstedt AE, Szpara ML, Kuhn B, Wang SSH, Enquist LW (2009) Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing
Pseudorabies Virus. PLoS ONE 4(9): e6923. doi:10.1371/journal.pone.0006923
Editor: Pedro R. Lowenstein, Cedars-Sinai Medical Center and University of California Los Angeles, United States of America
Received May 26, 2009; Accepted July 22, 2009; Published September 9, 2009
Copyright: ? 2009 Granstedt 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: A.E.G. was supported by the National Defense Science and Engineering Fellowship; this research was funded by NIH grants NS060699 (L.W.E.) and
NS045193 (S.S.-H.W.), a 2004 Keck Distinguished Young Scholar in Medical Research award to S.S.-H.W., and a New Jersey Commission on Spinal Cord Research
award to M.L.S. 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: email@example.com
Recording the activity of multiple neurons in intact circuits
remains challenging for in vivo study because current tools cannot
simultaneously report activity and connectivity. Electrophysiolog-
ical recordings and fluorescent calcium indicators can inform
about activity but not large-scale circuit connectivity. Genetic tools
offer complementary ways of distinguishing and targeting cell
types , but a combination of functional imaging and circuit
tracing is still missing. Conversely, commonly used chemical
tracers  and neurotropic viruses  can identify neuroanatom-
ical circuits but do not document activity. An ideal tool should
simultaneously report activity and connectivity with sensitivity and
reliability for long periods of time in vivo.
Transient increases in calcium [Ca2+] levels are a useful
correlate of neuronal activity. Calcium transients can be measured
by injected dyes or by genetically encoded fluorescent [Ca2+]
indicator proteins (FCIPs). G-CaMP2 is an FCIP composed of a
circularly permuted green fluorescent protein (GFP) linked to
calmodulin (CaM) and the CaM target sequence M13 . In the
presence of [Ca2+], CaM binds M13, resulting in increased
fluorescence [5,6]. The fluorescence intensity increases as a
function of [Ca2+] concentration. G-CaMP2 is a calcium sensor
that is stable at physiological pH and mammalian temperature .
Several publications have demonstrated the use of G-CaMP2 in
mammals in vivo [8,9].
Though FCIPs can reliably monitor neural activity, they need a
vehicle to cross synapses and thereby report connectivity.
Neurotropic viruses have proven useful to this end . In
particular, pseudorabies virus (PRV) has been used extensively
for elucidating neural circuits in the peripheral and central
nervous system in vivo [10,11]. One variant, PRV-Bartha, is an
attenuated, retrograde tracer that travels along chains of
synaptically connected neurons, and many PRV-Bartha deriva-
tives with fluorescent labels have been widely implemented for
circuit tracing . A recent publication reported the construction
of a gE-deleted PRV-Kaplan recombinant containing the ratio-
metric calcium indicator TN-L15, and demonstrated its use by
imaging explanted retina .
In PRV’s history of use as a neuroanatomical tracer in vivo,
studies have traditionally been done by infecting living animals
and subsequently studying fixed and labeled tissues. Advances in
imaging technology, such as two-photon microscopy , now
raise the possibility of using PRV to deliver fluorescent labels that
can be imaged in living animals. The submandibular ganglia
(SMG) present an opportunity to implement these methods: a
minor surgery allows optical access to these peripheral ganglia in
living mice . This preparation has recently been used to study
synaptic contacts and receptor dynamics over time in vivo . The
combination of a fluorescent calcium sensor, delivered virally to
optically accessible SMG neurons, provides a first opportunity to
image PRV live in vivo to explore both connectivity and activity.
By inserting the calcium sensor G-CaMP2 into the genome of
the classical PRV-Bartha tracing strain and monitoring activity in
vivo, we present the first use of PRV to express a fluorescent
calcium indicator protein and report fluorescence-based neuronal
activity in the living animal. We confirmed that this recombinant,
PRV369, could reliably detect single action potentials in vitro. We
PLoS ONE | www.plosone.org1 September 2009 | Volume 4 | Issue 9 | e6923
then studied fluorescence-based activity in vivo in the SMG. We
identified a window of 48 hours after inoculation in which
PRV369 can be used to look at calcium signals in these ganglia.
Infected neurons responded to external electrical and sensory
stimuli. After 72 hours we detected changes in intracellular [Ca2+]
concentrations and duration of [Ca2+] transients, indicating cell or
tissue responses to infection. PRV369 can be used for in vivo
investigation of physiological neuronal circuit activity and
subsequent effects of infection with single cell resolution.
PRV369 infected cells express G-CaMP2
PRV369 was constructed by homologous recombination and
replacement of mRFP1 in the glycoprotein G (gG) locus of
PRV614, a PRV-Bartha-derived tracing strain (Figure 1A). Viral
protein gG is not required for spread in vivo , and many of the
commonly used PRV viral tracing strains employ insertions at this
locus . The G-CaMP2 open reading frame is transcribed from
the cytomegalovirus immediate-early (CMV-IE) promoter (see
 for more details). Correct genomic insertion of the G-CaMP2
cassette was confirmed by Southern blot analysis (Figure 1B).
Western blots revealed no change in expression of upstream and
downstream proteins (data not shown) in comparison to the
parental PRV614 strain. PRV369 grows comparably to PRV-
Bartha in cultured epithelial cells (Figure 1C).
Fluorescence traces correlate with neuronal activity in
To test whether changes in fluorescence correlated with firing of
action potentials (APs), we infected dissociated mouse superior
cervical ganglion (SCG) neurons and recorded action potentials
with sharp electrodes while imaging corresponding fluorescence
changes with two-photon microscopy. A typical infected SCG
neuron is shown in Figure 2A. The electrical recording shows
action potentials and the optical recording displays the corre-
sponding [Ca2+] transients. We found that in all recordings, every
action potential correlated with a change in fluorescence
(representative trace in Figure 2A). This virally-delivered indicator
reliably reported single action potentials in vitro.
We analyzed the amplitude of fluorescence fluctuations in
response to action potential firing. Single action potentials were
common, whereas bursts of two or more action potentials were
scarce and irregular. Single, double, or triplet spikes could easily
be separated in the [Ca2+] transients by the maximal relative
fluorescence change (Figure 2B). In one cell we detected an
average fluorescence change of 8.461.6% for single action
potentials, 19.365.5% for spike doublets, and 32% for a triplet.
We defined spikes as doublets and triplets if the interspike interval
was less than 300 milliseconds (Figure 2B). Across four cells, we
compared the average maximal amplitude of fluorescence change
and found a statistically significant difference between fluores-
cence levels for one, two, and three action potentials (Figure 2C).
We then measured the t1/2from the peak, or the time it takes for
the fluorescence to decay halfway from peak amplitude
(Figure 2D). As expected, there was no statistically significant
difference between one, two, and three action potentials (one vs.
two, p=0.1; two vs. three, p=0.8), indicating that G-CaMP2 was
not saturated with calcium. These in vitro characterizations
ensured that this viral strain appropriately expressed and
delivered G-CaMP2 in infected cells. Therefore we progressed
to studying fluorescence-based activity under physiological
conditions in vivo.
Figure 1. PRV369 stably encodes G-CaMP2 in the gG locus. (A)
Map of the gG region of PRV, for the background strain PRV-Bartha, the
mRFP1-containing derivative PRV614, and the G-CaMP2-containing
derivative PRV369. The retrograde-limited spread phenotype of PRV
Bartha stems from a deletion in this region, which results in the fused
open reading frame of gI-Us2. The lines below the gene names are
marked with 1 kb spacing. Cross marks indicate regions of homologous
recombination used to generate PRV369. (B) Nucleocapsid DNA from
strains PRV Bartha, PRV614, and PRV369 were digested with Sal1 and
probed by Southern blot with a 1.9 kb fragment of Us3 and gG (gray
box below Bartha genome in (A)). Fragments observed were the
expected sizes, based on Sal1 cut sites denoted by arrows in (A): PRV
Bartha fragment of 2.6 kb, PRV614 fragments of 1.5 and 3.4 kb, and
PRV369 fragments of 1.6 and 3.5 kb. (C) Equivalent single-step growth
kinetics of PRV Bartha and PRV369 in epithelial cells in vitro. PK-15 cells
were infected at an MOI of 10, with input virus inactivated by a low pH
citrate wash after 1 hour. Each time point was performed in triplicate
and titered in duplicate. The average titer for each virus is plotted along
with the standard error of the mean (SEM) for each time point and virus.
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Infection by PRV369 enables visualization of
spontaneous activity in vivo
Using two-photon microscopy, we imaged the spontaneous
activity in a known neuronal circuit in vivo. The submandibular
ganglia (SMG) are peripheral parasympathetic ganglia that
innervate the salivary glands and receive input from the superior
salivatory nucleus in the brainstem; this circuit has already been
defined in viral tracing experiments [19,20]. We imaged the
SMG in the anesthetized mouse at 24, 48, and 72 hours after
PRV369 inoculation (Figure 3A-B and Supplemental Video S1).
The ganglia are located along the salivary duct and are accessible
for imaging in vivo. We perfused the area with warmed
mammalian Ringer’s solution and heated the animal to maintain
physiological temperature. We used a miniature platform to raise
ganglia away from surrounding tissues (Figure 3A), and only
ganglia that were within a few millimeters of the gland were able
to be lifted on the platform. Fluorescence changes related to
spontaneous activity were easily detectable in vivo (Figure 3B).
The number of infected neurons appeared to increase with time.
To count the total number of labeled neurons, we pooled data
from accessible ganglia in 3 animals at each time point. At
24 hours we counted a total of 65 labeled cells; at 48 hours, 133
Figure 2. PRV369 sensitively and accurately detects graded neuronal activity in dissociated neurons in vitro. (A) Sample recording of
an impaled neuron, with diagram indicating electrode position on an actual image of an SCG neuron (left). In response to every action potential
(bottom trace), a calcium event was detected in the fluorescence trace (top trace). (B) Analysis for one neuron of the percent fluorescence change for
single, double, or triple action potentials. Fluorescence increases proportionally to action potential number. Ticks indicate action potentials by
electrophysiology. Bold line indicates average. (C) Quantitative comparison across multiple neurons. The amplitude of the fluorescence peak is
significantly different between one, two, or three action potentials: the average DF/F was 7%, 19%, and 27%, respectively. (D) Comparison of
fluorescence decay from peak. There is no statistical difference in decay between one, two, or three action potentials. The average t1/2was 0.9, 1.1,
and 1.1 seconds, respectively. AP, action potential; DF/F, relative fluorescence change.
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cells; and at 72 hours, 280 cells. [Ca2+] signals were detectable at
a high signal-to-noise ratio because of the high relative
fluorescence change of G-CaMP2 upon [Ca2+]-binding .
These results show that infection by PRV369 was successful in
vivo and allowed optical analysis of an infected neuronal circuit in
a living animal.
Figure 3. Infection with PRV369 reports spontaneous activity in vivo. (A) Diagram of setup for in vivo imaging. SMG (light blue) are located
along the salivary duct (d) and send projections to the salivary glands (g) where the virus was injected. The platform (p) elevates the ganglia for
imaging under two-photon microscopy. (B) Traces of spontaneous activity at 24, 48, and 72 hours post inoculation (h.p.i.). The number of infected
cells increases with time, and there is also an increase in the number of calcium events. The average fluorescence change of these changes in vivo is
50%. Scale bar=20 mm. (C) The average duration of a calcium event increases with the time after inoculation, with 72 h.p.i. exhibiting the most
significant increase. The average duration is 7.661.1, 10.560.7, and 15.860.6 seconds for 24, 48, and 72 h.p.i., respectively. (D) The average percent
of time that a cell is in a state of elevated intracellular calcium concentration also increases with time after inoculation. The average percent
time was 1.060.3, 2.560.5, 6.360.5% for 24, 48, and 72 h.p.i., respectively. Labeled cells at 72 h.p.i. spend significantly more time in an elevated
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Time course of infection reveals changes in calcium
Infection by PRV-Bartha activates host defenses [21,22,23],
which may influence the behavior of neurons. Therefore we
sought to define an optimal window for using PRV369 to image
native network activity in the SMG. We observed the rate of
spontaneous calcium events at 24, 48, and 72 hours post
inoculation (h.p.i.) in the salivary glands (Figure 3C). Analysis of
the number of calcium events at each time point revealed a change
in event frequency as time after infection increased: At 24 and
48 h.p.i., the majority of infected neurons generated events
sparsely, but by 72 h.p.i., more cells were active and at higher
rates (example in Figure 3B). In addition, we observed a
statistically significant difference in the average duration of
calcium events at 72 h.p.i. compared to 24 or 48 h.p.i.
(Figure 3C). We further calculated the fraction of time that
labeled cells spent at elevated intracellular calcium levels and
observed a difference between 24 and 48 h.p.i., with a larger
difference occurring at 72 h.p.i. (Figure 3D, see also Supplemental
Figure S1). At 72 h.p.i. the cells displayed more complex patterns
in the calcium events, indicative of unusually large bursts of
activity (compare traces in Figure 3B). We conclude that in the
SMG, PRV369 is most useful for measuring circuit activity within
48 hours after inoculation. Since G-CaMP2 fluorescence of
infected SMG neurons appeared by 24 hours, there is a window
of about 24 hours in which to observe and analyze neuronal
activity with minimal effects of infection.
Labeled SMG neurons reveal responses to electrical and
sensory stimuli in vivo
We next used PRV369 infection to record stimulus-evoked (non-
spontaneous) activity in the SMG in vivo. We electrically stimulated
infected ganglia to evaluate the responsiveness of the infected
a wire electrode placed on the presynaptic axon bundle emanating
from the brainstem to innervate the SMG. This stimulus triggered
synchronouscalciumtransientsina subset ofthe neuronsinthe field
of view (Figure 4A). Occasionally, an axon initial segment was
clearly visible and also responded with a transient signal upon
stimulation (Figure 4A, dotted purple trace). As expected, the
calcium event triggered in the axon initial segment was of shorter
neuron (Figure 4A, solid purple trace). In general, DF/F values for
electrically stimulated calcium transients were on average smaller
and shorter-lasting than spontaneous transients, especially at later
time points of infection. Moving the electrode to different position
on the presynaptic axon bundle activated a different subset of
neurons (data not shown). Antidromic stimulation yielded similar
results, and neurons were responsive to electrical stimulation at 24,
48, and 72 hours after inoculation (data not shown).
We tested the sensory response of the SMG at 48 h.p.i. It has
been reported that hot (T=50uC) distilled water delivered to the
oral cavity elicits a salivatory reflex . We applied 40uC distilled
water to the oral cavity and observed a sensory-evoked response in
PRV369-labeled neurons. A subset of neurons exhibited a strong
response (Figure 4B and Supplemental Video S2) and sensory-
evoked events were of similar duration to spontaneous events.
Similar groups of neurons fired during repetitions of the sensory
stimulus (data not shown). In one animal, across three repeated
sensory stimulations, 12 out of 15 fluorescent cells showed sensory
responses; 2/3 of the responsive cells exhibited calcium transients
100% of the time (data not shown). Sensory responses were not
seen when the oral cavity was stimulated with distilled water at
room temperature (23uC) (data not shown), demonstrating that the
responses were temperature-specific. Taken together, these results
demonstrate the utility of PRV369 infection for analysis of
electrically evoked responses and physiologically relevant sensory
We have constructed a circuit-tracing PRV-Bartha derivative,
PRV369, that expresses the fluorescent [Ca2+] indicator G-
Figure 4. PRV369-infected SMG neurons respond to external electrical and sensory stimuli. (A) An electrical stimulus (arrow below traces)
elicited a sharp and transient response in a subset of neurons. The diagram indicates where the tungsten electrode (orange) was placed on the
innervating presynaptic axons from the brainstem (black bundle). Occasionally, the axon initial segment was visible (dotted purple trace) and
triggered a shorter calcium event compared to the cell body of the same neuron (solid purple trace). (B) When hot distilled water (T=40uC) was
delivered to the oral cavity (arrow below traces) as a natural sensory stimulus for salivation, SMG neurons produced a strong and synchronous
response. Scale bar=20 mm.
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CaMP2. This virus provides the capability to reliably detect
neuronal activity by fluorescent protein expression in intact
circuits in living animals. We demonstrated its use in vivo for
observing spontaneous and elicited neuronal activity in the
peripheral nervous system, with a useful window of at least
24 hours from the first appearance of labeled neurons. PRV369 is
isogenic with PRV152 (Bartha GFP)  and PRV614 (Bartha
RFP) , two of the most frequently used PRV-based viral circuit
tracers, facilitating the use of PRV369 in circuits already
elucidated by these tracers. PRV369 can also be used in dual-
[10,18,25]. The prior use of PRV in delineating central nervous
system (CNS) circuits (reviewed in  and ), and recent
advances in live imaging with other genetic calcium indicators in
the CNS (see for example  and ), suggest the potential
usefulness of PRV369 for revealing activity in synaptically
connected CNS neurons in vivo.
In contrast to PRV369, the recently published As1-PRV08
calcium sensor virus was constructed in a mutated PRV-Kaplan
strain background, which the authors found to have higher
infectivity and more cytotoxicity than several PRV-Bartha-derived
strains tested in their study . In addition, As1-PRV08
expresses the TN-L15 calcium sensor , a FRET-based
[Ca2+] indicator. In matched in vivo comparisons , G-CaMP2
was found to have faster kinetics than TN-L15, including a 4-fold
faster off-rate for calcium. In response to a strong stimulus,
Boldogkoi et al. found an average 13% increase in the citrine:CFP
fluorescence ratio in ganglion cells infected by As1-PRV08 . In
SCG neurons infected with PRV369 in vitro, the average
fluorescence change was 7% for a single action potential, and
27% for triplets. In the SMG in vivo, signals of up to 100% relative
fluorescence change were observed.
SMG neurons could not be impaled for intracellular recording
or bolus-loaded with calcium dyes in vivo without damaging the
neurons. The SMG is surrounded by extracellular connective
tissue, and each neuron is ensheathed by satellite cells . Most
studies have characterized SMG neuron activity with electrodes ex
vivo (for example, ) or extracellular recording in vivo . One
published method for intracellular recording of SMG neurons
under in vivo conditions necessitated a separate recording chamber
and partial extraction of tissue . PRV369 is thus noteworthy
for allowing characterization of neuronal activity in a circuit that is
otherwise difficult to access by previous recording methods. In the
future, PRV369 should prove useful for studying local circuits in
which multiple connected neurons are visible within the same field
of view, allowing noninvasive and simultaneous characterization of
network activity in multiple cells.
The endogenous activity of PRV369-labeled neurons can be
monitored for at least 24 hours with limited effects due to
infection. Most SMG neurons generated signals sparsely at 24 and
48 hours post inoculation into the salivary glands, consistent with
previous published data that spontaneous firing is rarely observed
from these cells . However, the number of infected cells and
firing frequency increased significantly by 72 hours. In addition,
the average duration of a [Ca2+] event lasted significantly longer at
late time points in infection. As suggested by cytological changes
observed in other circuits, the later time point may correlate with
the initiation of host defenses stimulated by infection . The
increased activity we observe at 72 hours post inoculation marks
the first live, in vivo observation of these long-suspected effects of
viral infection. One previous report monitored herpes simplex
virus infection in transgenic report mice using titer-dependent
bioluminescence techniques, but not at the level of single cells .
We envision that further experiments with PRV369 in infected
circuits in vivo will allow exploration into the nature of the host
response to infection. In summary, PRV369-labelling of connected
neurons allows for reliable and sensitive detection of endogenous
circuit activity early in infection, and will provide a long-sought
means to simultaneously reveal both connectivity and activity at
single cell resolution, in intact neural circuits in vivo.
Cells and virus construction
PRV 369 was constructed by homologous recombination
between a plasmid containing a G-CaMP2 expression cassette
and the gG locus of the PRV-Bartha genome. The plasmid pN1-
G-CaMP2 (pN1-RSET-mG1.6#X-1) was a gift of Dr. Junichi
Nakai , and contains the coding sequence of G-CaMP2 in place
of EGFP in the pEGFP-N1 plasmid, under the control of a
cytomegalovirus immediate-early (CMV-IE) promoter (Clontech;
GenBank Accession # U55762). A 990 bp fragment of PRV gG
was isolated by PstI and NdeI digestion of the previously described
pBB04 plasmid . This vector was cloned into the DraIII site of
pN1-G-CaMP2, downstream of the simian virus 40 (SV40)
polyadenylation sequences. This plasmid was linearized and co-
transfected into swine epithelial (PK15) cells with DNA from
PRV614, a PRV Bartha derivative with mRFP1 inserted into the
gG locus . Homologous recombination of the G-CaMP2
cassette into the gG locus of PRV614 occurred between the CMV-
IE promoter and the flanking gG sequence, replacing the original
2.3 kb mRFP cassette at this locus with the 2.5 kb G-CaMP2
cassette. Resulting recombinants were plaque-purified on PK15
cells by selection of non-red plaques. Southern blot analysis using a
1.9 kb piece of gG (EcoRI – HindIII fragment) as a probe
confirmed that the GCaMP2 cassette had recombined correctly
into the PRV genome, producing the recombinant PRV369.
Restriction fragment length polymorphism (RFLP) analysis using
BamHI, KpnI, and PstI was used to compare PRV Bartha, the
parental PRV614, and PRV369, confirming the correct integra-
tion of the G-CaMP2 cassette (data not shown). Western blot for
the upstream gene Us3 and downstream gene gD revealed no
changes in expression of these proteins relative to the parental
PRV614 strain (data not shown). PRV614 and related tracing
strains have a previously described decrease in Us3 expression
relative to PRV-Bartha .
Dissociated neuronal cultures of superior cervical ganglia
SCG neurons were used for in vitro characterization of PRV369
for ease of maintenance in dissociated culture. Methods for
harvesting and culturing SCGs have been described in detail
elsewhere . Briefly, SCG ganglia were harvested from mouse
embryos at embryonic day 15, dissociated, and allowed to settle on
tissue culture grade plates coated with 0.5 mg/ml poly-ornithine
and 10 mg/ml laminin. Neurons were cultured in NeurobasalTM
media containing, 1X Penicillin-Streptomycin-Glutamine solu-
tion, 1X B-27 supplement, and 50 ng/ml nerve growth factor (all
from Invitrogen), and allowed to mature for 10 days before using
Experiments in vitro were performed at room temperature. We
recorded spontaneous activity from 16 to 18 hours after infection.
The effect of PRV infection on dissociated superior cervical
ganglia neurons has been recently characterized (McCarthy,
Tank, Enquist, submitted). For intracellular recordings, quartz
micropipettes were pulled (P-2000, Sutter Instrument, Novato,
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CA) to a resistance of 40–60 MV and filled with 10 mM
fluorescein in 3 M KCl. Recordings were acquired at 5 kHz
(Axopatch 200B, Axon Instruments).
Infection of mouse submandibular ganglia (SMG)
All animal procedures were performed in accordance with the
guidelines of the National Institutes of Health and were approved by
local authorities (Princeton University Institutional Animal Care and
Use Committee). The model for mouse SMG infection was
previously described in detail . Briefly, mice aged 3–5 months
were anesthetized with a mixture of ketamine (100 mg/kg) and
xylazine (10 mg/kg) by intraperitoneal administration. A midline
incision was made along the neck, exposing the salivary glands. Using
a Hamilton syringe, concentrated PRV inoculum (1010pfu/ml) was
injected into both submandibularglands(5 ml/side).The incision was
sutured and the mice were given a dose of buprenorphrine (100 mg/
kg concentration) against post-surgical pain. The mice were allowed
to recover for various times post inoculation.
Calcium imaging in vivo
The protocol for imaging infected SMG in living mice was
adapted from Purves and Lichtman [15,40]. Mice were anesthe-
tized in an isofluorane chamber and then transferred in supine
positiontoa custom-builtstage with heatingpad, rectal temperature
probe (FHC Inc.),and nose cone for isoflurane inhalation. A ventral
midline incision was made along the neck and the skin was pulled
back with retractors. The SMG were exposed and lifted on a small
custom-made metallic platform, controlled by a micromanipulator.
The area was perfused with warmed mammalian Ringer’s solution
containing(in mM): 154 NaCl, 5.6KCl, 2.4NaHCO3, 2 Trisbuffer
pH 8.0, and 2.2 CaCl2, adjusted to pH 7.4 with HCl. The animal’s
temperature was maintained at 35uC.
Electrical and sensory stimulation
We used a tungsten electrode (1 MV resistance) to electrically
stimulate pre- and post-synaptic axons by 10 pulses of 60 V,
delivered every 0.1 ms at 100 Hz (ISO-Flex triggered by Master-
8, A.M.P.I.). For the sensory stimulation, we injected aliquots
(300 ml) of hot water (40uC at outlet) at a pressure of 8 psi to the
mouth by air-pressure from a picospritzer (Pressure System IIe,
Toohey Company) while simultaneously imaging the SMG. The
tubing was heated electrically.
Two-photon fluorescence imaging was performed on a custom-
built microscope with a tunable Mira 900 Titanium:sapphire laser
(Coherent) at 910 nm exciation wavelength and a GaAsP
photomultiplier tube (H7422P-40, Hamamatsu). The microscope
was controlled by CfNT software (M. Mu ¨ller, Max Planck Institute
for Medical Research, Heidelberg, Germany). We used a 40x, 0.8
N.A. water-immersion objective (HCX APO, Leica) to record
movies with 2566256 or 1286128 pixel at 2 ms/line scan rate.
The two-photon raw data was first opened in ImageJ (W. S.
Rasband, ImageJ, National Institutes of Health, Bethesda,
Maryland, http://rsb.info.nih.gov/ij/). We defined an infected
neuron as 30% brighter than background fluorescence from an
area outside of the ganglia in the same field of view. We
established this threshold because uninfected cells have punctate
autofluorescence intensities that range from 10–20% above
background. A region of interest was drawn around each infected
cell, and the mean intensity over time was saved and further
analyzed using Igor Pro 6 software (WaveMetrics, Inc., http://
www.wavemetrics.com). For DF/F measurements in plots, the
background fluorescence was calculated by fitting a curve. Peaks
were identified using a custom-written program in Python (Python
Software Foundation). The analysis program used algorithms
based on a previous publication . The program identified a
baseline and recognized peaks as points that were 0.75 intensity
units above the baseline and 10% above baseline noise ratio.
calcium event will last a certain length of time (in seconds) for 24,
48, or 72 hours post inoculation (h.p.i.). At 72 h.p.i., the
occurrence of long-lasting calcium events is more frequent than
earlier time points, and only at this time point do calcium events
last longer than 50 seconds.
Found at: doi:10.1371/journal.pone.0006923.s001 (0.01 MB
Probability distribution that the occurrence of a
neurons, expressing G-CaMP2 delivered by PRV369. Movies
were acquired at 2566256 pixels and 2 ms/line for 800 frames,
and played back at 40 frames per second (20 times sped up). The
movies were recorded at 24, 48, or 72 hours post inoculation, in
order of appearance.
Found at: doi:10.1371/journal.pone.0006923.s002 (7.96 MB
Spontaneous activity of submandibular ganglia (SMG)
neurons, expressing G-CaMP2 delivered by PRV369. Movies
were acquired at 2566256 pixels and 2 ms/line, for 50 frames in
electrical stimulus and 400 frames in sensory stimulus, and played
back at 20 and 40 frames per second (10 and 20 times sped up),
respectively. Both movies were recorded at 48 hours post
inoculation. The white rectangle indicates the time of stimulus.
To reduce the amount of movement, the sensory-evoked stimulus
movie was stabilized with ImageJ plugin: (http://www.cs.cmu.
Found at: doi:10.1371/journal.pone.0006923.s003 (2.98 MB
Stimulus-evoked activity revealed in labeled SMG
We thank T.D. Parker for her contribution in virus construction, E.M.
Granstedt for writing code to analyze calcium events, D.W. Tank for
building the imaging stage, C. Chiriac for technical support, J. Swan for
feedback on manuscript, and all members of the Enquist and Wang
laboratories for input and discussions.
Conceived and designed the experiments: AEG MLS BK SSHW LWE.
Performed the experiments: AEG MLS BK. Analyzed the data: AEG MLS
BK. Contributed reagents/materials/analysis tools: AEG MLS BK SSHW
LWE. Wrote the paper: AEG MLS BK SSHW LWE.
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