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INTRODUCTION |
Heart rate is determined
primarily by the centrally mediated tonic and reflex activity of
parasympathetic outflow to the heart (Heymans and Neil
1958
; Spyer 1981
). Cardiac vagal activity is diminished and unresponsive in many disease states, and a delay in the
inhibitory actions of this autonomic motor system following exercise is
a powerful predictor of overall mortality (Cole et al.
1999
; Eckberg et al. 1971
; Larovere et
al. 1988
; Vanoli et al. 1991
). The control of
heart rate is also intertwined with the generation of respiratory
rhythm within the CNS (Anrep et al. 1935
; Gilbey
et al. 1984
). In each respiratory cycle, the heart beats more
rapidly in inspiration and slows during postinspiration and expiration
(referred to as respiratory sinus arrhythmia) (Anrep et al.
1935
). The degree of respiratory sinus arrhythmia is often a
clinical indicator of cardiorespiratory function, particularly during
the neonatal and postnatal period (Hathorn 1978
;
Hrushesky 1991
). Despite the clinical importance of
respiratory modulation of heart rate, the neuronal circuit for this
cardiorespiratory interaction has not yet been identified. One possible
source of cardiorespiratory interactions may involve superior laryneal
neurons that are active in respiration and are colocalized with cardiac vagal neurons in the nucleus ambiguus. Here we demonstrate that superior laryngeal motorneurons project to cardiovagal neurons. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alphaherpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and
then travels transsynaptically to continue labeling the second- and
higher-order neurons in a time-dependent manner (Jansen et al.
1995
; Jons and Mettenleiter 1997
; Loewy
1998
). Superior laryngeal motorneurons were tested as likely
mediators of cardiorespiratory interaction because they are active in
the postinspiration phase of respiration and, apart from their
innervation of laryngeal muscles, they send axonal branches in close
proximity to cardiovagal neurons within the brain stem (Gauthier
et al. 1980
; Zheng et al. 1991
).
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METHODS |
Retrograde transneuronal virus and fluorescent labeling
The gene encoding enhanced GFP, a bioluminescent protein
of 238 amino acids originally isolated from the jellyfish
Aequorea victoria, was inserted in the nonessential
glycoprotein G (gG) gene of the transneuronal viral tracer Bartha-PRV.
The coding sequence was cloned in frame behind the first seven codons
of the gG gene under control of the strong gG promotor in the
Bartha-PRV. To label cardiovagal neurons and the neurons that project
to them, 0.5-20 µL of virus [titer =108
plaque forming units per milliliter (pfu/ml)] was injected into the
pericardial sac of methoxyflurane anesthetized Sprague Dawley rats
(p4-p13) of either sex. In control experiments, injections of
Bartha-PRV-GFP into the chest cavity, but outside the pericardial sac,
did not produce any labeling in the brain stem. In immunohistochemical and electrophysiological experiments, a bilateral stellectomy was
performed to remove the sympathetic innervation to the heart (Pardini et al. 1989
). To independently identify
superior laryngeal motorneurons and cardiovagal neurons, crystals of
the fluorescent tracer tetramethylindocarbocyanine perchlorate
(DiI) were placed onto the superior laryngeal nerve and
rhodamine (1% solution XRITC, 20-50 µL) was injected into the
pericardial sac.
Immunohistochemistry procedures
In immunohistochemistry studies, the animals were anesthetized
with methoxyflurane and perfused through the heart with 0.9% saline,
followed by 4% paraformaldehyde, stored for 12-24 h, and then the
brain stem was cut into 30-µm-thick sections using a cryostat. Tissue
sections were incubated for 1 h in 0.1 M PBS, 2% bovine serum
albumin (BSA), and 0.3% Triton X-100, washed, and incubated overnight
at 4°C in a 1:1000 dilution of GFP monoclonal antibody (Clontech),
followed by a 1:100 dilution of goat anti-mouse IgG fluorescein
isothiocyanate (FITC) conjugate secondary antibody (Sigma).
Specificity was determined by omitting the primary antibody from the
incubation or by omitting the secondary antibody. Slices that included
the nucleus ambiguus were imaged with a Bio-Rad (Hercules, CA) MRC-1000
confocal microscope.
Electrophysiological and in vivo studies
For electrophysiological experiments, the animals were
anesthetized with methoxyflurane and killed by cervical dislocation, and the brain was sectioned into 250-µm sections. Neurons were imaged
with infrared and fluorescent illumination. Standard patch-clamp electrophysiological techniques were used while the slices were continuously perfused (2-3 ml/min) with a perfusate of the following composition (in mM): 125 NaCl, 3 KCl, 2 CaCl2, 26 NaHCO3, 5 dextrose, and 5 HEPES, constantly
bubbled with 95% O2-5%
CO2, and maintained at pH 7.4. Patch pipettes
were filled with a solution consisting of (in mM): 130 KGluconate, 10 HEPES, 10 EGTA, 1 CaCl2, and 1 MgCl2, in perforate patch experiments nystatin
(258 U/ml) was included. Action potentials were recorded using the
perforated-patch access and current-clamp configuration. Voltage-gated
ionic currents were studied using the voltage-clamp configuration.
Studies of the baroreceptor reflex were conducted in age-matched
animals 10-12 days old (anesthetized with urethan, 1.3 mg/kg, IP)
using standard phenylephrine infusions to increase blood pressure and evoke the baroreflex decrease in heart rate. The baroreflex curves were
generated (Origin 5.0) using a sigmoidal logistic fit,
y = A2 + (A1
A2)/[1 + (x/x0)p]. Two
animals had a paradoxical increase in heart rate and were omitted from
analysis. A paradoxical response in rats this age has also been
observed by others (Kasparov and Paton 1997
). All
experiments were performed in compliance with institutional guidelines
at George Washington University.
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RESULTS |
To identify cardiovagal neurons and in particular the neurons that
synapse on them, we first performed a series of experiments in which
Bartha PRV-GFP was injected into the pericardial sac of Sprague-Dawley
rats with increasing survival periods. After two days of survival, only
cardiovagal neurons were labeled in the nucleus ambiguus (Fig.
1). The labeling pattern was identical to
the population of cardiovagal neurons labeled with conventional retrograde fluorescent tracers such as rhodamine (XRITC) (Fig. 1). To
label superior laryngeal motorneurons, the fluorescent tracer DiI was
applied to the superior laryngeal nerve 2 days prior to death. Superior
laryngeal motorneurons were in close proximity to cardiovagal neurons
labeled 2 days after injection of Bartha-PRV-GFP into the pericardial
sac, but no superior laryngeal motorneurons contained the virus (Fig.
1).

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Fig. 1.
Cardiovagal neurons were labeled with Bartha pseudorabies virus-green
fluorescent protein (PRV-GFP) 2 days after injection of the virus into
the pericardial sac. A: Bartha-PRV-GFP (green) was
present only in cardiovagal neurons identified with the traditional
fluorescent rhodamine tracer XRITC (red). All cardiovagal neurons
contained both virus and rhodamine (yellow, n = 6).
B: superior laryngeal motorneurons labeled with the
fluorescent tracer DiI (red) were in close proximity to, but were not
colocalized in, neurons labeled with the Bartha PRV-GFP (green) 2 days
after injection of the virus into the pericardial sac
(n = 7). Scale bar is 25 µm.
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The utility of Bartha-PRV-GFP as a transneuronal tracer in living
tissue was examined electrophysiologically 2 days after virus injection
into the pericardial sac. Cardiovagal neurons could be easily
visualized by both a traditional fluorescent tracer (XRITC) and the GFP
fluorescent signal in an in vitro 250-µm slice of tissue 2 days after
injection of the virus and XRITC into the pericardial sac (Fig.
2). The electrophysiological properties of both XRITC-labeled and PRV-infected cardiovagal neurons were analyzed by patch-clamp recordings. The voltage-gated currents and
firing properties in Bartha PRV-GFP or XRITC-labeled cardiovagal neurons were indistinguishable. Cardiovagal neurons were silent with a
typical resting membrane potential of
70 mV. On injection of
depolarizing current cardiovagal neurons had a pattern of action potential firing with little delayed excitation or spike frequency adaptation (Fig. 2). Depolarizations to potentials more positive than
50 mV evoked a rapidly activating and inactivating sodium current
that has been shown previously to be tetrodotoxin resistant, requiring
1 µM tetrodotoxin for complete block (Mendelowitz 1996
, 1999
). Immediately following the sodium current transient
outward and long-lasting potassium currents were elicited which consist of an A-type and a delayed rectifier potassium current, respectively (Mendelowitz 1996
, 1999
).

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Fig. 2.
Bartha-PRV-GFP is compatible with normal electrophysiological
recordings in vitro. A: cardiovagal neurons could be
identified by the GFP fluorescent signal (green), visualized using
infrared wavelengths (middle) as well as the traditional
fluorescent tracer XRITC (red) in an in vitro 250-µm slice of tissue
2 days after injection of the virus and XRITC into the pericardial sac.
Scale bar is 10 µm. B: the voltage-gated currents,
absence of firing at rest, and the depolarization evoked firing
activity of cardiovagal neurons identified with Bartha PRV-GFP
(left, n = 11) were identical to the
properties of cardiovagal neurons labeled with rhodamine
(right, n = 12). Scale bars in
current traces are 1 nA and 500 ms.
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After establishing that Bartha PRV-GFP could be used to identify
neurons in specific functional circuits for subsequent
electrophysiological experiments, a second series of
immunohistochemical experiments was performed in which animals were
killed 3 days after injection of virus into the pericardial sac. In
addition DiI was applied to the superior laryngeal nerve 3 days prior
to sacrifice. After 3 days, the labeling was still present in
cardiovagal neurons and advanced to other neurons in the nucleus
ambiguus as well as additional brainstem regions including the nearby
periambigual area and the nucleus tractus solitarius. Within the
nucleus ambiguus, superior laryngeal motorneurons were colabeled with
both DiI and Bartha-PRV-GFP, demonstrating that they innervate
cardiovagal neurons (Fig. 3). Superior
laryngeal motorneurons transsynaptically labeled 3 days after injection
of Bartha-PRV-GFP into the pericardial sac could also be readily
visualized by GFP fluorescence in an in vitro slice of tissue (Fig. 3).
The traditional retrograde tracer DiI also identified these neurons.
Superior laryngeal motorneurons typically fire spontaneously and
continuously at a frequency of 5-7 Hz. The firing properties and
individual action potentials in Bartha PRV-GFP- or DiI-labeled superior
laryngeal motorneurons were identical.

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Fig. 3.
Superior laryngeal motorneurons project to cardiovagal neurons.
Three days after injection of Bartha-PRV-GFP into the pericardial sac,
virus-labeled neurons included cardiovagal neurons and the neurons that
project to them. A: these neurons included superior
laryngeal motorneurons (green), which were also identified by the
retrograde fluorescent tracer DiI (red). Double-labeled superior
laryngeal motorneurons (yellow) contained both the traditional
fluorescent tracer DiI and the trans-synaptic tracer
Bartha-PRV-GFP (n = 6). Scale bar is 25 µm.
B: superior laryngeal motorneurons were identified in
vitro by the presence of the virus (green), visualized with infrared
wavelengths (middle) and by the traditional tracer DiI
(red, n = 5). Scale bar is 15 µm.
C: electrophysiological recordings in superior laryngeal
motorneurons labeled with the virus (n = 5) and DiI
(n = 14) were indistinguishable and are
electrophysiologically normal 3 days after injection of Bartha-PRV-GFP
into the pericardial sac.
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Since the electrophysiological studies indicate both cardiac vagal
neurons and superior laryngeal neurons labeled with the virus were
electrophysiologically indistinguishable from these neurons identified
with traditional tracers, we examined whether the baroreceptor reflex
was altered in animals 3 days after injection of the virus into the
pericardial sac. Phenylephrine was infused to increase blood pressure
and evoke baroreflex mediated decreases in heart rate. The baroreflex
responses in animals labeled with PRV-GFP were not different from
sham-labeled animals (Fig. 4).

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Fig. 4.
Increases in blood pressure evoke baroreflex mediated decreases in
heart rate. The reflex responses were unaltered in animals
(n = 7) labeled with virus compared with
sham-operated controls (n = 7). Initial heart rate,
midpoint of the reflex, minimal heart rate, or gain of the reflex were
not statistically different in virus compared with control animals.
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DISCUSSION |
It is well known that the neuronal projections from the brain to
the heart strongly influence cardiac function, and an abnormal cardiovagal activity has been implicated in diseases such as cardiac arrhythmia (Spyer 1981
). Respiratory sinus arrhythmia is
present in healthy fetuses, newborns, and mature animals and humans
(Elghozi et al. 1991
). However, in distressed
fetuses, as well as partially asphyxiated newborns, a slowing of
the heart rate and diminished respiratory sinus arrhythmia is strongly
correlated with low postnatal Apgar scores (a quick clinical assessment
of overall newborn well being) and subsequent neonatal mortality such
as in Sudden Infant Death syndrome (Meny et al. 1994
;
Schechtman et al. 1992
). This study, using the
Bartha-PRV-GFP transneuronal retrograde tracer has identified a pathway
that can mediate the normal interactions between the respiratory system
and the control of cardiac function that may be altered in diseases of
the cardiorespiratory systems. The projection from superior laryngeal
motorneurons to cardiovagal neurons may be responsible for respiratory
modulation of heart rate. The nonbursting spontaneous activity in
superior laryngeal neurons is unlikely to be responsible for generating
respiratory rhythms but is more likely involved in coordinating motor
activity to the heart and respiratory muscles. Superior laryngeal
neurons may provide an excitatory input to cardiac vagal neurons during postinspiration. In addition to this pathway, other work has shown cardiac vagal neurons are inhibited by a GABAergic input during inspiration (Gilbey et al. 1984
). The use of the PRV-GFP transneuronal retrograde tracer may provide an opportunity to study this and other
pathways that are also involved in cardiorespiratory interactions.
A very recent report has examined the utility of using a PRV that
also expresses enhanced GFP in an in vitro study of retinal pathways.
Transsynaptically labeled neurons in the suprachiasmatic nucleus and
retinal ganglia retained their major excitatory and inhibitory inputs,
suggesting this virus does not interfere with the synaptic function in
retinal pathways (Smith et al. 2000
). This study,
however, did not determine whether PRV-GFP caused any changes in firing
properties or voltage-gated currents in the labeled neurons or evoked
any functional changes in the retinal pathways. The present work
demonstrates that the firing properties and voltage-gated currents in
PRV-GFP-labeled cardiorespiratory neurons are unaltered by PRV-GFP.
Moreover, this study demonstrates the function of the brain stem
reflexes in the labeled pathway that control heart rate are maintained
and have normal arterial baroreflex characteristics. This study also
identifies a previously undescribed synaptic pathway from superior
laryngeal neurons to cardiac vagal neurons that may be involved in
mediating cardiorespiratory rhythms.
The present work, and that of Smith et al. (2000)
,
indicates fluorescent viral transneuronal tracers, such as Bartha
PRV-GFP are useful tools for in vitro physiological studies,
particularly since in the early phases of the infection (in these
studies 2-3 days), the electrophysiological properties of the neurons
appear normal. In addition, the synaptic input to neurons and the
reflex control of the labeled pathways are not altered by these
viruses. It is anticipated that this virus can be used in the
cardiorespiratory system to identify other neurons that synapse on
cardiac vagal neurons and neurons involved in other cardiorespiratory
functions, which can then be studied electrophysiologically. It is also
likely this virus can be utilized to introduce gene products other than GFP to deliberately alter the function of specific neurons in CNS
pathways that control cardiorespiratory function.
This work was supported by National Heart, Lung, and Blood
Institute Grants HL-49965 and HL-59895 to D. Mendelowitz, HL-25449 to
A. Loewy, and Deutsche Forschungsgemeinschaft Grant Me854/4 to T. Mettenleiter.
Address for reprint requests: D. Mendelowitz, Dept. of Pharmacology,
George Washington University, 2300 Eye St. NW, Washington, DC 20037 (E-mail: dmendel{at}gwu.edu).