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The Journal of Neuroscience, October 1, 2000, 20(19):7446-7454
Definition of Neuronal Circuitry Controlling the Activity of
Phrenic and Abdominal Motoneurons in the Ferret Using Recombinant
Strains of Pseudorabies Virus
I.
Billig1,
J. M.
Foris1,
L. W.
Enquist4,
J. P.
Card2, 3, and
B. J.
Yates1, 2
Departments of 1 Otolaryngology,
2 Neuroscience, and 3 Psychiatry, University of
Pittsburgh, Pittsburgh, Pennsylvania 15213, and
4 Department of Molecular Biology, Princeton University,
Princeton, New Jersey 08544
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ABSTRACT |
During a number of behaviors, including vomiting and some postural
adjustments, activity of both the diaphragm and abdominal muscles
increases. Previous transneuronal tracing studies using injection of
pseudorabies virus (PRV) into either the diaphragm or rectus abdominis
(RA) of the ferret demonstrated that motoneurons innervating these
muscles receive inputs from neurons in circumscribed regions of the
spinal cord and brainstem, some of which have an overlapping
distribution in the magnocellular part of the medullary reticular
formation (MRF). This observation raises two possibilities: that two
populations of MRF neurons provide independent inputs to inspiratory
and expiratory motoneurons or that single MRF neurons have
collateralized projections to both groups of motoneurons. The present
study sought to distinguish between these prospects. For this purpose,
recombinant isogenic strains of PRV were injected into these
respiratory muscles in nine ferrets; the strain injected into the
diaphragm expressed  -galactosidase, whereas that injected into RA
expressed green fluorescent protein. Immunofluorescence localization of
the unique reporters of each virus revealed three populations of
infected premotor neurons, two of which expressed only one virus and a
third group that contained both viruses. Dual-infected neurons were
predominantly located in the magnocellular part of the MRF, but were
absent from both the dorsal and ventral respiratory cell groups. These
data suggest that coactivation of inspiratory and expiratory muscles
during behaviors such as emesis and some postural adjustments can be
elicited through collateralized projections from a single group of
brainstem neurons located in the MRF.
Key words:
pseudorabies virus; diaphragm; abdominal muscle; emesis; dorsal and ventral respiratory groups; medullary reticular formation; raphe nuclei; dual-labeling immunofluorescence
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INTRODUCTION |
The diaphragm and abdominal muscles
typically contract out of phase as they induce inspiration and
expiration, respectively. These contractions are regulated by the
brainstem dorsal and ventral respiratory groups, which generate the
respiratory rhythm and also impose that rhythm on respiratory
motoneurons (Feldman, 1986 ). However, the diaphragm and abdominal
muscles also contract in phase during a number of behaviors, including
emesis (Gold and Hatcher, 1926; McCarthy and Borison, 1974), some
postural adjustments (Grillner et al., 1978), and in response to
vestibular stimulation (Yates et al., 1993; Rossiter et al., 1996).
Brainstem respiratory group neurons are not responsible for eliciting
simultaneous increases in inspiratory and expiratory muscle activity
during at least some of these responses. For example, bulbospinal
inspiratory neurons in the dorsal and ventral respiratory groups are
inhibited and mainly silent during emesis (Bianchi and Grèlot,
1989; Miller et al., 1990). Furthermore, vestibular stimulation can
activate respiratory muscles without modulating the firing of
respiratory group neurons (Yates et al., 1994; Wood-ring and Yates,
1997), and lesions of the main respiratory groups do not abolish
vestibulo-respiratory reflexes (Yates et al., 1995; Rossiter et al.,
1996; Shiba et al., 1996; Woodring and Yates, 1997).
Previous neuroanatomical studies in the rat employing the transneuronal
transport of pseudorabies virus (PRV) injected into the diaphragm have
revealed the locations of inspiratory neurons in the brainstem and
spinal cord of this species (Dobbins and Feldman, 1994). It was
reported that rat inspiratory premotor neurons were mainly located in
the dorsal and ventral respiratory groups, although a few labeled
neurons were present in the raphe nuclei, medial reticular formation,
and parabrachial nucleus. Nevertheless, differences in the organization
of neurons presynaptic to phrenic motoneurons were recently
demonstrated in an emetic species, the ferret. In contrast to the rat,
the ventral portion of the ferret medial medullary reticular formation
(MRF), particularly the magnocellular division, contained a substantial
number of infected neurons after the injection of PRV into the
diaphragm, although both species exhibited infection in the region of
the ventrolateral reticular formation known to contain the ventral respiratory group (Yates et al., 1999). In another group of
experiments, neurons in the MRF were also infected by transynaptic
passage of PRV from the ferret rectus abdominis (RA) muscle, along with neurons in portions of the ventrolateral reticular formation known to
contain the ventral respiratory group, the nucleus retroambiguus, and
the raphe nuclei (Billig et al., 1999). The overlapping distribution of
labeled MRF neurons after injection of PRV into either the diaphragm or
RA raises two possibilities: that single neurons in this area could
have collateralized projections to both inspiratory and expiratory
motoneurons or that two populations of neurons with overlapping
distributions provide parallel inputs to inspiratory and expiratory
motoneurons, as is the case for cells in the ventral respiratory group
(Feldman, 1986).
In the present study, two antigenically distinct recombinant strains of
PRV were used in dual injection paradigms to distinguish between these
two prospects. This experimental approach is based on the ability of
the two recombinant viruses to coinfect neurons with common synaptology
(Fig. 1A). Recent
studies have demonstrated the utility of this approach in mapping
autonomic and visual circuitry (Jansen et al., 1995; Levatte et al.,
1998; Ueyama et al., 1999) while also identifying the factors that may
contribute to the generation of false negatives (Kim et al., 1999;
Mabon et al., 1999). Although these data reveal the need to be
conservative in interpreting negative findings, the demonstrated
ability of two recombinants to coinfect neurons in these dual infection
paradigms provides a powerful means of addressing issues of
collateralization that cannot be achieved with other anatomical
methods. In the present study we have adapted this experimental
approach to determine if a single population of brainstem neurons is
presynaptic to two distinct populations of motoneurons involved in the
control of respiration and emesis in the ferret.
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Figure 1.
A, The experimental strategy used
in this study. Two recombinant strains of pseudorabies virus were used
to produce retrograde transynaptic infection of neurons synaptically
linked to motoneurons innervating the diaphragm and rectus abdominis
muscles. Each viral strain expressed a unique reporter that could be
detected with the use of monospecific antisera. B, The
genomic organization of the recombinant viruses used in this study. The
genome of PRV contains unique long (UL) and unique short
(US) regions. The parental strain used to produce the
recombinants used in this study was an attenuated vaccine strain known
as PRV-Bartha. PRV-Bartha has several well characterized mutations and
deletions that distinguish it from the wild-type virus (PRV-Becker).
These alterations are mapped on the diagram. All recombinants involved
insertion of the transgene at the gG locus of the viral genome. In
PRV-BaBlu, gG was replaced with the gene encoding -galactosidase. In
PRV-152 and PRV-154, the gene encoding green fluorescent protein was
inserted into the gG gene, either alone (PRV-152) or as part of a
fusion protein (PRV-154). C, The distribution of the
reporters within two infected neurons that were both double-labeled.
-galactosidase expression in neurons infected with PRV-Bablu
provided staining of neuronal perikarya and dendrites (red
fluorescence, panels B and E).
Two EGFP-expressing viruses (PRV-152 and PRV-154) labeled cells
differently. PRV-152 produced staining of perikarya and dendrites
comparable to that produced by PRV-Bablu (green
fluorescence, C). In contrast, the fusion
protein produced by PRV-154 (EGFP+Us9) was
differentially concentrated in the Golgi and rough endoplasmic
reticulum of infected cells (green fluorescence,
F). The majority of experiments reported here
used PRV-152. Neurons containing both PRV-Bablu and PRV-152 or
-154 appeared as having yellow fluorescence
(A and D).
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MATERIALS AND METHODS |
Animals. Experiments were conducted in 17 adult male
ferrets (obtained from Marshall Farms, North Rose, NY). Animals were housed singly and allowed a minimum of 1 week acclimation to the animal
facility before being injected with PRV. The experimental procedures
used in this study conformed to regulations stipulated in the United
States Department of Health and Human Services publication CDC 88-8395
and were approved by the University of Pittsburgh Institutional Animal
Care and Use Committee.
Recombinant viruses. The experimental strategy is
illustrated schematically in Figure 1A, and the
organization of the genome of the recombinants used is shown in Figure
1B. Three recombinants of the Bartha strain of PRV
(PRV-Bartha) that express either -galactosidase (-gal) or
enhanced green fluorescent protein (EGFP) were used in this analysis.
PRV-Bartha is an attenuated strain of PRV developed as a vaccine
(Bartha, 1961). It has been used widely for transneuronal tracing (cf.,
Enquist et al., 1999; Card, 2000 for recent reviews), including in
previous studies that provided the foundation for the present
investigation (Billig et al., 1999; Yates et al., 1999).
Preparation of PRV-BaBlu, a recombinant Bartha strain that expresses
-gal, has been described previously (Standish et al., 1995; Kim et
al., 1999). Briefly, this strain contains the lacZ gene at the gG locus
and produces -gal under the control of the viral gG promoter.
PRV-152 expresses EGFP. This virus carries an insertion at the gG locus
such that EGFP is constitutively expressed using the cytomegalovirus
immediate early promoter. The cell body, nucleus, and processes of
cells infected with PRV-152 are filled with EGFP. PRV-154 expresses a
novel membrane-anchored form of EGFP such that the
trans-Golgi network is the only part of the infected cell
that contains EGFP. This virus carries an insertion of a Us9-EGFP
hybrid gene at the gG locus such that the fusion protein is expressed
using the cytomegalovirus immediate early promoter (Brideau et al.,
2000). PRV-Bartha, PRV-BaBlu, and PRV-152 do not express the Us9 gene;
however, PRV-154 expresses two novel antigens, EGFP and Us9, that are
localized predominately in the trans-Golgi network.
All viruses were grown in pig kidney (PK15) cells. The final
titers in plaque-forming units (pfu) determined in PK15 cells were:
PRV-BaBlu and PRV-152 = 1 × 108
pfu/ml, PRV-154 = 3 × 108
pfu/ml. Viruses were aliquoted at 100 µl/tube and stored at  80°C. Individual aliquots of virus were thawed immediately before
injection. Excess virus was inactivated with Clorox and discarded.
Injection procedures. Initial experiments were conducted
using eight animals in which a single virus, either PRV-Bablu or PRV-152, was injected into the left RA to determine if the two recombinants were transported at the same rate. Animals were then killed after survival times of 3 (n = 1), 4 (n = 1), 5 (n = 4), or 6 d
(n = 2). Subsequently, nine additional experiments were conducted in which PRV-Bablu was injected into the diaphragm, and
PRV-152 or PRV-154 was injected into the RA muscle of the same animal.
These animals were killed 4 (n = 1), 4.5 (n = 1), 5 (n = 5), or 5.5 (n = 2) d after inoculation. Further detail regarding
the strain and volume of viruses injected in these experiments is
provided in Table 1.
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Table 1.
Parameters of injections in experiments involving
injection of PRV-Bablu into the diaphragm and PRV-152 or -154 into rectus abdominis
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The injection procedures conformed to those detailed in our previous
viral transneuronal analyses of premotor circuits that modulate the
activity of diaphragm and RA motoneurons (Billig et al., 1999; Yates et
al., 1999). Animals were initially anesthetized using a mixture of
ketamine (25 mg/kg) and xylazine (2.5 mg/kg) injected intramuscularly;
anesthesia was supplemented with 0.5-1% isoflurane, which was
vaporized in O2 and administered through a face
mask to maintain areflexia. In cases in which the diaphragm was
injected with PRV, a midline incision was made through the linea alba,
and the ventral surface of the left diaphragm was exposed by retracting
the viscera. PRV-Bablu was injected beneath the peritoneal lining of
the diaphragm using a 10 µl Hamilton syringe equipped with a 26 gauge
needle. Injections of virus (1-2 µl/injection) were made at multiple
sites in both the costal and crural regions of the diaphragm
ipsilaterally. The total volume of virus injected was 60-100 µl.
Similar procedures were used to inject 60 µl of PRV-152 or PRV-154
into the left RA; these injections were made beneath the connective
tissue sheath surrounding the muscle and were restricted to the region
within 3 cm of the diaphragm. After the injections, the abdominal
musculature and skin were closed using sutures, and animals were
maintained under Biosafety level II conditions for the balance of the
survival period.
Tissue preparation. After their respective survival times,
animals were deeply anesthetized using ketamine (35 mg/kg) and xylazine
(5 mg/kg) injected intramuscularly, and then perfused transcardially
with saline followed by paraformaldehyde-lysine-periodate (PLP)
fixative (McLean and Nakane, 1974), as previously described (Billig et
al., 1999; Yates et al., 1999). The brainstem and cervical, thoracic,
and lumbar cord segments were removed, post-fixed 4-5 hr or overnight
at 4°C in PLP, and cryoprotected by immersion in a 30%
sucrose-PBS solution at 4°C for 2 d. Transverse 50 µm sections of the spinal cord and brainstem were cut with a freezing microtome, collected sequentially in four wells of cryopreservant (Watson et al., 1986), and stored at 20°C until processed for immunohistochemical localization of viral antigens or reporter proteins.
Immunohistochemistry: single virus injection experiments. In
experiments in which a single viral strain was injected (into RA),
neurons infected with PRV-Bablu or PRV-152 were visualized using
immunoperoxidase procedures described in detail elsewhere (Enquist and
Card, 1996; Card and Enquist, 1999). Briefly, one well of sections was
incubated in rabbit polyclonal anti-pseudorabies virus antiserum
(Rb-133) diluted to a final concentration of 1:10,000. Thereafter,
sections were processed using the avidin-biotin modification of the
peroxidase anti-peroxidase procedure (Hsu et al., 1981), which used
affinity-purified biotinylated donkey anti-rabbit IgG (Jackson
ImmunoResearch, West Grove, PA) and Vectastain reagents (Elite kit;
Vector Laboratories, Burlingame, CA) for immunoperoxidase localization
of viral antigen. On completion of the immunohistochemical processing,
the tissue was mounted on gelatin-coated slides, dehydrated, cleared,
and coverslipped using Cytoseal 60 (VWR Scientific, West Chester, PA).
Immunohistochemistry: double virus injection experiments. In
experiments in which different recombinants were injected into the
diaphragm and RA, dual-labeling immunofluorescence techniques were used
to visualize infected neurons. One bin of free-floating sections
(section frequency of 200 µm) was incubated for 2 d at 4°C in
a combination of mouse anti--galactosidase (Sigma, St. Louis, MO;
1:1,500) and rabbit anti-green fluorescent protein (Clontech, Palo
Alto, CA; 1:1000 or Molecular Probes, Eugene, OR; 1:250) to localize
PRV-Bablu or either PRV-152 or PRV-154, respectively. Sections were
then washed thoroughly in PBS before being incubated in
affinity-purified secondary antibodies raised in donkey that were
conjugated to either the CY3 (red) or CY2 (green) carbocyanine (Jackson
ImmunoResearch). As a convention in this study, secondary antibodies
conjugated to CY3 (concentration of 1:500) were used to visualize
PRV-BaBlu, and secondary antibodies conjugated to CY2 (concentration of
1:300) were used to visualize PRV-152 and -154. The incubations in
these species-specific secondary antibodies were conducted
simultaneously for 2 hr at room temperature before the sections were
washed and mounted on gelatin-coated slides, and then dehydrated,
cleared, and coverslipped with Cytoseal 60. An adjacent bin of
brainstem tissue was stained for Nissl substance and fiber tracts using
a modified Kluver-Barrera procedure (Kiernan, 1990), so that the
precise boundaries of neuronal structures could be determined.
Several controls were included in the analysis to establish the
efficiency of transgene expression and the subcellular localization of
the reporter proteins. These included: (1) dual labeling
immunofluorescence localization of viral antigens with either
-galactosidase or EGFP to demonstrate that the transgenes were
efficiently expressed in all infected neurons, (2)
immunoperoxidase localization of -galactosidase or EGFP in sections
adjacent to those processed for immunofluorescence, (3)
immunoperoxidase localization of infected neurons in adjacent sections
of brainstem with a rabbit polyclonal antiserum (Rb133) raised against
acetone-inactivated PRV, and (4) localization in PRV-154 infected
neurons of EGFP to the Golgi apparatus and not to the processes or
nucleus. Collectively, these localizations demonstrated that the
distribution of infected neurons produced by each recombinant
recapitulated the projection-specific distribution of neurons revealed
in our previous studies (Billig et al., 1999; Yates et al., 1999).
Importantly, these data also demonstrated that the distribution of
neurons expressing transgenes was coextensive with the distribution of
infected neurons revealed by the rabbit polyclonal antiserum. Thus,
these data demonstrate that transgene expression provides an accurate
measure of the full extent of viral replication and transynaptic passage.
The subcellular localization of reporter proteins differed among the
recombinants. The -gal and EGFP expressed by PRV-BaBlu and PRV-152,
respectively, produced extensive staining of the somata (cell nucleus
and cytoplasm) and dendrites of infected neurons (Fig. 1C,
panels B, C, E). In contrast, the
Us9-EGFP fusion protein produced by PRV-154 was differentially
concentrated within the Golgi complex and perinuclear membranes of
infected neurons; the nucleus was not labeled (Fig. 1C,
panel F). The differential localization of the
Us9-EGFP fusion protein is consistent with the demonstration by Brideau
et al. (1998) that Us9 localizes to the trans-Golgi network
and endocytotic pathways in PRV-infected neurons, but not to the
endoplasmic reticulum or nuclear membranes. The extensive cellular
staining produced by PRV-152 had the added advantage of providing
detail on the morphology of infected neurons. For this reason, we used
PRV-152 in the majority of the dual infection studies. However, the
cell body and non-nuclear staining exhibited by PRV-154 was useful in
identifying neurons that were co-infected with PRV-BaBlu, and thus this
virus was used in a limited number of cases.
Tissue analysis. The primary analysis was done on sections
that were spaced 200 µm apart, both in animals injected with a single
virus or two viruses. Previous studies in the ferret have shown that
this frequency is sufficient for an accurate localization of all cell
groups synaptically linked to motoneurons innervating the diaphragm
(Yates et al., 1999) or the RA muscle (Billig et al., 1999). Two
additional series of thoracic spinal cord tissue from all double
virus-injected animals were processed using immunoperoxidase procedures
to determine whether labeling was present in sympathetic preganglionic
neurons located in the intermediolateral column (IML), and an
additional group of brainstem sections was processed to determine the
extent of infection of parasympathetic pathways. In addition to
addressing the potential contribution of autonomic pathways to
brainstem infection, this analysis provided a more comprehensive
analysis of the distribution of brainstem circuitry involved in the
transynaptic infection.
Sections were examined and photographed using a Zeiss Axioplan
photomicroscope equipped with epifluorescence and differential interference contrast (DIC) optics. Immunoperoxidase preparations were
photographed using transmitted light and fluorescence material was
photographed using epifluorescence in combination with filters that
selectively excited CY2 or CY3. In the dual-labeling immunofluorescence analysis, each field was photographed in single exposures that recorded
the location of cells harboring each reporter protein and in double
exposures that revealed the cellular localization of both viruses (Fig.
1C). Verification that yellow fluorescence reflected the
colocalization of both the CY2 and CY3 fluorophors and was not
attributable to the presence of overlapping cells that each contained
one of the fluorophors, was established using a 40× objective. Images
were also digitized using a Dage MTI 3CCD camera (Mutech, Billerica,
MA) and a Simple32 image analysis system (Compix, Lake Oswego, OR).
Images were prepared for publication using Adobe Photoshop software.
Individual images were adjusted for size and contrast, but color
balance was not altered.
In the dual virus injection experiments, only a qualitative analysis of
the locations of neurons expressing immunoreactivity to one or both
viruses was attempted. As noted in the introductory remarks, infection
of a neuron by one strain of PRV can lower its susceptibility to
infection by a second strain (Kim et al., 1999). Thus, it is possible
that some neurons with divergent projections to both diaphragm and RA
motoneurons may not have been double-labeled in these experiments. The
prospect of false negatives mandates a conservative interpretation of
the data but does not detract from the significance of dual infected
neurons. However, the presence of false negatives could produce
misleading results regarding the relative proportion of a cell group
that contains neurons that collateralize to innervate both populations
of motoneurons. Therefore, we have refrained from making quantitative
determinations of the number of dual-infected neurons. Nevertheless, it
should be emphasized that the patterns of infection described and
illustrated in this report are representative of all animals included
in the analysis.
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RESULTS |
Involvement of autonomic pathways
A thorough analysis of both the thoracic spinal cord and brainstem
revealed only minor infection within autonomic pathways after injection
of PRV into the diaphragm and RA. No infected parasympathetic
preganglionic neurons were observed in the brainstem of any of the
animals. Varying degrees of infection were observed in the IML of the
thoracic spinal cord. Infected sympathetic preganglionic neurons were
observed in all experimental animals, but the number of cells was small
and the intracellular distribution of viral antigens detected with the
rabbit polyclonal anti-PRV antiserum was characteristic of early stages
of infection. Specifically, only scattered labeled neurons were
observed in a subset of sections through the IML and, in those cases,
viral immunoreactivity was largely restricted to the nucleus,
cytoplasm, and proximal dendrites of the infected neurons, as
illustrated in Figure 2. This restricted distribution of viral antigens in these neurons was in marked contrast
to the extensive distribution observed within infected motoneurons from
the same animals (Fig. 3). Even at the
earliest survival intervals examined, infected motoneurons
characteristically exhibited dense staining throughout the
somatodendritic compartment, including distal branches of the dendritic
arbor. Because the intracellular distribution of viral antigens is a
reflection of the timing of infection (i.e., early stages of infection
are associated with a restricted distribution of immunoreactivity)
(Card, 1995; Card and Enquist, 1999), these data indicate that the
replication and transynaptic transport of virus through motor circuits
was more advanced than that in autonomic circuitry. Further evidence that brainstem labeling was not attributable to transneuronal passage
of virus from sympathetic preganglionic neurons lies in the
distribution of infected neurons, which differed substantially from
that documented in viral transynaptic analysis of autonomic circuitry
(Loewy, 1990; Jansen et al., 1995).
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Figure 2.
Photomicrographs illustrating restricted infection
of sympathetic preganglionic neurons after injection of PRV into
respiratory muscles. A, B, Infected neurons in the
intermediolateral cell column of the T8 spinal segment
after injection of PRV-152 into rectus abdominis; the section was
immunoprocessed using immunoperoxidase. B is a higher
magnification view of the region in A indicated by a
box. Note that the infection was confined to the
nucleus, cell body, and proximal dendrites of the labeled sympathetic
preganglionic neurons. C, Infected neurons in the
T11 spinal cord segment after the combined injection of
PRV-Bablu into the diaphragm and PRV-152 into rectus abdominis. The
section was immunoprocessed using immunofluorescence and photographed
under illumination that excited both the CY2 and CY3 fluorophors. Note
that the labeling of sympathetic preganglionic neurons in the
intermediolateral cell column, which is indicated by a
box, is much weaker than that of motoneurons located in
the ventral horn. Scale bars, 100 µm.
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Figure 3.
Photomicrographs of presumed motoneurons infected
4 and 4.5 d after injection of PRV-Bablu into the diaphragm and
PRV-152 into rectus abdominis. A, B, Large presumed
motoneurons immunostained with the red CY3 fluorophor
(A) or immunoprocessed with peroxidase
(B) after an injection of PRV-Bablu into the
diaphragm. The presumed phrenic motoneurons were located in the
ventromedial ventral horn of the C6 spinal cord segment
ipsilateral to the side of the injection. These large neurons
characteristically exhibited fasciculated bundles of dendrites that
extended toward the central canal (CE) and sometimes
crossed the midline to the contralateral side. C, D,
Examples of a cluster of presumed motoneurons, immunostained with the
green CY2 fluorophor (C) or immunoprocessed with
peroxidase (D) after an injection of
PRV-152 into the rectus abdominis. The cells were observed in
ipsilateral lamina VIII of the T10 spinal cord segment.
These large neurons exhibited large dendritic arbors, with some
processes crossing the midline or reaching the central canal
(CE). Scale bars, 200 µm.
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Patterns of neuronal infection
The distribution of infected neurons revealed by injection of the
recombinant viruses into the diaphragm and RA duplicated the
projection-specific patterns of transport that resulted from individual
injection of the parental virus into these muscles (Billig et al.,
1999; Yates et al., 1999). This occurred irrespective of whether the
recombinants were injected individually or in dual injection paradigms.
After injection of virus into the diaphragm, infected large presumed
motoneurons were present in the ventral horn of segments
C5 through C7 (but mainly
C6) ipsilateral to the injection, whereas
injection of virus into the proximal 3 cm of RA-infected neurons
concentrated largely in the ventral horn of the
T10 to T14 segments on the
ipsilateral side. Examples of infected motoneurons are illustrated in
Figure 3. The morphology and disposition of these neurons within the
ventral horn also differed depending on the injected muscle. Infected
diaphragm motoneurons were confined to a tight column in the
ventromedial portion of the ventral gray and gave rise to fasiculated
bundles of dendrites that exhibited a polarized trajectory toward the area immediately subjacent to the central canal. In contrast, infected
RA motoneurons were more widely dispersed throughout the central and
lateral portions of the ventral gray and gave rise to dendrites that
extended radially from the somata. In both cases, the neuronal
perikarya were 40-60 µm in widest diameter and exhibited
morphological features classically defined for motoneurons using
classical retrograde tracers. The number of infected cells exhibiting
this morphology increased with advancing survival, but the segmental
distribution and disposition of the cells in the ventral horn remained
projection-specific throughout the longest postinoculation intervals.
At postinoculation intervals extending to 5.5 d, transynaptic
passage and replication of the recombinant viruses led to the appearance of infected neurons in the spinal cord and brainstem. The
distribution of these neurons correlated with that demonstrated in our
previous reports of this circuitry derived from injections of the
parental virus into the diaphragm or RA (Billig et al., 1999; Yates et
al., 1999). The infected neurons included local circuit neurons at
multiple levels of the spinal cord (Fig.
4) as well as reproducible groups of
neurons in the caudal brainstem (Figs.
5,6). The
brainstem labeling was first apparent 4 d after injection of
either muscle, and the number of infected neurons increased
progressively through 5.5 d. Neurons were observed bilaterally with an ipsilateral propensity. In both paradigms, some infected neurons were observed in brainstem regions known to contain the dorsal
and ventral respiratory groups in a variety of mammalian species
(Feldman, 1986), and substantial numbers of neurons were observed in
the MRF and along the midline in the vicinity of the raphe obscurus and
raphe pallidus.
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Figure 4.
Photomicrographs of presumed spinal cord
interneurons infected 4.5 and 5 d after PRV-Bablu injection into
the diaphragm and PRV-152 injection into rectus abdominis. These
neurons were dually immunostained with the red CY3 and the green CY2
fluorochromes, indicating that they made synaptic connections with both
phrenic and abdominal motoneurons. The cells were located in lamina VII
of the T8 (A) and lamina VIII of the
L2 (B) spinal cord segments,
ipsilateral to the injections. A1-B2 show the cell
under illumination that excites one of the two fluorophors, whereas
A and B were photographed under
illumination that excites both fluorophors. Scale bars, 200 µm.
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Figure 5.
Examples of premotor neurons in regions of
the brainstem known to contain the dorsal and ventral respiratory
groups, which were infected 4.5 d after PRV injections into the
diaphragm and rectus abdominis. A-D show
photomicrographs of transverse brainstem sections located ~1 mm
caudal to the obex (A, B) or at the level
of the obex (C, D); these sections were
stained with the use of a modified Kluver-Barrera method.
Boxes on the photomicrographs indicate the locations of
the infected neurons illustrated in the middle column;
these sections were immunoprocessed using immunofluorescence. The
right column illustrates infected neurons at the same
location in sections immunoprocessed using immunoperoxidase.
A1 and A2 show neurons in nucleus
retroambiguus that were infected after injecting PRV-152 into rectus
abdominis on the contralateral side. The neurons were immunostained
with the green CY2 fluorophor (A1) or with peroxidase
(A2). B1 and B2 show
premotor neurons in nucleus retroambiguus infected after injecting
PRV-Bablu into the diaphragm on the contralateral side; these neurons
were labeled with the red CY3 fluorophor (B1) or through
immunoperoxidase processing (B2). C1 and
C2 illustrate infected premotor neurons in the vicinity
of nucleus ambiguus and the retrofacial nucleus that were immunostained
with the red CY3 fluorophor (C1) or with peroxidase
(C2) after injections of PRV-Bablu into the
contralateral diaphragm. D1 and D2 show
infected neurons in the ventrolateral portion of the nucleus of the
solitary tract, which were immunostained with the red CY3 fluorophor
(D1) or with peroxidase (D2) after
injecting PRV-Bablu into the ipsilateral diaphragm. Scale bars, 400 µm. 12, Hypoglossal nucleus; 5SL,
laminar spinal trigeminal nucleus; 5SP, alaminar spinal
trigeminal nucleus; A, nucleus ambiguus;
AP, area postrema; CE, central
canal; DMV, dorsal motor nucleus of the vagus;
FTL, lateral tegmental field; GRR,
rostral division of gracile nucleus; IO, inferior
olivary nucleus; LRI, internal division of lateral
reticular nucleus; NTS, nucleus of the solitary tract;
P, pyramid; RA, nucleus retroambiguus;
Rob, nucleus raphe obscurus; S, solitary
tract; SM, medial nucleus of solitary tract;
V4, fourth ventricle.
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Figure 6.
Location of premotor neurons in the MRF infected
after injection of PRV into the diaphragm and rectus abdominis.
A and B show infected neurons after a
4.5 d survival time, whereas C-E illustrate
infection after a 5 d survival. In addition, A and
D show labeling at ~2.5 mm rostral to the obex,
whereas B and E show labeling at ~2.0
mm rostral to the obex, and C illustrates labeling at
~3.0 mm rostral to the obex. Left column,
Photomicrographs of brainstem sections (on the side ipsilateral to the
injections) stained with a modified Kluver-Barrera method.
Boxes surround the area containing infected neurons
illustrated in the photomicrographs immediately on the
right. Middle and right
columns, Photomicrographs of infected neurons observed in the
MRF. Red cells were only infected by retrograde transynaptic
passage of PRV-Bablu from the diaphragm, green cells were
only infected by retrograde transynaptic passage of PRV-152 from rectus
abdominis, and yellow cells contain both viruses.
Photomicrographs in the right column are a magnification
of those in the middle column. Note that after a 5 d survival time, labeling is more prevalent and extends further
rostrally than after a 4.5 d survival. Scale bars, 400 µm.
Abbreviations are the same as in Figure 4, with the following
additions: FTG, gigantocellular tegmental field;
FTM, magnocellular tegmental field; INT,
nucleus intercalatus; Rm, nucleus raphe magnus;
Rpa, nucleus raphe pallidus.
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Dual infection experiments
Immunofluorescence analysis with filters selective for the CY2 and
CY3 fluorophors and with a wide-band Omega filter that excites both
fluorophors allowed a detailed analysis of neurons infected with one or
both of the recombinant viruses. As noted in Materials and Methods, the
CY2 fluorophor was used to identify neurons infected with either
PRV-152 or PRV-154, whereas CY3 was used to localize the
-galactosidase reporter expressed by PRV-BaBlu. Additionally,
PRV-BaBlu injection was restricted to the diaphragm, and the
EGFP-expressing recombinants were only injected into RA. Thus, neurons
that were a dedicated part of polysynaptic circuits selectively
innervating the diaphragm appeared red, those infected selectively by
transynaptic passage of virus from RA appeared green, and neurons that
collateralized to innervate motoneurons to both muscles appeared
yellow. Each of the dual-injected animals that survived  4.5 d
contained neurons that were replicating both recombinants. Furthermore,
sections that contained dual-infected neurons always also contained
neurons selectively labeled with -galactosidase or EGFP.
Consequently, we are confident that both recombinants had replicated
and passed transynaptically into comparable levels of the brainstem in
all experimental animals. Nevertheless, we cannot preclude the
possibility that previous infection of some neurons by one strain of
virus made those cells refractory to replication of the second strain,
as was recently demonstrated by Kim et al. (1999). However, it is
noteworthy that the interference in viral replication demonstrated by
Kim et al. (1999) was observed in animals in which one strain of virus
was more virulent and transported at a faster rate than the other
strain. In this study, both recombinants were produced from the same
attenuated virus (PRV-Bartha) and exhibited the same relative rate of transport.
As noted above, some infected neurons were observed in brainstem
regions known to contain the dorsal and ventral respiratory groups.
Further immunofluorescence analysis revealed that these cells consisted
of two projection-specific populations, and no cells contained
reporters for both recombinants. Neurons selectively expressing either
-galactosidase or EGFP immunoreactivity were observed in the
vicinity of the ventral respiratory group, whereas most neurons in the
vicinity of the dorsal respiratory group expressed -galactosidase
immunoreactivity (Fig. 5). This observation contrasted with the
labeling observed in the more rostrally situated sections through the
MRF of the same animals. In each animal, three populations of neurons
were observed in the magnocellular tegmental field in the MRF: those
replicating only one recombinant and those that contained the reporter
proteins of both recombinants (i.e., dual-infected neurons were
intermixed with cells that only expressed the EGFP or -galactosidase
reporters), as illustrated in Figure 6. However, it was uncommon to
observe dual-labeled neurons along the midline in the vicinity of the
raphe cell groups, although neurons expressing immunoreactivity to one
of the two reporters were present in this area. Figure
7 shows the distribution of infected
neurons at selected levels of the brainstem in two animals.
View larger version (53K):
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Figure 7.
Locations of neurons infected 4.5 and 5 d
after PRV injection into the diaphragm and rectus abdominis. Neuronal
locations are plotted on camera lucida drawings of transverse brainstem
sections. Only labeling at selected levels of the brainstem in two
animals is illustrated; the approximate rostrocaudal distance of each
section from the obex is indicated. Neurons infected by injection of
PRV-Bablu into the diaphragm are represented by open
squares, those infected after injecting PRV-152 or PRV-154 into
rectus abdominis are represented by open circles; dually
infected neurons are represented by shaded triangles.
Scale bar, 5 mm. Abbreviations are the same as in Figures 4 and 5, with
the following additions: 5SM, magnocellular alaminar
spinal trigeminal nucleus; CX, external cuneate nucleus;
IFT, infratrigeminal nucleus; PR,
paramedian reticular nucleus; RFN, retrofacial nucleus;
VMN, medial vestibular nucleus; VIN,
inferior vestibular nucleus.
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DISCUSSION |
This study provides the first direct evidence that a population of
neurons in the brainstem, located mainly in the magnocellular part of
the MRF, provides inputs to both inspiratory and expiratory motoneurons
in the spinal cord. Furthermore, the patterns of dual infection of
brainstem neurons reported here support the conclusion that cells
synaptically linked to both the diaphragm and RA exhibit a functional
segregation from neurons that provide inputs to only one of these
muscles. This functional parcellation is characterized by segregation
of circuitry responsible for the generation of the respiratory rhythm
from that responsible for coordinating cocontraction of multiple
respiratory muscles during behaviors such as emesis.
Two caveats must be considered when interpreting the results of this
study. First, after the injection of the recombinant viruses into the
diaphragm and rectus abdominis the infection of neurons in the dorsal
and ventral respiratory groups was not as extensive as when the
parental stain, PRV-Bartha, was used (Billig et al., 1999; Yates et
al., 1999). It is possible that the recombinant viruses were not
transported as rapidly as the parental strain and that a longer
transport time would have resulted in a more extensive infection of the
respiratory groups. Nonetheless, the distribution of labeling produced
by injection of PRV-Bartha and the recombinants PRV-152, PRV-154, and
PRV-Bablu into respiratory muscles was similar, and there is no
indication that the general results would have been different from
those in the present experiments had longer survival times been used. A
second caveat is that a limited amount of infection of sympathetic
nervous system neurons did occur in these studies, raising the prospect
that some of the labeling of brainstem neurons observed in this study
was attributable to transneuronal passage of virus from sympathetic
preganglionic neurons in the thoracic spinal cord. Although we cannot
eliminate this possibility, two lines of evidence raised in Results
support our conclusion that the MRF neurons were infected predominantly by virtue of their collateralized projections to motoneurons
controlling the diaphragm and rectus abdominis muscles.
Although previous experiments using PRV injections have shown that
overlapping populations of MRF neurons influence RA and diaphragm
activity (Billig et al., 1999; Yates et al., 1999), these conventional
transneuronal tracing studies using one virus could not distinguish
whether parallel projections from two groups of neurons or
collateralized projections from a single group provided inputs to
inspiratory and expiratory motoneurons. Furthermore, the present
findings have physiological importance, because they shed light on
lesion studies showing that inactivation of the MRF abolishes
simultaneous increases in diaphragm and abdominal muscle activity
during emesis (Miller et al., 1996). The current data demonstrate that
individual MRF neurons may simultaneously activate inspiratory and
expiratory motoneurons during vomiting, opening the possibility that
pharmacological agents acting on this single neuronal population could
abolish this behavior. Nevertheless, additional electrophysiological
and lesion studies will be required to determine the role of MRF
neurons in coordinating the contractions of the diaphragm and abdominal
muscles during vomiting (Gold and Hatcher, 1926; McCarthy and Borison,
1974), as well as during postural adjustments (Grillner et al., 1978),
and reaction to vestibular stimulation (Yates et al., 1993; Rossiter et
al., 1996). In addition, it remains to be determined whether abdominal
muscles other than RA receive substantial inputs from MRF premotor
neurons, including those that also influence diaphragm activity.
It seems likely that the methodology used in this study can be used to
determine whether any two muscles or muscle groups receive common
influences from particular CNS regions. However, several
limitations are apparent in this approach. Because infection of a
neuron by one virus can lower its susceptibility to be infected by a
second virus (Kim et al., 1999), it is only possible to interpret positive results (i.e., the presence of double-labeled neurons) when
using this method. Furthermore, because the immune system of the ferret
rapidly and effectively compartmentalizes and eliminates neurons that
are infected with PRV (Billig et al., 1999; Yates et al., 1999), the
temporal window during which infected neurons can be detected is
limited. As a result, experiments incorporating injection of two virus
tracers cannot be used in isolation to determine neural pathways that
are involved with coordinating movements. Nonetheless, these
experiments may provide clues regarding potential neural pathways that
can be investigated in subsequent lesion, electrophysiological, and
conventional neuroanatomical studies.
In conclusion, the present demonstration of dual infections of MRF
neurons by antigenically distinct recombinants injected into the
diaphragm and RA provides novel insights into the functional organization of brainstem circuitry that controls respiratory muscles.
Furthermore, comparison of these data with those obtained from previous
studies in rat (Dobbins and Feldman, 1994) suggests that the
organization of this circuitry is species-specific. Finally, the
spatial separation of cell groups involved in the generation of the
respiratory rhythm from those in the MRF that collateralize to
innervate inspiratory and expiratory motoneurons suggests a functional
segregation in brainstem premotor respiratory neurons that can be
explored experimentally.
| |
FOOTNOTES |
Received May 9, 2000; revised June 21, 2000; accepted July 21, 2000.
This work was supported by National Institutes of Health Grants R01
DC00693, R01 DC03732, and P01 DC03417 (B.J.Y.) and R01 NS33506
(L.W.E.). We thank Lucy Cotter and Kristine Hartge for excellent
technical support during the course of these experiments.
Correspondence should be addressed to Dr. Bill Yates, University of
Pittsburgh, Department of Otolaryngology, Room 106, Eye and Ear
Institute, Pittsburgh, PA 15213. E-mail: byates{at}pitt.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
