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The Journal of Neuroscience, June 1, 2003, 23(11):4657-4666
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Brainstem Substrates of Sympatho-Motor Circuitry Identified Using Trans-Synaptic Tracing with Pseudorabies Virus Recombinants
Ilan A. Kerman,1,2,3 Lynn W. Enquist,4 Stanley J. Watson,3 andBill J. Yates1,2 1 Department of Neuroscience University of Pittsburgh, Pittsburgh, Pennsylvania 15213, 2 Department of Otolaryngology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, 3 Mental Health Research Institute, Department of Psychiatry, University of Michigan, Ann Arbor, Michigan 48109, and 4 Department of Molecular Biology, Princeton University, Princeton New Jersey 08544
Abstract
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Abstract
Introduction
Previous physiological investigations have suggested the existence of a neural circuit that coordinates activation of motor and autonomic efferents before or at the onset of exercise. Traditionally these circuits have been postulated to involve forebrain areas. However, overlapping populations of medullary reticular formation neurons that participate in motor or autonomic control have been described previously, suggesting that individual pontomedullary reticular formation neurons may coordinate both motor and autonomic responses. We tested this hypothesis by conducting transneuronal retrograde tracing of motor and sympathetic nervous system pathways in rats using recombinant strains of pseudorabies virus (PRV). A PRV strain expressing the green fluorescent protein (PRV-152) was injected into the left gastrocnemius muscle, which was surgically sympathectomized, whereas another recombinant (PRV-BaBlu) was injected into the left adrenal gland. Immunofluorescence methods using monospecific antisera and distinct fluorophores identified neurons infected with one or both of the recombinants. Brainstem neurons coinfected with both PRV recombinants, which presumably had collateralized projections to both adrenal sympathetic preganglionic neurons and gastrocnemius motoneurons, were observed in several areas of the pontomedullary reticular formation. The largest number of such neurons was located in the rostral ventromedial medulla within the ventral gigantocellular nucleus, gigantocellular nucleus pars alpha, raphe obscurus, and raphe magnus. These neurons are candidates for relaying central command signals to the spinal cord.
Key words: pseudorabies virus; rats; dual-labeling immunofluorescence; central command; stress; exercise; motor pathway; sympathetic nervous system; raphe nuclei; pontomedullary reticular formation; rostral ventromedial medulla
Introduction
A wide range of human and animal behavior is characterized by coordinated and complementary activation of motor and autonomic outflows. These behaviors are often part of a broader response to a perturbation in the organism's external environment, such as exposure to a noxious stimulus or encountering a predator or rival and preparing to fight or flee to ensure survival. Alternatively, some behavioral responses during conditions that are not stressful, such as ingesting a meal or sleep, also require synchronized activation of motor and autonomic efferents. Rapid eye movement sleep is characterized by postural muscle atonia that accompanies sympathetic nervous system hypoactivity. Locomotion, as well as other forms of exercise, requires rapid and immediate increases in respiratory function, heart rate, stroke volume, cardiac output, peripheral resistance, and blood pressure (Waldrop et al., 1996). Physiologists have debated for more than a century about the origin of the neural inputs that regulate the concomitant motor and autonomic changes during exercise (Rowell, 1993
Traditional views on the central control of motor and autonomic efferent systems have been quite disparate. Motor function has long been thought to be primarily under volitional control and mediated essentially via direct corticospinal and rubrospinal projections (Kandel et al., 2000
On the basis of physiological evidence suggesting the existence of central command circuits, we hypothesized that there is a neural pathway dedicated to the coordinated activation of motor and autonomic efferents. Because previous anatomic data have suggested that neurons with dual (motor and sympathetic) innervation targets may be located at the pontomedullary level of the brainstem, we focused our analysis on this portion of the nervous system. These experiments were conducted by injecting distinct recombinants of the transneuronal tracer pseudorabies virus (PRV) into the rat gastrocnemius muscle and the adrenal gland, with subsequent determination of the location of brainstem neurons synaptically connected to each target.
Materials and Methods
All of the procedures regarding animal use in this study conformed to the Guide for the Care and Use of Laboratory Animals (1996, National Academy of Sciences) and were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.Overview of experimental procedures
In the present studies we used transgenic recombinants of an attenuated PRV strain, PRV-Bartha, for transneuronal tracing of multisynaptic pathways providing inputs to gastrocnemius motoneurons and adrenal sympathetic preganglionic neurons (SPNs). PRV has preferential tropism for axonal terminals (Vahlne et al., 1978-galactosidase (
Forty-three male rats (Sprague Dawley, Hilltop, NY) weighing 210–476 gm were used in these studies. Initial experiments were aimed at optimizing viral infection of motor and sympathetic targets in the periphery. In two of the animals, a monosynaptic retrograde tracer, the
Mapping of gastrocnemius motoneurons
The monosynaptic retrograde tracer CTUse of pseudorabies virus for transneuronal tracing
In six preliminary experiments, different volumes of PRV-152 were injected into the left gastrocnemius muscle to determine the volume required to produce maximal motoneuronal infection. For this purpose, the muscle was isolated as described above, and a series of up to 30 injections of 1 µl volume were made directly into the muscle belly using a Hamilton syringe. In five rats, 30 µl injections of PRV-152 were made into their left gastrocnemius muscles, and the same volume of PRVBaBlu was delivered into the contralateral gastrocnemius muscle. Spinal cord tissue harvested from this group of animals was used to verify specificity of our immunofluorescence detection methods (see below). The potential for spread of virus to non-target tissues was evaluated in three rats, in which as much as 30 µl of PRV-152 was applied on top (but not injected into the belly) of the gastrocnemius muscle and 4 µl of PRVBaBlu was injected directly over the viscera surrounding the adrenal gland. No motoneuron labeling by PRV-152 was observed in the spinal cord after these injections. Similarly, only a few intermediolateral cell column (IML) neurons were infected by PRV-BaBlu injected adjacent to the adrenal gland in one of these animals, and in the other two cases there was no infection in the spinal cord. Thus, it seems likely that CNS labeling produced by injection of PRV recombinants into gastrocnemius muscle or the adrenal gland was caused by infection of the target neurons and did not result from spread of virus to non-target tissues.Hindlimb sympathectomy. In addition to motor efferents, the hindlimb is also innervated by sympathetic efferents that project to blood vessels, sweat glands, and other smooth muscle targets (Jänig and McLachlan, 1992
Double-virus injections. To determine the location of neurons coordinating muscle contraction and autonomic activity, 27 rats received injections of PRV-152 into their left gastrocnemius muscle and injections of PRV-BaBlu into their left adrenal gland. The gastrocnemius muscle was injected with a 30 µl total volume of PRV-152, which was divided into multiple injections of 0.5–1.0 µl. The ipsilateral adrenal gland was dissected via a left flank incision and gently separated from surrounding viscera and fat; the adjacent connective tissue was then retracted to facilitate access to the gland. Two or three injections of PRV-BaBlu were then made into the gland through a glass pipette attached to a Hamilton syringe; the total volume of virus injected was 2–4 µl. After each injection the gland was swabbed with a cotton-tip applicator to decrease nonspecific viral spread.
In 14 rats both gastrocnemius and the adrenal gland were injected with PRV on the same day. Most of these animals (n = 10) survived 96 hr after the injections, whereas the rest of the rats (n = 4) survived 120 hr after virus injections. To determine the locations of SPNs, three of the animals that survived 96 hr after virus injections received 0.6 or 1.0 ml intraperitoneal injections of 0.5% Fluorogold solution (FG; Fluorochrome, Boulder, CO) 5 d before virus injections. FG labels all of somatic motoneurons and sympathetic preganglionic neurons; it does not interfere with PRV infectivity and propagation when injections of FG and PRV are temporally separated (Papka et al., 1995
In another 13 animals, PRV-152 injections into gastrocnemius preceded PRV-BaBlu injections into the adrenal gland by 24 hr to provide for matching of the infection produced by the two recombinants. Three of the rats survived 96 hr after gastrocnemius muscle injections and 72 hr after adrenal gland injections, whereas 10 survived 120 hr after gastrocnemius muscle injections and 96 hr after adrenal gland injections.
Tissue processing
After transcardial perfusion with PLP (see above), the entire brain was extracted, the thoracic spinal cord was removed in two blocks (T1–T7 and T8–T13), and individual lumbar spinal cord segments were harvested. In some animals the S1 and S2 spinal levels were also removed, and in six cases the cervical cord was extracted as a single block (C1–C8). Brains were postfixed in PLP for 2–4 hr, whereas spinal cord tissue was left in the fixative overnight. Blocks of thoracic and cervical spinal cord tissue were sectioned in the horizontal plane at a thickness of 35 or 40 µm, and sections were separated into three bins. Lumbar spinal cord segments were sectioned coronally at 35 or 40 µm and collected into three bins, whereas brains were sectioned coronally at a thickness of 40 µm and collected into six bins. Tissue was stored at -20°C in cryoprotectant (30% sucrose, 30% ethylene glycol, 1% polyvinyl-pyrrolidine) until immunohistochemical processing was conducted.Immunohistochemical procedures
Tissue was initially rinsed with 0.1 M PBS several times at room temperature. It was then soaked in a 0.5% sodium borohydrate solution for 10–30 min and washed thoroughly in 0.1 M PBS before sections were incubated in primary antibody solution.CT
In initial parametric experiments, neurons infected with PRV-152 or PRV-BaBlu were identified with immunoperoxidase procedures that used a rabbit polyclonal antibody (Rb-133, concentration of 1:10,000) that recognizes all major viral envelope and capsid proteins (Card and Enquist, 1994
Specificity of the primary and secondary antibodies used in our double-labeling experiments has been documented previously (Billig et al., 2000
Tissue analysis
Tissue was examined using a Leica DMRD photomicroscope. The rat atlas of Paxinos and Watson (1986Infected neurons were digitized using a Sony DXC-970MD color camera and MCID Elite 6.0 imaging software (Imaging Research, St. Catharines, Ontario). Different color fluorophores were detected using specific filter sets (Chroma Technology, Brattleboro, VT) with the following respective excitation and emission ranges: FG, 300–400 nm and 440–490 nm (blue fluorescence); BODIPY-FL, 460–500 nm and 510–560 nm (green fluorescence); Cy3, 560–680 nm and 660–740 nm (red fluorescence). Use of such specific filter sets ensured that we did not detect any false-positive results. Images of neurons where obtained using each of the filter sets individually, and the presence of multiple fluorophores within a given cell was determined by digitally overlaying the images. Brightness and contrast of images were optimized for presentation purposes, whereas color balance and content were not altered in any of the photomicrographs. Digital images were imported into Canvas 6.0 (Deneba Systems, Miami, FL) and Photoshop 6.0 (Adobe Systems, San Jose, CA) software for preparation of figures.
Statistical analysis
Linear regression analyses of data were performed using Microsoft (Redmond, WA) Excel 2002 Software for Windows. Significance level was set as p < 0.05.
Results
Spinal cord labeling
Identification of gastrocnemius motoneurons
The location of gastrocnemius motoneurons was determined in two rats by retrograde transport of CT200 µm apart. Gastrocnemius motoneurons were located within the lateral ventral horn on the side ipsilateral to the injection site. The neurons were confined to a column within lamina 9 at the L4 and L5 spinal levels. As illustrated in Figure 1A, CT
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Figure 1. Labeling of gastrocnemius motoneurons produced by injecting CT
In subsequent experiments, different numbers of 1 µl injections of PRV-152 were made into the left gastrocnemius muscle to determine the total injection volume required to produce a consistent infection of the entire motoneuron pool. The total volumes of PRV injected were as follows: 10 µl (n = 1 rat), 15 µl (n = 1), 20 µl (n = 2), and 30 µl (n = 7). Figure 1B illustrates examples of motoneurons infected by injection of PRV-152 into gastrocnemius that were visualized using immunoperoxidase, whereas Figure 1C shows infected motoneurons that were labeled using immunofluorescence. As indicated in Figure 2A, when 30 µl injection volumes were used a reliable infection of gastrocnemius motoneurons was achieved; smaller volumes were less effective in producing labeling. There was also a strong negative correlation between the weight of the animal and the extent of motoneuron infection in the lumbar spinal cord. Figure 2B illustrates that animals weighing <300 gm had the most robust infections of gastrocnemius motoneurons. Conversely, infection of a large number of gastrocnemius motoneurons was less likely in animals that weighed ≥400 gm. For example, the largest animal used in this experiment (476 gm) exhibited only a few labeled motoneurons after injection of 30 µl of PRV into gastrocnemius (Fig. 2A, arrow). Importantly, the number of motoneurons labeled per section after 30 µl injections of PRV in animals weighing <300 gm (average of 5.6 ± 0.6, SD) was similar to the number of motoneurons labeled after CT
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Figure 2. Factors influencing the infection of motoneurons and the subsequent transneuronal infection of brainstem neurons after injection of PRV-152 into the gastrocnemius muscle.A, Effect of increasing the total volume of virus injected on the number of motoneurons infected.In general, injection of 30 µl of PRV consistently produced infection of a substantial number of motoneurons; the only exceptional case (identified by arrow) was the largest animal (weight, 476 gm) used in this study. A linear regression revealed a significant relationship between the volume of virus injected and the number of infected motoneurons(r2=0.58;p=0.01);the regression line is included in the figure. B, A strong negative correlation was also noted between the animal's weight and the total number of motoneurons infected(r2= 0.41; p < 0.001). C, The total number of brainstem neurons infected with PRV-152 was correlated directly with the number of gastrocnemius motoneurons infected. This linear correlation had a steeper slope in animals surviving 120 hr after viral inoculations (r2 = 0.89; p < 0.01) as compared with those killed at 96 hr (r2 = 0.93; p < 0.01).
To evaluate the effectiveness of the hindlimb sympathectomy, thoracic and upper lumbar spinal cord sections were examined for the presence of IML neurons infected by PRV-152. In sham-operated animals, large numbers of thoracic and lumbar IML neurons were infected with PRV-152. Labeling of small and medium neurons was also present near the central canal in laminas 7 and 10 of upper lumbar spinal segments. Additionally, in sham-sympathectomized animals, considerable numbers of large neurons were observed within laminas 8 and 9 of the upper lumbar spinal cord segments, rostral to the gastrocnemius motor pool. These presumed motoneurons were apparently infected via direct or multisynaptic connections with IML neurons because this labeling was absent in sympathectomized rats. Furthermore, similar ventral horn labeling was observed after PRV-BaBlu injections into the adrenal gland, suggesting the existence of intraspinal connections between motor and sympathetic outflows.
Labeling of SPN and interneurons
To determine the location of SPNs in horizontal spinal cord sections, the retrograde tracer FG was injected intraperitoneally (Joshi et al., 1995
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Figure 3. Identification of SPNs in horizontal sections of the T5–T7 spinal cord. A, Top, SPNs infected with PRV-BaBlu were visualized with Cy3-conjugated antibodies and appeared red. These neurons were observed at their expected location in the IML at the junction of the gray matter and the lateral funiculus (LF). A, Middle, Systemic injections of FG were made in the same animal to identify all of the sympathetic preganglionic neurons. FG, a blue-emitting fluorophore, was visualized with a specific UV fluorescence filter. A, Bottom, When immunofluorescences of both FG and Cy3 were overlaid, a subset of cells was identified as containing both markers (indicated with arrows). These cells were classified as adrenal preganglionic neurons. Neurons containing Cy3 but not FG fluorescence were most likely second-order neurons labeled through the trans-synaptic transmission of PRV from SPNs, whereas those neurons containing only FG were classified as SPNs projecting to non-adrenal targets. IML neurons were also infected with PRV-152 after its injection into the gastrocnemius muscle of sham-sympathectomized rats (B). Most of these cells were located ipsilateral to the injected muscle (Bii), although some infected neurons were also observed contralaterally(Bi).In contrast, PRV-152 infection of IML neurons was absent in sympathectomized rats (C). Scale bars: low-power images, 250 µm; high-power images (insets), 200 µm. CC, Central canal; LF, lateral funiculus.
Brainstem labeling
The animals that received PRV-152 injections into the left gastrocnemius muscle and PRV-BaBlu injections into the ipsilateral adrenal gland were divided into three groups on the basis of the success of the hindlimb sympathectomy. Group 1 rats (n = 7) received a sham sympathectomy and were used as negative controls. Animals in group 2 (n = 12) received a successful hindlimb sympathectomy, as indicated by a lack of neurons immunopositive for PRV-152 in the IML along with the presence of a similar number of infected motoneurons as in group 1 rats. Rats in group 3(n = 8) were those in which the sympathectomy resulted in only a partial reduction of PRV-152-containing IML neurons. Because infection of sympathetic efferents with PRV injected into gastrocnemius would confound interpretation of our data, these animals were excluded from further analysis, and the data will not be discussed.Of the 12 group 2 (successful sympathectomy) animals, 5 were euthanized 96 hr after the injection of PRV into both the adrenal gland and the gastrocnemius muscle, and the remaining 7 survived 120 hr after the gastrocnemius injection and 96 hr after the adrenal injection. In the latter animals, injections into gastrocnemius and the adrenal gland were separated by 24 hr to balance the temporal pattern of brainstem infection with both recombinants. Previous work has demonstrated that infection of a neuron with one viral strain can preclude subsequent infection with another recombinant (Kim et al., 1999
The initial analysis focused on parameters influencing transmission of PRV-152 to the brainstem. As illustrated in Figure 2C, the number of PRV-152-infected brainstem neurons was correlated significantly with the number of gastrocnemius motoneurons that were infected with this recombinant at both the 120 hr (r2 = 0.89; p < 0.01) and the 96 hr (r2 = 0.93; p < 0.01) survival periods. There appeared to be a threshold number of gastrocnemius motoneurons that were required to be infected with PRV-152 before appreciable labeling was observed in the brainstem. For the 120 hr survival period this number was
Examination of the brainstem in sympathectomized and sham-operated rats revealed a consistent presence of neurons infected with one or both of the viral recombinants. These neurons were observed in the rostral ventrolateral medulla (RVLM), ventral gigantocellular nucleus (GiV), gigantocellular nucleus pars alpha (GiA), raphe pallidus (Rpa), raphe obscurus (Rob), raphe magnus (RMg), the A5 and A7 noradrenergic cell groups, locus coeruleus (LC), and subcoeruleus (subCA). The numbers of neurons infected with PRV-152 in the RVLM was much greater in sham than in sympathectomized animals. However, both the total number of PRV-152-positive brainstem neurons and the number of double-infected neurons in particular brainstem regions differed among animals. Of the 12 animals with complete hindlimb sympathectomies, 5 displayed double-labeled neurons in RVLM, 10 inGiV, 7 in ROb, 5 in RPa, 6 RMg, 8 in GiA, 6 in A5, 7 in LC, 6 in subCA, and 8 in A7. The most prominent source of this variability appeared to be infection of an insufficient number of gastrocnemius motoneurons to produce transneuronal spread of virus to the brainstem. This factor could be controlled for in six animals in which >50 gastrocnemius motoneurons were infected at 120 hr after injection or >150 gastrocnemius motoneurons were infected at 96 hr after injection. All six of these animals exhibited double-infected neurons within GiV, GiA, ROb, RMg, LC, subCA, and A7. In addition, five of these rats displayed double-labeled neurons in RVLM, RPa, and A5 (Fig. 4).
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Figure 4. Location of double-labeled brainstem neurons. Medullary and pontine neurons infected with only one of the viral recombinants were identified with either green-emitting (PRV-152) or red-emitting (PRV-BaBlu) fluorophores, whereas neurons that contained both recombinants appeared yellow. Drawings in the left column indicate the locations of anatomical regions shown to the right. Numbers next to the drawings represent the approximate distance in millimeters from calamus scriptorius, defined as the point of convergence of the fourth ventricle to form the central canal. Photomicrographs located in the middle column (immediately to the right of the drawings) represent data from an animal allowed to survive 96 hr after injection, whereas those on the far right were taken from an animal that survived 120 hr after PRV-152 injection into the left gastrocnemius muscle and 96 hr after PRV-BaBlu injection into the left adrenal gland. Note the increase in the numbers of double-labeled neurons after the longer survival time. Scale bars, 200 µm. 7g, Facial nerve genu; 7n, facial nerve; 7nu, facial nucleus; 12nu, hypoglossal nucleus; GiA, gigantocellular nucleus parsalpha; GiV, ventral gigantocellular nucleus; IO, inferior olivary nucleus; LC, locus coeruleus; MCP, middle cerebellar peduncle; MVe, medial vestibular nucleus; NA, nucleus ambiguus; NTS, nucleus tractus solitarius; PrH, prepositus hypoglossi; Pr5, principal sensory trigeminal nucleus; py, pyramidal tracts; RMg, raphe magnus; ROb, raphe obscurus; RPa, raphe pallidus; RVLM, rostral ventrolateral medulla; s5, sensory root of the trigeminal nerve; SCP, superior cerebellar peduncle; Sp5, trigeminal nucleus and tract; subCA, subcoeruleus nucleus.
The presence of double labeling was confirmed by examining the tissue under illumination that excited each of the fluorophores alone, to ensure that the labeling was not caused by the overlapping of two cells that were each infected by a single viral recombinant. Infected neurons contained viral immunoreactivity in cell nuclei and cytoplasm. Viral antigen in the somatodendritic compartment also extended into dendrites to varying degrees (Fig. 5). The tissue also included fluorescent signal representing portions of neurons and processes that were not clearly connected to a cell body within the same section. Such neuronal fractions were excluded from further analyses.
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Figure 5. High-power photomicrographs of regions containing substantial numbers of infected neurons at different pontomedullary levels. A–C, Images taken from an animal that survived 96 hr after viral injection into the left gastrocnemius muscle and the left adrenal gland; D–F, images taken from an animal that survived 120 hr after PRV-152 injection into the left gastrocnemius muscle and 96 hr after PRV-BaBlu injection into the left adrenal gland. Labeling is shown in the following regions: A, raphe obscurus;B, raphemagnus;C, A7;D, ventral gigantocellular nucleus;E, gigantocellular nucleus pars alpha;F, locus coeruleus.Scale bars, 100 µm.
The number of double-infected neurons in different anatomical locations was determined in the six animals with the most extensive motoneuron labeling. Cells were counted only if the soma and processes were clearly defined. Furthermore, care was taken to analyze the same number of coronal brainstem sections in each animal; the sections were cut at 40 µm thickness, and every sixth section was examined (spacing between sections of 240 µm). As shown in Figure 4, the RVLM was defined as the triangular region at the ventral surface of the brainstem ventral to nucleus ambiguus and lateral to the inferior olive. Four consecutive sections were used to quantify RVLM labeling, with the caudalmost section located
Double-labeled neurons were distributed bilaterally but were slightly more prevalent ipsilateral to injections; labeling on both sides was counted to quantify the distribution of infected cells. The number of double-labeled neurons appeared to be greater within the ventromedial medullary areas, GiV, GiA, ROb, and RMg, than in the other regions (Fig. 6).
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Figure 6. Mean number of neurons coinfected with both PRV-152 and PRV-BaBlu in different brainstem regions. The number of neurons infected with both recombinants was quantified in 10 medullary and pontine regions of 12 rats in which the hindlimb was surgically sympathectomized, and PRV-152 was injected into the gastrocnemius muscle, whereas PRV-BaBlu was injected into the ipsilateral adrenal gland. Numbers represent total number of double-labeled cells (quantified across multiple sections) in each region; see Results for details of the quantification procedure. Double-labeled neurons were distributed bilaterally but were slightly more prevalent ipsilateral to injections; labeling on both sides was counted to quantify the distribution of infected cells. GiA, Gigantocellular nucleus pars alpha; GiV, ventral gigantocellular nucleus; LC, locus coeruleus; RMg, raphemagnus; ROb, raphe obscurus; RPa, raphepallidus; RVLM, rostral ventrolateral medulla; subCA, nucleus subcoeruleus.
Discussion
This study exploited the ability of PRV to infect chains of synaptically linked neurons and is the first direct examination of the locations of CNS neurons that innervate both sympathetic and motor outflows. It seems likely that these neurons play a role in concomitant regulation of sympathetic and motor efferent activities during behaviors that require such coordination, such as exercise. Our data show that neurons that participate in coordinated motor and autonomic regulation exist in several brain regions but are concentrated in the dorsolateral pons (A7, LC, and subCA), the rostral ventromedial medulla (raphe nuclei, GiV, and GiA), and the lateral brainstem (RVLM and the A5 cell group).Among the areas described above, the region of the rostral ventromedial medulla near the pontomedullary junction contained the greatest numbers of neurons that were double-infected with the viral recombinants injected into both the adrenal gland and the gastrocnemius muscle. In addition, double-infected neurons were detected in this region at the shortest survival times that resulted in brainstem labeling, suggesting that these double-labeled cells are part of the reticulospinal projection system and that their axons likely collateralize to innervate both adrenal SPNs and gastrocnemius motoneurons. This finding is supported by previous studies using monosynaptic tracers that demonstrated extensive, collateralized projections from the rostral ventromedial medulla to the spinal cord in various species (Martin et al., 1979a
The dorsolateral pons also contained neurons that were dually infected by the viral recombinants injected into gastrocnemius and the adrenal gland, although the cells were less numerous than those in the rostral ventromedial medulla. This finding is not unexpected, because studies using monosynaptic tracers have suggested that LC, subCA, and the A7 cell group neurons, including noradrenergic cells located in these regions, have collateralized projections to the spinal cord (Nygren and Olson, 1977
The number of motoneurons infected by injection of PRV-152 into the gastrocnemius muscle as well as the number of brainstem neurons trans-synaptically infected by this virus varied inversely with an animal's weight. One possibility is that this effect was caused by the size of the muscle. Presumably the gastrocnemius muscle enlarges as the animal gets older and heavier, thus dispersing motor end-plates and motoneuron terminals over a greater area. This could effectively decrease the viral titer at each motoneuron terminal, thereby decreasing the probability of PRV entry into a particular terminal. An alternative explanation of this finding is that it may be related to the animal's age: younger animals may be more susceptible to infection with PRV. Consistent with this notion, it has been reported that swine are most vulnerable to developing Aujezsky's disease after exposure to PRV when they are young (Gustafson, 1975
Although this study revealed that neurons in several brainstem regions, most prominently the rostral ventromedial medulla, are likely to participate in both autonomic and motor control, available data do not permit a precise definition of the hierarchy of connections. This is attributable in large part to the demonstration that the onset of viral replication within a circuit is connection dependent (Card et al., 1999
Brainstem neurons with projections to both SPNs and motoneurons could play a number of different functional roles. These cells could serve to simultaneously adjust the excitability of motor and autonomic output pathways, so that both pathways would be more or less responsive to specific commands relayed through other neural circuits. For example, inhibitory bulbospinal neurons could potentially globally reduce both autonomic and motor activity during behaviors such as sleep (Futuro-Neto and Coote, 1982
Footnotes
Received Dec. 26, 2002; revised Mar. 17, 2003; accepted Mar. 17, 2003.This work was supported by National Institutes of Health Grants R01 DC00693 (B.J.Y.) and NS33506 (L.W.E.). We thank Kristine Cloonan, Ryan Mori, and Jen-Shew Yen for their excellent technical assistance. We are grateful to Dr. Stephen DiCarlo for his advice on surgical sympathectomy. We thank Dr. Pat Card for critical reading of an earlier version of this manuscript and for his advice and encouragement.
Correspondence should be addressed to I. A. Kerman, 205 Zina Pitcher Place, Mental Health Research Institute, Department of Psychiatry, University of Michigan, Ann Arbor, MI 48109. E-mail: kerman{at}umich.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/234657-10$15.00/0
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