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A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections
Brain Research, 491 (1989) 156-162 Elsevier
156 BRE 23562
A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections A.M. Strack 1, W.B. Sawyer t, J.H. Hughes 1, K.B.
Platt 2 a n d A . D . L o e w y ~
1Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110 (U.S.A.) and 2Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, 1A 50011 (U.S.A.) (Accepted 7 March 1989)
Key words: A5 cell group; Adrenal gland; Central gray matter; Celiac ganglion; Lateral hypothalamic area; Lumbar paravertebral ganglion; Paraventricular hypothalamic nucleus; Pseudorabies virus; Raphe nucleus; Rostral ventrolateral medulla; SteUate ganglion; Superior cervical ganglion; Transneuronal viral cell body labeling; Vasopressin; Ventral medulla; Viral transport
Pseudorabies virus (PRV) injections of various sympathetic ganglia and the adrenal gland were made in rats. These produced immunohistochemically detectable retrograde viral infections of ipsilateral sympathetic preganglionic neurons (SPNs) and transneuronal infections of the specific sets of second order neurons in the spinal cord and brain that innervate the infected SPNs. Five cell groups in the brain appear to regulate the entire sympathetic outflow: the paraventricular hypothalamic nucleus (PVH), A5 noradrenergic cell group, caudal raphe region, rostral ventrolateral medulla, and ventromedial medulla. In addition, local interneurons in laminae VII and X of the spinal cord are also involved. Other CNS areas also became transneuronally labeled after infections of certain sympathetic ganglia, most notably the superior cervical and stellate ganglia. These areas include the central gray matter and lateral hypothalamic area. The zona incerta was uniquely labeled after stellate ganglion infections. The cell body labeling was specific. This specificity was demonstrated in the PVH where the neurons of the parvocellular PVH that form the descending sympathetic pathway were labeled in a topographic fashion. Finally, we demonstrate that the retrograde transneuronal viral cell body labeling method can be used simultaneously with either neuropeptide transmitter or transmitter synthetic enzyme immunohistochemistry.
In 1938, Sabin 23 d e m o n s t r a t e d that neurotropic viruses are t r a n s p o r t e d in a retrograde transneuronal fashion. Several investigators have taken advantage of this p h e n o m e n o n and applied it to n e u r o a n a t o m ical studies 7'8"16'22'27. H o w e v e r , one difficulty in using neurotropic viruses is that most of these viruses are e x t r e m e l y virulent and can p r o d u c e uncontrolled and non-specific CNS infections. This p r o b l e m may be circumvented by the use of attenuated strains of neurotropic viruses 8'27. H o w e v e r , another practical p r o b l e m is that m a n y of these viruses infect humans and thus, present potential health hazards. Pseudorabies virus ( P R V ) , a herpes virus that is endemic to pigs and cattle, is an ideal virus for neuroanatomical studies in standard l a b o r a t o r y animals because it has b e e n shown to be t r a n s p o r t e d in a retrograde
transneuronal fashion in rats 16'23'27 and does not infect humans (e.g. ref. 3, however, see ref. 17). In the present study, we have used an a t t e n u a t e d strain of P R V - - B a r t h a ' s K strain 19 - - to study the general pattern of the central organization of the cell groups of the brain and spinal cord that regulate the sympathetic outflow. Using an operating microscope with a precalibrated reticule, 30-60 nl injections of a suspension of p s e u d o r a b i e s virus ( B a r t h a ' s K strain; titer = 1.03.0 × 106 pfu/ml) were m a d e via glass pipettes into either the adrenal gland (n = 12), the superior cervical (n = 8), the stellate (n = 8), the celiac (n = 7), or the L5 sympathetic ganglia in p e n t o b a r b i t a l anesthetized (50 mg/kg) S p r a g u e - D a w l e y rats (male, wt = 120-200 g, S A S C O , O ' F a l l o n , M O ) . A s
Correspondence: A.D. Loewy, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8108, St. Louis, MO 63110, U.S.A. 0006-8993/89/$03.50 (~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
157 the pipette was withdrawn, a drop of liquid sealant was applied to prevent viral leakage (Newskin, MedTech Labs, Cody, WY). Two days later, a lateral cerebroventricular injection of colchicine (100 #g/10 /~1 saline) was made, and then, after an additional 2 days (4 total), the rats were perfused with saline followed by 4% paraformaldehyde in 0.1 M NaPO4 buffer (pH = 7.4). The entire CNS was removed from the animals, placed in fixative for 24 h, and then transferred to 30% sucrose/phosphate buffer for 2 days. Brains were cut in the transverse plane at 40 pm on a freezing microtome. A 1-in-5 series was reacted using a 1:500 dilution of a pig anti-PRV polyclonal antibody (NC332) and visualized with an avidinbiotin-horseradish peroxidase complex using a Vectastain ABC kit (Vector Labs, Burlingame, CA), mounted on slides and coverslipped using DPX mountant (BDH, Poole, U.K.). Control sections were reacted with a preimmune pig serum (NC474); no cell body staining occurred. Spinal segments were cut in the horizontal plane at 50/~m and stained by the ABC procedure. To visualize putative neurotransmitters in PRVinfected neurons, a double fluorescence immunohistochemical procedure was used. Free floating tissues were reacted simultaneously at 4 °C for 12-16 h with a rabbit antibody to a neuropeptide or a transmitter synthesizing enzyme and a pig antibody to PRV. For visualization of the putative neurotransmitters or related enzymes, polyclonal antibodies were made in rabbits: rabbit anti-lysine vasopressin (Incstar, Stillwater, MN), at a dilution of 1:2000; rabbit antioxytocin (Incstar, Stillwater, MN), at a dilution of 1:2000; rabbit anti-tyrosine hydroxylase (East Acres Biologicals, Southbridge, MA), at a dilution of 1:1000; rabbit anti-phenylethanolamine-N-methyl transferase (Incstar, Stillwater, MN), at a dilution of 1:100; or rabbit anti-dopamine-fl-hydroxylase (Eugene Tech, Allendale, NJ), at a dilution of 1:50. These were used simultaneously with a pig polyclonal PRV antibody (NC332) at a dilution of 1:250. The tissues were incubated for 2 h in a 1:200 dilution of biotinylated goat anti-rabbit IgG (Vector Labs, Burlingame, CA) containing 2% goat serum/KPBS, rinsed, incubated for 2 h in 1:150 dilution of fluorescein isothiocyanate (FITC)-conjugated streptavidin (Jackson Labs, West Grove, PA), simulta-
neously with 1:50 dilution of rhodamine isothiocyahate (RITC)-conjugated goat anti-swine IgG (Jackson Labs, West Grove, PA) in 2% goat serum/KPBS, rinsed, mounted on gelatin-dipped slides, air-dried, and coverslipped with glycerol mountant (Citifluor Ltd., London). Sections were mapped for the double antibody labeling procedure using a Zeiss fluorescence microscope equipped with a BP450-490 excitation filter for visualizing FITC-stained cells and a BP546/12 excitation filter for visualizing RITC-stained cells with an X-Y plotter attached to an MD1 microscope digitizer (Minnesota Datametrics Corp., St. Paul, MN). Representative sections were then photographed using Kodak Tri-X (ASA 1600) film and developed with Agfa Rodinal. A topographic map of the PVH was constructed. To do this, PRV-infected vasopressin immunoreactive cell bodies from several different experiments (n = 2 for each sympathetic ganglion) were compared by making projection drawings based on a common landmark. This landmark was the densely clustered group of vasopressin neurons found in the posterior magnocellular subnucleus in the PVH 24. For each animal studied, 4 sections (40 pm thick) were taken from a 400-/~m-thick interval through this region from an individual animal and then compiled on a single drawing at the level of the PVH where oxytocin immunoreactive cells form a ring around the densely packed vasopressin neurons. The pattern of cell body labeling in each rat could then be compared to the pattern in other experiments by aligning the lateral and dorsal edges of the magnocellular nucleus of the PVH as shown by the straight dotted lines in Fig. 4. PRV cell body infections common to all the experiments were found in 5 places in the brain: paraventricular hypothalamic nucleus (PVH), A5 cell group, caudal raphe nuclei, rostral ventrolateral medulla and ventromedial medulla (Figs. 1 and 2). While each of these nuclei are known to project to the intermediolateral cell column 1°-12,15, it has not been known that each of these CNS nuclei may influence all levels of the sympathetic outflow. Spinal interneurons also regulate the sympathetic outflow at the local spinal levels. This was deduced from the observation that large numbers of small neurons in laminae VII and X of the spinal cord were
158 labeled in the same segments that contained infected sympathetic preganglionic neurons. When cell counts were made and compared to prevkmsly published Fluorogold retrograde cell body labeling data 26, we observed a marked increase in percentage and actual distribution of the labeled cells in the intercalated and central autonomic nuclei as compared to the intermediolateral cell column. Preliminary cell counts revealed that the total number of infected spinal neurons exceeded the number of comparable retrogradely labeled cells observed in Fluorogold experiments 26 by at least a 2:1 ratio. In addition to these 5 cell groups, other areas of the brain were also labeled. For example, after
Medulla .= Nuclei
Fig. 1. The general pattern of innervation of the sympathetic outflow as demonstrated by transneuronal cell body labeling after PRV infections of the sympathetic ganglia or adrenal gland. This pattern was seen after PRV infections of the superior cervical, stellate, celiac, and L5 sympathetic ganglia, as well as the adrenal gland. Other areas like the central gray matter, lateral hypothalamic area, and zona incerta were also labeled after infections of the stellate ganglion (see text). Abbreviations: IO, inferior olivary nucleus; MeV, medial vestibular nucleus; NTS, nucleus tractus solitarii; PB, parabrachial nucleus; SpV, spinal trigeminal nucleus; VMH, ventromedial hypothalamic nucleus; III, third ventricle. Drawings modified from ref. 18.
superior cervical ganglion (SCG) or stellate ganglion PRV injections, cell body infections were found in the mesencephalic central gray matter and the lateral hypothalamic area (LHA); however, a much larger number of infected cells was seen in the L H A after stellate ganglion experiments. The ventral zona incerta region (subincertal nucleus described in ref. 18) was labeled only after stellate ganglion injections. These uniquely infected cell groups may subserve specialized functions. For example, the central gray matter appears to be a critical site coordinating the defense reaction 1"2"4and the ventral zona incerta is involved in regulation of heart rate 25. We modified the transneuronal retrograde cell body labeling technique by using an attenuated PRV strain and combining it with a double immunohistochemical procedure for simultaneous detection of viral labeling and of putative neurotransmitters or their synthetic enzymes. Using this method we observed that after adrenal gland infections - 7 0 % of the PRV infected cells in the A5 region contain tyrosine hydroxylase immunoreactivity. Since the neurons in this area stained with an antibody directed against dopamine-fl-hydroxylase, but not phenylethanolamine-N-methyl transferase, these cells are presumably noradrenergic neurons. The spinal projections from the PVH arise from different neurochemicalty coded neurons 24. We studied the vasopressin (VP)-containing subclass in detail. Our experiments show both a differential topography of transneuronal retrograde cell body labeling of this set of neurons as well as a quantitative difference in the density of the innervation of the different sympathetic outflows. After SCG viral injections - 2 2 % of the PRV-infected PVH neurons contained VP immunoreactivity (Fig. 3), after stellate ganglion infections - 1 0 % of the PRV cells exhibited VP immunoreactivity, after celiac ganglion and adrenal gland infections less than 2% of the PRV-infected PVH cells contained VP immunoreactivity. The parvocellular PVH spinally projecting neurons are organized in a topographic fashion which correlates with the rostrocaudal distribution of the ganglion-specific SPNs (Fig. 4). The PVH neurons controlling the upper thoracic sympathetic outflow lies medially and those projecting to the mid- and lower thoracic levels lie dorsolaterally. In addition, we observed a 3:2 ratio of ipsilateral to
159
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Fig. 2. Top panels: tyrosine hydroxylase immunoreactive cell bodies in the A5 cell group become transneuronally infected after a PRV viral infection of the adrenal gland. (A) is a photomicrograph of FITC labeled tyrosine hydroxylase labeled immunoreactive A5 neurons. (B) is a photomicrograph of PRV infected neurons as revealed by RITC indirect immunohistochemistry. Note each of the A5 neurons was infected. Bar = 50/zm. Bottom panels: neurons in the rostral ventrolateral medulla and raphe pallidus become infected after PRV infections of the stellate ganglion. (A) is a photomicrograph of avidin-biotin-diaminobenzidine stained PRV infected neurons in the rostral ventrolateral medulla. (B) is a photomicrograph of similar material to show PRV-infected neurons in the raphe pallidus nucleus. Bar = 50/zm. The numbers given in the line drawings indicate the level relative to the calamus scriptorius. Abbreviations: same as Fig. 1 with the addition of LC, locus coeruleus; MoV, motor trigeminal nucleus; NA, nucleus ambiguus; PrH, prepositus hypoglossal nucleus; PrV, principal sensory trigeminal nucleus; SO, superior olivary nucleus; SpVe, spinal vestibular nucleus; VII, facial nerve.
160 not be applicable to all neuronal systems. Not all neurons may be virally infected with PRV. Examples of this refractiveness are the neurons of the mesencephalic nucleus of the trigeminal nerve (but not the trigeminal ganglion) and the neurons in the olfactory
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Fig. 3. (A) Drawing showing the cytoarchitectonic divisions of the paraventricular hypothalamic nucleus of a rat based on ref. 24. Square indicates the location where the photographs were taken. (B) Photomicrograph demonstrating vasopressin immunoreactive neurons (FITC). (C) Two PVH vasopressin neurons (arrows) exhibited viral infection after a PRV injection in the superior cervical ganglion (RITC). Bar = 50/ira.
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thalamic (PVH) neurons that project to different sympathetic outflows as determined after viral infections of the various sympathetic ganglia and the adrenal gland. (A) Pattern of cell body infections seen after PRV infections of the superior cervical ganglion and adrenal gland. (B) Pattern of cell body labeling after injections in the stellate and celiac sympathetic ganglia. (C) Spinal segmental distribution of sympathetic preganglionic neurons labeled after viral infection of the various sympathetic ganglia for comparison with the cell body labeling in the PVH.
161 strated here and elsewhere 27, it may not be possible to use it for demonstrating all putative neurotransmitters, because herpes viruses, and PRV in particular, are known to shut off host protein synthesis in infected cells6'2°. Therefore, to histochemically demonstrate some cellular markers in viraily infected cells may be impossible if their turnover is rapid. However, this does not seem to be a problem for the demonstration of a variety of neuropeptides and catecholamine transmitter synthetic enzymes used in this and a recent study 27. Third, our overall success rate was approximately 20% and this may relate to both the weak virus used as well as genetic variations of the immune response to viral infections 13A4"21. The rats we used were an out-bred strain and thus might account for this variability. The use of rodents with particular genetic variations or the use of immunosuppressive drugs may have enhanced this result. A final important issue affecting the interpretation of these experiments is the specificity of the technique. It is important to emphasize that we examined the second order cell body labeling in selected brains only if the labeling of the sympathetic preganglionic neurons exhibited the same ipsilateral segmental distribution as seen with a conventional fluorescent dye retrograde neuroanatomical marker 26. This is a critical factor. Since this is a very sensitive technique wherein a few aberrantly labeled cells will result in erroneous second order labeling, careful analysis of the first order neurons is necessary. In addition, we avoided any histological material that had evidence that the SPNs had undergone cell lysis. In our experience, when a large number of the first order neurons showed cell lysis, it was likely that the infection could spread to non-related neural systems. However, with Bartha's K strain of PRV, unlike the wildtype strain, we did not observe cell lysis or evidence of non-specific viral immunoreactive material in the neuropi127. This seems to be a critical factor in the specificity of this method. In addition, it is extremely unlikely that the viruses were taken up by fibers of passage since cell body infections occurred only in cell groups known to project to the intermediolaterai cell column (IML) 1°-12"15. If non-specific uptake had occurred, then various areas of the medullary and pontine reticular formation as well as the locus coeruleus
would have been labeled because these areas project to the intermediate spinal gray matter which lie in close proximity to the SPNs 5. No labeled cells were found in these areas except when diffuse spinal viremias occurred which spread beyond the SPNs and such material was not used in our analysis. Two additional lines of evidence support the contention that specific transsynaptic labeling occurred. First, we observed certain CNS cell groups labeled only after infections of particular sympathetic ganglia. Most notably, labeling of the region of the ventral zona incerta was seen only after stellate ganglion infections. Since the first order SPNs labeled after stellate ganglion infection have a similar spinal segmental distribution as that seen after SCG infection (Fig. 4), if non-specific transneuronal labeling occurred, then the zona incerta would have been labeled after SCG infections. This did not occur. Second, Martin and Dolivo ~6 reported that PRV injections in the anterior chamber of the eye resulted in intermediolateral cell column labeling only in T1-T2 spinal segments. This observation is important because the SCG is a good site for determining viral specificity. Since sympathetic ganglionic neurons innervating the iris are intermingled with other ganglionic neurons receiving inputs from the Cs-T 5 SPNs 9, any viral diffusion after eye injection would have labeled the Cs-T 5 levels of the IML. This did not occur, thus supporting our contention that the PRV was transported specifically. In summary, our results suggest that there is a commonality of CNS cell groups in the brain controlling the sympathetic outflow to the head, thoracic viscera, gut, adrenal gland, and limbs. Certain nuclei like the PVH are organized in a topographic fashion but other nuclei may not be organized this way. They may project throughout all levels of the intermediolateral cell column and function to orchestrate global sympathetic changes that occur during the 'fight or flight' reaction or in the sleep-wakefulness cycle. This work was supported by National Heart, Lung, and Blood Institute of the National Institutes of Health (HL25449), the Diabetes Research and Training Center, and the Iowa Livestock Health Advisory Committee. A.M.S. is supported by the
162
M a r k e y F o u n d a t i o n and the M c D o n n e l l C e n t e r for
assistance and D a v i d G o t t l i e b , J a m e s K r a u s e and
the Studies of H i g h e r Brain F u n c t i o n . J . H . H .
J e f f L i c h t m a n for constructive c o m m e n t s on the
is
s u p p o r t e d by the M S T P training grant. We t h a n k
manuscript.
X a y Van N g u y e n and M i c h e l e Solis for technical
preparing the manuscript.
W e t h a n k Sue E a d s for her h e l p in
1 Bandler, R., Induction of ~rage' following microinjections of glutamate into midbrain but not hypothalamus of cats, Neurosci. Lett., 30 (1982) 183-188. 2 Bandler, R. and Carrive, P., Integrated defence reaction elicited by excitatory amino acid microinjection in the midbrain periaqueductal grey region of the unrestrained cat, Brain Research, 439 (1988) 95-106. 3 Gustafson, D.P., Pseudorabies. In A.D. Leman et al. (Eds.), Diseases of Swine, Iowa State Univ. Press, Ames, IA, 1975, pp. 209-223. 4 Hilton, S.M. and Redfern, W.S., A search for brainstem cell groups integrating the defence reaction in the rat, J. Physiol. (Lond.), 378 (1986) 213-228. 5 Jones, B.E. and Yang, T.-Z., The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat, J. Comp. Neurol.. 242 (1985) 56-92. 6 Kaplan, A.S., Shimono, H. and 13en-Porat, T., Synthesis of proteins in ceils infected with herpesviruses. III. Relative amino acid content of various proteins formed after infection, Virology, 40 (1970) 90-101. 7 Kristensson, K., Nennesmo, I., Persson, L. and Lycke, E., Neuron to neuron transmission of herpes simplex virus. Transport of virus from skin to brainstem nuclei, J. Neurol. Sci., 54 (1982) 149-156. 8 Kucera, P., Dolivo, M., Coulon, P. and Flamand, A., Pathways of the early propagation of virulent and avirulent rabies strains from the eye to the brain, J. Virol.. 55 (1985) 158-162. 9 Lichtman, J.W., Purves, D. and Yip, J.W., On the purpose of selective innervation of guinea-pig superior cervical ganglion cells, J. Physiol, (Lond.), 292 (1979) 69-84. 10 Loewy, A.D., Raphe pallidus and raphe obscurus projections to the intermediolateral cell column in the rat, Brain Research, 222 (1981) 129-133. 11 Loewy, A.D., McKellar, S. and Saper, C.B., Direct projections from the A5 catecholamine cell group to the intermediolateral cell column, Brain Research, 174 (1979) 309-314. 12 Loewy, A.D., Wallach, J.H. and McKellar, S., Efferent connections of the ventral medulla oblongata in the rat, Brain Res. Rev., 3 (1981) 63-80. 13 Lopez, C., Genetics of natural resistance to herpesvirus infections in mice, Nature (Lond.), 258 (1975) 152-153. 14 Lopez, C., Resistance to HSV-1 in the mouse is governed by two major, independently segregating, non-H-Z loci,
lmmunogenetics, 11 (1980) 87-92. 15 Luiten, P.G.M., ter Horst, G.J., Karst, H. and Steffens, A.B., The course of paraventricular hypothalamic efferents to autonomic structures in medulla and spinal cord, Brain Research, 329 (1985) 374-378. 16 Martin, X. and Dolivo, M., Neuronal and transneuronal tracing in the trigeminal system of the rat using the herpes virus suis, Brain Research, 273 (1983) 253-276. 17 Mravik, S., Bienzle, O., Feldmeier, H., Hampl, H. and Habermehl, K.-C., PRV in man, Lancet, 1 (1987) 501-502. 18 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney, 1986. 19 Platt, K.B., Mar6, C.J. and Hinz, P.N., Differentiation of vaccine strains and field isolates of PRV (Aujeszky's disease) virus: thermal sensitivity and rabbit virulence markers, Arch. Virol., 60 (1979) 13-23. 20 Roizman, 13. and Batterson, W., Herpesviruses and their replication. In B.N. Fields et al. (Eds.), Virology, Raven, New York, 1985, pp. 497-526. 21 Rosenstreich, D.L., Weinblatt, A.C. and O'Brien, A.D., Genetic control of resistance to infection in mice, CRC Crit. Rev. lmmunol., 3 (1982) 263-330. 22 Rouiller, E.M., Capt, M., Dolivo, M. and De Ribapierre, E, Tensor tympani reflex pathways studied with retrograde horseradish peroxidase and transneuronal viral tracing techniques, Neurosci. Lett., 72 (1986) 247-252. 23 Sabin, A.B., Progression of different nasally instilled viruses along different nervous pathways in the same host, Proc. Soc. Exp. Biol. Med.. 38 (1938) 270-275. 24 Sawchenko, P.E. and Swanson, L.W., Immunohistochemical identification of neurons in the paraventricutar nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat, J, Comp. Neurol., 205 (1982) 260-272. 25 Spencer, S.E., Sawyer, W.B. and Loewy, A.D., L-Glutamate stimulation of the zona incerta in the rat decreases heart rate and blood pressure, Brain Research, 458 (1988) 72-81. 26 Strack, A.M., Sawyer, W.B., Marubio, L.M. and Loewy, A.D., Spinal origin of sympathetic preganglionic neurons in the rat, Brain Research, 455 (1988) 187-191. 27 Strack, A.M., Sawyer, W.B., Platt, K.B. and Loewy, A.D., CNS cell groups regulating the sympathetic nervous outflow to adrenal gland as revealed by transneuronal cell body labeling with pseudorabies virus, Brain Research, 000 (1989) 000-000.
156 BRE 23562
A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections A.M. Strack 1, W.B. Sawyer t, J.H. Hughes 1, K.B.
Platt 2 a n d A . D . L o e w y ~
1Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110 (U.S.A.) and 2Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, 1A 50011 (U.S.A.) (Accepted 7 March 1989)
Key words: A5 cell group; Adrenal gland; Central gray matter; Celiac ganglion; Lateral hypothalamic area; Lumbar paravertebral ganglion; Paraventricular hypothalamic nucleus; Pseudorabies virus; Raphe nucleus; Rostral ventrolateral medulla; SteUate ganglion; Superior cervical ganglion; Transneuronal viral cell body labeling; Vasopressin; Ventral medulla; Viral transport
Pseudorabies virus (PRV) injections of various sympathetic ganglia and the adrenal gland were made in rats. These produced immunohistochemically detectable retrograde viral infections of ipsilateral sympathetic preganglionic neurons (SPNs) and transneuronal infections of the specific sets of second order neurons in the spinal cord and brain that innervate the infected SPNs. Five cell groups in the brain appear to regulate the entire sympathetic outflow: the paraventricular hypothalamic nucleus (PVH), A5 noradrenergic cell group, caudal raphe region, rostral ventrolateral medulla, and ventromedial medulla. In addition, local interneurons in laminae VII and X of the spinal cord are also involved. Other CNS areas also became transneuronally labeled after infections of certain sympathetic ganglia, most notably the superior cervical and stellate ganglia. These areas include the central gray matter and lateral hypothalamic area. The zona incerta was uniquely labeled after stellate ganglion infections. The cell body labeling was specific. This specificity was demonstrated in the PVH where the neurons of the parvocellular PVH that form the descending sympathetic pathway were labeled in a topographic fashion. Finally, we demonstrate that the retrograde transneuronal viral cell body labeling method can be used simultaneously with either neuropeptide transmitter or transmitter synthetic enzyme immunohistochemistry.
In 1938, Sabin 23 d e m o n s t r a t e d that neurotropic viruses are t r a n s p o r t e d in a retrograde transneuronal fashion. Several investigators have taken advantage of this p h e n o m e n o n and applied it to n e u r o a n a t o m ical studies 7'8"16'22'27. H o w e v e r , one difficulty in using neurotropic viruses is that most of these viruses are e x t r e m e l y virulent and can p r o d u c e uncontrolled and non-specific CNS infections. This p r o b l e m may be circumvented by the use of attenuated strains of neurotropic viruses 8'27. H o w e v e r , another practical p r o b l e m is that m a n y of these viruses infect humans and thus, present potential health hazards. Pseudorabies virus ( P R V ) , a herpes virus that is endemic to pigs and cattle, is an ideal virus for neuroanatomical studies in standard l a b o r a t o r y animals because it has b e e n shown to be t r a n s p o r t e d in a retrograde
transneuronal fashion in rats 16'23'27 and does not infect humans (e.g. ref. 3, however, see ref. 17). In the present study, we have used an a t t e n u a t e d strain of P R V - - B a r t h a ' s K strain 19 - - to study the general pattern of the central organization of the cell groups of the brain and spinal cord that regulate the sympathetic outflow. Using an operating microscope with a precalibrated reticule, 30-60 nl injections of a suspension of p s e u d o r a b i e s virus ( B a r t h a ' s K strain; titer = 1.03.0 × 106 pfu/ml) were m a d e via glass pipettes into either the adrenal gland (n = 12), the superior cervical (n = 8), the stellate (n = 8), the celiac (n = 7), or the L5 sympathetic ganglia in p e n t o b a r b i t a l anesthetized (50 mg/kg) S p r a g u e - D a w l e y rats (male, wt = 120-200 g, S A S C O , O ' F a l l o n , M O ) . A s
Correspondence: A.D. Loewy, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8108, St. Louis, MO 63110, U.S.A. 0006-8993/89/$03.50 (~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
157 the pipette was withdrawn, a drop of liquid sealant was applied to prevent viral leakage (Newskin, MedTech Labs, Cody, WY). Two days later, a lateral cerebroventricular injection of colchicine (100 #g/10 /~1 saline) was made, and then, after an additional 2 days (4 total), the rats were perfused with saline followed by 4% paraformaldehyde in 0.1 M NaPO4 buffer (pH = 7.4). The entire CNS was removed from the animals, placed in fixative for 24 h, and then transferred to 30% sucrose/phosphate buffer for 2 days. Brains were cut in the transverse plane at 40 pm on a freezing microtome. A 1-in-5 series was reacted using a 1:500 dilution of a pig anti-PRV polyclonal antibody (NC332) and visualized with an avidinbiotin-horseradish peroxidase complex using a Vectastain ABC kit (Vector Labs, Burlingame, CA), mounted on slides and coverslipped using DPX mountant (BDH, Poole, U.K.). Control sections were reacted with a preimmune pig serum (NC474); no cell body staining occurred. Spinal segments were cut in the horizontal plane at 50/~m and stained by the ABC procedure. To visualize putative neurotransmitters in PRVinfected neurons, a double fluorescence immunohistochemical procedure was used. Free floating tissues were reacted simultaneously at 4 °C for 12-16 h with a rabbit antibody to a neuropeptide or a transmitter synthesizing enzyme and a pig antibody to PRV. For visualization of the putative neurotransmitters or related enzymes, polyclonal antibodies were made in rabbits: rabbit anti-lysine vasopressin (Incstar, Stillwater, MN), at a dilution of 1:2000; rabbit antioxytocin (Incstar, Stillwater, MN), at a dilution of 1:2000; rabbit anti-tyrosine hydroxylase (East Acres Biologicals, Southbridge, MA), at a dilution of 1:1000; rabbit anti-phenylethanolamine-N-methyl transferase (Incstar, Stillwater, MN), at a dilution of 1:100; or rabbit anti-dopamine-fl-hydroxylase (Eugene Tech, Allendale, NJ), at a dilution of 1:50. These were used simultaneously with a pig polyclonal PRV antibody (NC332) at a dilution of 1:250. The tissues were incubated for 2 h in a 1:200 dilution of biotinylated goat anti-rabbit IgG (Vector Labs, Burlingame, CA) containing 2% goat serum/KPBS, rinsed, incubated for 2 h in 1:150 dilution of fluorescein isothiocyanate (FITC)-conjugated streptavidin (Jackson Labs, West Grove, PA), simulta-
neously with 1:50 dilution of rhodamine isothiocyahate (RITC)-conjugated goat anti-swine IgG (Jackson Labs, West Grove, PA) in 2% goat serum/KPBS, rinsed, mounted on gelatin-dipped slides, air-dried, and coverslipped with glycerol mountant (Citifluor Ltd., London). Sections were mapped for the double antibody labeling procedure using a Zeiss fluorescence microscope equipped with a BP450-490 excitation filter for visualizing FITC-stained cells and a BP546/12 excitation filter for visualizing RITC-stained cells with an X-Y plotter attached to an MD1 microscope digitizer (Minnesota Datametrics Corp., St. Paul, MN). Representative sections were then photographed using Kodak Tri-X (ASA 1600) film and developed with Agfa Rodinal. A topographic map of the PVH was constructed. To do this, PRV-infected vasopressin immunoreactive cell bodies from several different experiments (n = 2 for each sympathetic ganglion) were compared by making projection drawings based on a common landmark. This landmark was the densely clustered group of vasopressin neurons found in the posterior magnocellular subnucleus in the PVH 24. For each animal studied, 4 sections (40 pm thick) were taken from a 400-/~m-thick interval through this region from an individual animal and then compiled on a single drawing at the level of the PVH where oxytocin immunoreactive cells form a ring around the densely packed vasopressin neurons. The pattern of cell body labeling in each rat could then be compared to the pattern in other experiments by aligning the lateral and dorsal edges of the magnocellular nucleus of the PVH as shown by the straight dotted lines in Fig. 4. PRV cell body infections common to all the experiments were found in 5 places in the brain: paraventricular hypothalamic nucleus (PVH), A5 cell group, caudal raphe nuclei, rostral ventrolateral medulla and ventromedial medulla (Figs. 1 and 2). While each of these nuclei are known to project to the intermediolateral cell column 1°-12,15, it has not been known that each of these CNS nuclei may influence all levels of the sympathetic outflow. Spinal interneurons also regulate the sympathetic outflow at the local spinal levels. This was deduced from the observation that large numbers of small neurons in laminae VII and X of the spinal cord were
158 labeled in the same segments that contained infected sympathetic preganglionic neurons. When cell counts were made and compared to prevkmsly published Fluorogold retrograde cell body labeling data 26, we observed a marked increase in percentage and actual distribution of the labeled cells in the intercalated and central autonomic nuclei as compared to the intermediolateral cell column. Preliminary cell counts revealed that the total number of infected spinal neurons exceeded the number of comparable retrogradely labeled cells observed in Fluorogold experiments 26 by at least a 2:1 ratio. In addition to these 5 cell groups, other areas of the brain were also labeled. For example, after
Medulla .= Nuclei
Fig. 1. The general pattern of innervation of the sympathetic outflow as demonstrated by transneuronal cell body labeling after PRV infections of the sympathetic ganglia or adrenal gland. This pattern was seen after PRV infections of the superior cervical, stellate, celiac, and L5 sympathetic ganglia, as well as the adrenal gland. Other areas like the central gray matter, lateral hypothalamic area, and zona incerta were also labeled after infections of the stellate ganglion (see text). Abbreviations: IO, inferior olivary nucleus; MeV, medial vestibular nucleus; NTS, nucleus tractus solitarii; PB, parabrachial nucleus; SpV, spinal trigeminal nucleus; VMH, ventromedial hypothalamic nucleus; III, third ventricle. Drawings modified from ref. 18.
superior cervical ganglion (SCG) or stellate ganglion PRV injections, cell body infections were found in the mesencephalic central gray matter and the lateral hypothalamic area (LHA); however, a much larger number of infected cells was seen in the L H A after stellate ganglion experiments. The ventral zona incerta region (subincertal nucleus described in ref. 18) was labeled only after stellate ganglion injections. These uniquely infected cell groups may subserve specialized functions. For example, the central gray matter appears to be a critical site coordinating the defense reaction 1"2"4and the ventral zona incerta is involved in regulation of heart rate 25. We modified the transneuronal retrograde cell body labeling technique by using an attenuated PRV strain and combining it with a double immunohistochemical procedure for simultaneous detection of viral labeling and of putative neurotransmitters or their synthetic enzymes. Using this method we observed that after adrenal gland infections - 7 0 % of the PRV infected cells in the A5 region contain tyrosine hydroxylase immunoreactivity. Since the neurons in this area stained with an antibody directed against dopamine-fl-hydroxylase, but not phenylethanolamine-N-methyl transferase, these cells are presumably noradrenergic neurons. The spinal projections from the PVH arise from different neurochemicalty coded neurons 24. We studied the vasopressin (VP)-containing subclass in detail. Our experiments show both a differential topography of transneuronal retrograde cell body labeling of this set of neurons as well as a quantitative difference in the density of the innervation of the different sympathetic outflows. After SCG viral injections - 2 2 % of the PRV-infected PVH neurons contained VP immunoreactivity (Fig. 3), after stellate ganglion infections - 1 0 % of the PRV cells exhibited VP immunoreactivity, after celiac ganglion and adrenal gland infections less than 2% of the PRV-infected PVH cells contained VP immunoreactivity. The parvocellular PVH spinally projecting neurons are organized in a topographic fashion which correlates with the rostrocaudal distribution of the ganglion-specific SPNs (Fig. 4). The PVH neurons controlling the upper thoracic sympathetic outflow lies medially and those projecting to the mid- and lower thoracic levels lie dorsolaterally. In addition, we observed a 3:2 ratio of ipsilateral to
159
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160 not be applicable to all neuronal systems. Not all neurons may be virally infected with PRV. Examples of this refractiveness are the neurons of the mesencephalic nucleus of the trigeminal nerve (but not the trigeminal ganglion) and the neurons in the olfactory
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thalamic (PVH) neurons that project to different sympathetic outflows as determined after viral infections of the various sympathetic ganglia and the adrenal gland. (A) Pattern of cell body infections seen after PRV infections of the superior cervical ganglion and adrenal gland. (B) Pattern of cell body labeling after injections in the stellate and celiac sympathetic ganglia. (C) Spinal segmental distribution of sympathetic preganglionic neurons labeled after viral infection of the various sympathetic ganglia for comparison with the cell body labeling in the PVH.
161 strated here and elsewhere 27, it may not be possible to use it for demonstrating all putative neurotransmitters, because herpes viruses, and PRV in particular, are known to shut off host protein synthesis in infected cells6'2°. Therefore, to histochemically demonstrate some cellular markers in viraily infected cells may be impossible if their turnover is rapid. However, this does not seem to be a problem for the demonstration of a variety of neuropeptides and catecholamine transmitter synthetic enzymes used in this and a recent study 27. Third, our overall success rate was approximately 20% and this may relate to both the weak virus used as well as genetic variations of the immune response to viral infections 13A4"21. The rats we used were an out-bred strain and thus might account for this variability. The use of rodents with particular genetic variations or the use of immunosuppressive drugs may have enhanced this result. A final important issue affecting the interpretation of these experiments is the specificity of the technique. It is important to emphasize that we examined the second order cell body labeling in selected brains only if the labeling of the sympathetic preganglionic neurons exhibited the same ipsilateral segmental distribution as seen with a conventional fluorescent dye retrograde neuroanatomical marker 26. This is a critical factor. Since this is a very sensitive technique wherein a few aberrantly labeled cells will result in erroneous second order labeling, careful analysis of the first order neurons is necessary. In addition, we avoided any histological material that had evidence that the SPNs had undergone cell lysis. In our experience, when a large number of the first order neurons showed cell lysis, it was likely that the infection could spread to non-related neural systems. However, with Bartha's K strain of PRV, unlike the wildtype strain, we did not observe cell lysis or evidence of non-specific viral immunoreactive material in the neuropi127. This seems to be a critical factor in the specificity of this method. In addition, it is extremely unlikely that the viruses were taken up by fibers of passage since cell body infections occurred only in cell groups known to project to the intermediolaterai cell column (IML) 1°-12"15. If non-specific uptake had occurred, then various areas of the medullary and pontine reticular formation as well as the locus coeruleus
would have been labeled because these areas project to the intermediate spinal gray matter which lie in close proximity to the SPNs 5. No labeled cells were found in these areas except when diffuse spinal viremias occurred which spread beyond the SPNs and such material was not used in our analysis. Two additional lines of evidence support the contention that specific transsynaptic labeling occurred. First, we observed certain CNS cell groups labeled only after infections of particular sympathetic ganglia. Most notably, labeling of the region of the ventral zona incerta was seen only after stellate ganglion infections. Since the first order SPNs labeled after stellate ganglion infection have a similar spinal segmental distribution as that seen after SCG infection (Fig. 4), if non-specific transneuronal labeling occurred, then the zona incerta would have been labeled after SCG infections. This did not occur. Second, Martin and Dolivo ~6 reported that PRV injections in the anterior chamber of the eye resulted in intermediolateral cell column labeling only in T1-T2 spinal segments. This observation is important because the SCG is a good site for determining viral specificity. Since sympathetic ganglionic neurons innervating the iris are intermingled with other ganglionic neurons receiving inputs from the Cs-T 5 SPNs 9, any viral diffusion after eye injection would have labeled the Cs-T 5 levels of the IML. This did not occur, thus supporting our contention that the PRV was transported specifically. In summary, our results suggest that there is a commonality of CNS cell groups in the brain controlling the sympathetic outflow to the head, thoracic viscera, gut, adrenal gland, and limbs. Certain nuclei like the PVH are organized in a topographic fashion but other nuclei may not be organized this way. They may project throughout all levels of the intermediolateral cell column and function to orchestrate global sympathetic changes that occur during the 'fight or flight' reaction or in the sleep-wakefulness cycle. This work was supported by National Heart, Lung, and Blood Institute of the National Institutes of Health (HL25449), the Diabetes Research and Training Center, and the Iowa Livestock Health Advisory Committee. A.M.S. is supported by the
162
M a r k e y F o u n d a t i o n and the M c D o n n e l l C e n t e r for
assistance and D a v i d G o t t l i e b , J a m e s K r a u s e and
the Studies of H i g h e r Brain F u n c t i o n . J . H . H .
J e f f L i c h t m a n for constructive c o m m e n t s on the
is
s u p p o r t e d by the M S T P training grant. We t h a n k
manuscript.
X a y Van N g u y e n and M i c h e l e Solis for technical
preparing the manuscript.
W e t h a n k Sue E a d s for her h e l p in
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