- Email: [email protected]
Lacrimal preganglionic neurons form a subdivision of the superior salivatory nucleus of rat: transneuronal labelling by pseudorabies virus
Journal of the Autonomic Nervous System 77 Ž1999. 45–54
Lacrimal preganglionic neurons form a subdivision of the superior salivatory nucleus of rat: transneuronal labelling by pseudorabies virus Ida E. Toth ´ a
a,)
, Zsolt Boldogkoi ˝ a, Istvan ´ Medveczky b, Miklos ´ Palkovits
a
Joint Research Laboratory of Neuromorphology, Semmelweis UniÕersity of Medicine and Hungarian Academy of Sciences, Tuzolto ˝ ´ utca 58, Budapest H-1450, Hungary b Department of Microbiology and Infectious Diseases, UniÕersity of Veterinary Science, Hungaria krt. 23-25, Budapest H-1143, Hungary Received 10 August 1998; received in revised form 23 March 1999; accepted 5 May 1999
Abstract Transneuronal viral tracing was applied to localize preganglionic parasympathetic neurons in the brainstem which innervate the extraorbital lacrimal gland in the rat. The Bartha strain of pseudorabies virus was injected into the lacrimal gland, and after different survival times, the superior cervical and Gasserian ganglia, the upper thoracic spinal cords and the brainstems were immunostained by antiviral antiserum. Virus-labelled neurons appeared in the ganglia and in the ventrolateral part of the ipsilateral brainstem at the pontomedullary junction 45 h after inoculation. The virus-labelled brainstem neurons comprised a subgroup of the superior salivatory nucleus ŽSSN. located between the root fibers of the facial nerve and the nuclei of the superior olive, and were clearly distinguished from the tyrosine hydroxylase ŽTH.-immunopositive, A5 catecholaminergic neurons by double immunostaining. The number of infected cells in the ipsilateral SSN was increased by 72 h, and labelled neurons appeared in the intermediolateral cell column ŽIML. of the ipsilateral thoracic spinal cord. In rats with cervical ganglionectomy prior to the virus injection in the lacrimal gland, virus-infected cells appeared in the SSN, but not in the thoracic spinal cord, indicating that preganglionic SSN cells were infected via parasympathetic axons of the facial nerve. A double-virus tracer labelling technique was applied to determine the topographical relationship between the preganglionic parasympathetic neurons of the lacrimal gland and those of the submandibular gland within the SSN. Simultaneous injection of Bartha strain of pseudorabies virus into the submandibular gland, and a lacZ gene-containing Bartha-derived virus strain into the lacrimal gland Žand vice versa. demarcated a ventral lacrimal and a dorsal submandibular subgroup in the SSN. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Transneuronal viral tracing; Lacrimal gland; Immunohistochemistry
1. Introduction The superior salivatory nucleus ŽSSN. is a group of parasympathetic preganglionic neurons innervating the lacrimal, submandibular and sublingual glands, in addition to the nasal and palatine mucosa, through the pterygopalatine Žalso called sphenopalatine. and submandibular ganglia. In the rat, in contrast with humans, primates and many other mammalian species, early studies involving anatomical Žretrograde degeneration. and electrophysiolog-
AbbreÕiations: Ba-NeutLac, b-galactosidase expressing Bartha strain of pseudorabies virus; IML, intermediolateral cell column; PB, phosphate buffer; pfu, plaque-forming unit; PrV-Ba, Bartha strain of pseudorabies virus; SSN, superior salivatory nucleus; TH, tyrosine hydroxylase ) Corresponding author. Tel.: q36-1-215-6920r3660; fax: q36-1218-1612; e-mail: [email protected]
ical approaches led to controversial results concerning the exact topographical localization of the SSN in the lower brainstem. Recent studies with retrograde tracers resulted in descriptions and names for different subdivisions of the SSN depending on the approach applied. Axons of preganglionic neurons innervating the lacrimal gland and the nasal and palatine mucosa contribute to the greater superficial petrosal nerve, and terminate in the pterygopalatine ganglion. Other preganglionic neurons in the SSN which innervate the submandibular and sublingual glands contribute axons to the submandibular ganglion through the chorda tympani. Retrograde viral transneuronal tracer injected into the submandibular gland ŽJansen et al., 1992., or horseradish peroxidase injected into the chorda tympani ŽHiura, 1977; Contreras et al., 1980; Mitchell and Templeton, 1981; Nicholson and Severin, 1981. revealed neurons in the dorsomedial part of the SSN. Injections of retro-
0165-1838r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 9 . 0 0 0 3 2 - 6
46
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
kept under standard laboratory conditions Žroom temperature, 22 " 18C, with a 12-h light–dark cycle, with lights on at 0600 h.. Regular rat chow and tap water were provided ad libitum. The experimental procedures were approved by the local Animal Care and Use Committee. 2.2. Viruses
Fig. 1. Infection of the extraorbital lacrimal gland. Ž1. Parasympathetic fibers from the SSN ŽS., with synaptic transfer in the pterygopalatine ŽSP. ganglion. Ž2. Sympathetic fibers from the IML in the thoracic spinal cord, with synaptic transfer in the superior cervical ganglion ŽCS.. Ž3. Sensory fibers through the trigeminal nerve ŽV. and ganglion ŽG. to the nucleus of the spinal trigeminal tract ŽSV..
grade tracers ŽInsausiti and Gonzalo, 1980; Senba et al., 1987; Suzuki et al., 1990; Nemoto et al., 1995. or virus ŽSpencer et al., 1990. into the pterygopalatine ganglion or horseradish peroxidase into the greater petrosal nerve ŽHosoya et al., 1984, 1990. resulted in neuronal labelling in the ventrolateral part of the SSN. Reflecting the function of these cells, this subdivision of the SSN is also referred to as the lacrimo-nasopalatine ŽSenba et al., 1987., lacrimo-muconasal ŽInsausiti and Gonzalo, 1980. or lacrimal ŽContreras et al., 1980. nucleus. In the present study, we set out to localize the preganglionic neurons innervating the lacrimal gland by using a viral tracer technique. This technique is capable of revealing multisynaptic connections via specific transynaptic propagation of the virus Žfor details, see Card, 1995; Ugolini, 1995; Toth ´ and Palkovits, 1997.. The neurotropic viruses infect all types of neurons, e.g. parasympathetic, sympathetic and sensory ones, and thus, we studied these three possible pathways through which viruses may enter the brain ŽFig. 1.. Through the application of the Bartha strain of pseudorabies virus ŽPrV-Ba, Aujeszky, 1902; Bartha, 1961. and a genetically engineered b-galactosidase expressing Bartha-derived strain as transneuronal tracer, a subdivision of the SSN projecting exclusively to the extraorbital lacrimal gland of rat was distinguished. We determined the topographical relationship between the lacrimal and submandibular subdivisions of SSN by application of a double virus tracer system, e.g. by injection of immunohistochemically separable virus tracers into these two glands.
2. Experimental procedures 2.1. Animals Twenty-four adult rats of the Sprague–Dawley and Wistar strains were utilized in this study. The animals were
Two pseudorabies viruses: Ži. PrV-Ba ŽAujeszky, 1902; Bartha, 1961. and Žii. a genetically engineered, Bartha-derived strain, expressing Escherichia coli b-galactosidase ŽBa-NeutLac., were used in this study. Ba-NeutLac was constructed by the insertion of the lacZ gene to a ‘neutral’ segment Žplaying no role in the viral virulence. of the Bartha genome ŽBoldogkoi ˝ et al., 1998.. The aim of the insertion of the lacZ gene to the viral DNA was to obtain a marker for discrimination of viruses inoculated into two different peripheral organ. In order not to alter the spreading properties of the lacZ gene-containing virus compared with that of PrV-Ba, the reporter gene was inserted to a genomic location of the parental virus ŽPrV-Ba. which was demonstrated to be neutral with respect to the virulence of PrV ŽBoldogkoi ˝ et al., 1998.. The details on the construction of this virus will be described elsewhere. The viruses were propagated in monolayers of porcine kidney ŽPK-15. cells, and assayed by a standard plaque assay Žfor details, see Boldogkoi ˝ et al., 1996.. The virus suspension was stored at y708C, and before inoculation, it was quickly thawed to 378C in a water bath and kept on ice until used. Aliquots containing 10 8 pfu Žplaque-forming unit.rml, 5 = 10 8 pfurml or 10 9 pfurml were used in the experiments. Immediately, after each inoculation, the titer of the virus suspension was determined. ŽWe observed no decrease in the titer of the virus stock during the storage for several months.. In two rats receiving the highest dose Ž10 9 pfurml., a compact nucleus was observed in the ipsilateral ventrolateral medulla at the pontomedullary junction at a survival time of 72 h. One of the animals receiving the lowest dose of the virus injection was not infected at all, and only 2–3 neuronsrsection were observed in the brainstem in the other animal, at a survival time of 72 h. Thus, in the course of further experiments, 10 9 pfurml titer was used. However, in the double virus tracer experiment, 5 = 10 8 pfurml titer was used for both virus strains since the concentration of Ba-NeutLac was 5 = 10 8 pfurml. 2.3. Injection of Õirus The animals were anesthetized with ketamine Ž80 mgrkg. and xylazine Ž15 mgrkg.. The right extraorbital lacrimal gland was exposed and 10 ml Ž2 = 5 ml. of the virus suspension was injected with a Hamilton syringe Ž5 ml or 10 ml.. The needle was slowly advanced superficially into the gland and kept in situ for 10 min after completion of the injection. The surface of the gland was
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
then rinsed with physiological saline twice, and the incisure was closed. A long-acting penicillin ŽTardomyocell w . was given i.m. to prevent bacterial infection. The rats were sacrificed 20 h Ž n s 2., 45 h Ž n s 2., or 72–76 h Ž n s 4. after the viral injection. In a second experiment, the left superior cervical ganglion was removed under a dissecting microscope, and the next day, the virus was injected into the ipsilateral extraorbital lacrimal gland. These animals were anesthetized, and the brains and the spinal cords were processed for immunohistochemistry following a survival period of 72 h Ž n s 2.. In double-virus tracer experiment, 2 = 5 ml Ba-NeutLac was injected into the right lacrimal gland, and 2 = 5 ml PrV-Ba was injected into the right submandibular gland, and vice versa Ž n s 4 rats.. The titer of both virus stocks was 5 = 10 8 pfurml. The rats were anesthetized and perfused 3 days later, and the brain was then processed for immunohistochemistry. In order to investigate whether the two virus strains can reach the same target neurons, the following experiment was performed. A mixture Ž1:1. of the two strains Ž2 = 5 ml. was injected into the submandibular gland, then 3 days later, the rats Ž n s 2. were perfused and the brainstems were processed for immunohistochemical detection of the virus-infected neurons. The consecu-
47
tive coronal sections were stained for b-galactosidase, for the viral protein, or for double-immunostaining of both epitopes. No significant differences were observed in the number of the infected neurons stained with different manner, suggesting that the two different viruses can reach the same target neurons. In the control experiment, the skin above the lacrimal gland was incised and 20 ml of the PrV-Ba Ž10 9 pfurml. virus suspension was injected onto the surface of gland without opening its connective tissue capsule Ž n s 5 rats.. The incisure was closed 10 min later. Three animals Žtwo with single-virus and one with double-virus injection. were anesthetized 3 days later, and their brains were processed for immunohistochemistry. Virus-labelled neurons were not detected in the brain. The remaining two animals were observed for 12 days. They did not show any sign of illness during this period, except anxiety and mild aggressiveness by day 12, and thus they were sacrificed. The virus-infected animals were kept separated, and all other precautions necessary in work with infective agents were applied. The rats were inspected twice a day. We observed transient sneezing Ždays 2 and 3. when the injection with the highest titer of the virus was made into the lacrimal or submandibular glands. No other symptoms were observed during the 3-day survival period. The rats
Fig. 2. ŽA–D. Virus-labelled ganglion cells Žarrows. in the ipsilateral Gasserian ŽA, C. and superior cervical ŽB, D. ganglia, 45 ŽA, B. and 72 ŽC, D. h after viral injection into the lacrimal gland. Scale bars s 100 mm.
48
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
took food and water and exhibited normal activity and behavior. 2.4. Immunohistochemistry After various survival periods, the rats were anesthetized again and perfused transcardially with 0.9% NaCl Ž50 ml., followed by fixative solution Žabout 350 ml. containing 4% paraformaldehyde and 15% Žvrv. saturated picric acid in 0.1 M phosphate buffer ŽPB. at pH 7.4. Heparin Ž0.5 ml. was given intracardially before the start of the perfusion. The virus-infected cells were visualized by immunoperoxidase staining of cryosections Ž40–50 mm thick. through the brainstem Žcoronal sections., spinal cord Žfrontal sections, thoracic segments 1–4. and both ipsiand contralateral superior cervical and Gasserian ganglia Žsagittal and horizontal sections, respectively.. The tissue samples were pretreated with 30% sucrose, and the superior cervical ganglia were embedded in agar–agar before sectioning. After washing three times in PB for 10 min, the sections were preincubated in 3% H 2 O 2 in methanol for 20 min, and then, after several washings in PB, treated with 10% normal goat serum in PB for 1 h. After rinsing with PB, the sections were incubated with the primary antiserum for 24–48 h at 48C. The polyclonal antiviral antibody Žraised in rabbit, code number Rb134, courtesy of R.R. Miselis, Philadelphia, see Card et al., 1990. was used in a dilution of 1:5000 in 0.1 M PB containing 0.1% bovine serum albumin and 0.05% sodium azide. For double immunostaining, anti-tyrosine hydroxylase ŽTH. antibody Žmonoclonal, INCSTAR. in a dilution of 1:1000, or anti-b-galactosidase Žmonoclonal, Boehringer Mannheim. in a dilution of 1:2500, with a protein content of 4 ngrml. was combined with antiviral antibody staining. The antibodies were visualized with avidinrbiotin peroxidase staining by the ABC technique ŽVectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA.. The immunoperoxidase reaction was developed with 0.02% H 2 O 2 , using 0.05% 3,3X-diaminobenzidine as a chromogen, or intensified with 0.02% 3,3X-diaminobenzidine in 0.5% ammonium nickel sulfate in 0.05 M Tris–HCl, pH 7.6. This intensification was applied to stain the first primary antibody in the double labelling procedure. The sections were washed several times in Tris–HCl buffer, mounted on gelatin-coated slides, air-dried, rinsed in xylene and coverslipped with depex. Occasionally, the sections were coun-
Fig. 3. ŽA–B. Virus-labelled preganglionic neurons in the thoracic IML ipsilateral to the injection site in the lacrimal gland. Post-inoculation time: 72 h. The same cells are indicated by arrows in ŽA. and ŽB. at low and high magnification. Scale barss100 mm ŽA. and 600 mm ŽB..
terstained with Cresyl violet or Kernechtrot before covering the slides.
3. Results 3.1. Kinetics of labelling No virus-infected cells were seen either in the brain or in the ganglia 20 h after inoculation. A few labelled neurons Ž1–3rsection. appeared in the ipsilateral Gasserian ŽFig. 2A. and superior cervical ŽFig. 2B. ganglia 45 h after the injection. Their number was slightly increased by 72 h ŽFig. 2C,D.. No labelled neurons were observed in any contralateral ganglia. In the brain, infected neurons became visible first in the pontomedullary junction of the brainstem at 45 h ŽFig. 4A.. Progressive labelling was observed in the preganglionic parasympathetic and sympathetic neurons in the survival period between 45 and 72 h
Fig. 4. ŽA–C. Virus-labelled cells in the SSN Žarrows.. ŽA. Forty-five h after infection: a few labelled neurons in the SSN, only ipsilateral to the injection. ŽB. Seventy-two h after injection: extended neuronal labelling, but only in the ipsilateral SSN. ŽC. Virus-labelled cells in the ipsilateral SSN 72 h after inoculation in a rat which had undergone prior ipsilateral superior cervical ganglionectomy. Ganglionectomy did not influence the viral infection of the SSN neurons Žarrow.. Inserts show parts of the SSN at higher magnification. The arrows denote the same neurons in the figures. VII s facial nerve; PY s pyramidal tract; nerve; nVII s facial nucleus; SV s nucleus of the spinal trigeminal tract; T s spinal tract of the trigeminal nerve. Scale bars s 220 mm.
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
ŽFig. 4B.. By 72 h after inoculation, few labelled cells Ž0–3rsection. were seen in the oral part of the trigeminal
49
sensory nucleus. These second-order neurons appeared only ipsilateral to the site of the injection.
50
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
No label was noted in the intermediolateral cell column ŽIML. at a survival time of 45 h, although some postganglionic cells had been infected in the ipsilateral superior cervical ganglion ŽFig. 2B.. Virus-labelled cells appeared first in the IML at a survival time of 72 h, when 3–5 infected cellsrsection were seen in the first and second thoracic segments ŽFig. 3A,B.. Infected neurons were not detected on the contralateral side, or in other thoracic segments.
By 72 h after inoculation, a few surrounding glial cells were also labelled around the preganglionic parasympathetic neurons in the brainstem. 3.2. Topography of lacrimal preganglionic neurons in the SSN A few labelled neurons Ž2–5rsection. were observed in the brainstem, ipsilateral to the site of the injection, at a
Fig. 5. ŽA–B. A double-virus tracing. Ba-NeutLac was injected into the lacrimal, and PrV-Ba into the submandibular glands. b-Galactosidase ŽA. and polyclonal antiviral antiserum Žcode no. Rb134. immunostaining of coronal consecutive sections. Cresyl violet counterstaining. The Ba-NeutLac-infected neurons occupy the ventromedial part of the superior salivatory nucleus Žarrows., while the PrV-Ba-labelled cells Žarrowheads. are located dorsomedially. VII s facial nerve; g s genu of the facial nerve; nVII s facial nucleus; PY s pyramidal tract. Scale bars s 220 mm.
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
survival time of 45 h ŽFig. 4A.. These neurons were located in the ventrolateral part of the caudal pons and rostral medulla oblongata, medial to the root fibers of the facial nerve and lateral to the superior olive, or more caudally, to the motor facial nucleus. The number of labelled neurons increased to a maximum of about 35– 40rsection at 72 h ŽFig. 4B., and extended both in dorsoventral and in rostrocaudal directions. The maximal extension of the labelled neurons was about 1.1–1.2 mm rostrocaudally Ž10.1–11.2 mm posterior to the level of the bregma.. The density of labelled cells was slightly variable, depending on the degree of the infection of the animal, but their location was identical in all rats. The area Ža subdivision of the SSN. of virus-labelled lacrimal preganglionic neurons is immediately adjacent to the A5 catecholaminergic cell group. By double immunostaining, involving virus vs. TH, these two groups of cells could be easily distinguished. The smaller, fusiform cells of the virus-labelled preganglionic neurons were always TH-negative, and were situated immediately dorsal to the TH-positive larger, polygonal catecholaminergic cells. No strict borderline could be drawn between the two cell groups since virus-labelled cells were intermingled with TH-positive cells. It is noteworthy that TH-immunopositive fibers occasionally formed a nest around the viruslabelled cells. 3.3. Superior cerÕical ganglionectomy No virus-labelled cells were found in the upper thoracic spinal cord in rats which underwent ipsilateral cervical ganglionectomy prior to virus inoculation into the lacrimal gland. This surgery did not alter the topography and density of the virus-labelled cells in the SSN ŽFig. 4C.. 3.4. Topographical relationship between the lacrimal and submandibular preganglionic neurons in the SSN In order to visualize lacrimal and submandibular preganglionic neurons separately in the SSN, Ba-NeutLac was injected into the lacrimal and PrV-Ba into the submandibular glands of the same animal. The two populations of infected neurons constituted an uninterrupted cell group, the SSN. Ba-NeutLac-infected neurons appeared in the ventrolateral one-third of the SSN, as shown in the previous Žsingle-virus application. experiment in this study. The densest cell population was located 10.60–10.85 mm posterior to the level of the bregma ŽFig. 5A.. PrV-Ba-labelled neurons occupied a more dorsal and somewhat medial position ŽFig. 5B.. The dorsal two-thirds of the SSN contained exclusively PrV-Ba-infected neurons. Thus, cells in the ventral third of the nucleus project to the lacrimal, while others in the dorsal two-thirds project to the submandibular glands. Neurons projecting to the submandibular gland extended more caudally than the lacrimal neurons Žabout 11.75 mm caudal to the level of the bregma.
51
and completely occupied the most caudal portion of the SSN. Although the two subdivisions of the SSN can be clearly separated, their neurons were intermingled at the border of the subdivisions; 3–5 neuronsrsection occurred in the other region. A similar subdivisional distribution was found when the two viruses were injected in the opposite manner.
4. Discussion 4.1. Methodological considerations The retrograde transneuronal labelling technique has already been proved to be a powerful tool for investigations of neuronal connections in the central and peripheral nervous systems ŽDolivo, 1980; Card et al., 1990; Spencer et al., 1990; Jansen et al., 1992; Card, 1995; Enquist, 1995; Sams et al., 1995; Ugolini, 1995; Jasmin et al., 1997; Toth ´ and Palkovits, 1997.. There are two approaches as concerns the titer and dose of the injected herpes viruses to achieve specific and significant transneuronal transfer of the virus from a peripheral organ to the brain. Lower titers Že.g. 2 = 10 5 pfurml. and small doses of injection units Že.g. 10 3 pfu. result in a specific but low rate of injection ŽSams et al., 1995.. When such a low dose and titer are used, the first-order neurons may not be labelled and, certainly, there will be an underrepresentation of labelling in second- and third-order neurons ŽUgolini, 1995.. A high titer Že.g. 5 = 10 8 pfurml. and a higher dose Že.g. 10 6 pfu. per injection are required to achieve extensive labelling and full infectivity with pseudorabies virus ŽCard, 1995; Ugolini, 1995; Toth ´ and Palkovits, 1997.. In the present study, a high dose and a high titer were applied since preliminary experiments showed that significant and effective labelling could be obtained only with a high dose and a high titer of PrV-Ba. The disadvantage of using a high-dose viral concentration is the possible lysis of the infected neurons. In that case, the viruses could be dispersed in the neuropil and could result in nonspecific nonviral labelling of the second- and third-order neurons ŽSams et al., 1995.. In the present study, neuronal chains were followed along to the first-order neurons, i.e. to the synaptical localization of preganglionic neurons which might not be influenced by high-dose-induced cell lysis. 4.2. Retrograde and anterograde labelling Following inoculation into the lacrimal gland, the viruses enter nerve endings and are transported retrogradely to the ipsilateral parasympathetic Žpterygopalatine., sympathetic Žsuperior cervical. and sensory ŽGasserian. ganglia. After replication in these ganglia, the virus travels to the next hierarchical level at the neuronal chain, e.g. it enters the spinal cord and the lower brainstem. It has been observed that the virus may not be equally transported in all neu-
52
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
ronal systems. The interval that is required for the replication, release and transneuronal transfer of viruses may be highly variable, depending on the type of neurons. In the present study, the virus may use three different avenues. 4.2.1. Retrograde tracing of the Õirus in parasympathetic ganglionic and preganglionic neurons Virus injected into the lacrimal gland enters the terminal of the lacrimal nerve which arises in the pterygopalatine ganglion after transfer by the zygomatic nerve. The virus is replicated in the ganglion cells and transsynaptically infects the facial preganglionic neurons having axons in the greater superficial petrosal nerve. Retrograde cell body labelling was then detected in the ipsilateral ventrolateral medulla oblongata in a region that has been termed the SSN Žsee below..
for visualization in sensory neurons. In the present study, the virus was taken up by the trigeminal nerve terminals around the capsule of the lacrimal gland or in the surrounding tissue, infected the cells in the Gasserian ganglion, and subsequently transported to the sensory trigeminal nucleus. 4.3. Lacrimal preganglionic parasympathetic neurons in the SSN The preganglionic parasympathetic neurons which control the activity of the lacrimal gland are located in the ventrolateral brainstem, at a level corresponding to the caudal part of the superior olive down to the level of the rostral third of the motor facial nucleus in the medulla oblongata ŽFig. 6.. The majority of these neurons are
4.2.2. The second route used by the Õirus to enter the brain is the sympathetic chain Neurons in the superior cervical ganglion are infected retrogradely through axons innervating the lacrimal gland. The related preganglionic sympathetic neurons are located in the upper thoracic spinal cord in the IML at the first and second thoracic segments. These preganglionic neurons receive fairly rich neuronal inputs from different brain areas, including cells in the ventrolateral part of the medulla oblongata ŽSuzuki et al., 1990; Sun, 1995.. Theoretically, these brainstem neurons, including those in the SSN, might be infected retrogradely by spinal cord neurons. Cervical ganglionectomy performed before inoculation prevents the labelling through this avenue: in these animals, there was an absence of label in the IML, whereas the labelling in the SSN was like that in the control rats. This finding, together with the observation that infected cells appear in the SSN prior to the IML, clearly indicate that PrV-Ba travels from the lacrimal gland to the SSN through the parasympathetic pathway. 4.2.3. Anterograde transneuronal transfer through sensory trigeminal neurons Despite some suggestive histological and immunohistochemical evidences ŽNikkinen et al., 1984., the existence of the sensory innervation of the lacrimal gland in the rat has not yet been completely established ŽJansen et al., 1992.. Although it has been suggested that pseudorabies virus travels only by retrograde axonal transport in the central nervous system ŽJasmin et al., 1997., viral labelling in the Gasserian ganglionic cells and sensory trigeminal neurons indicates that, under certain conditions, when a high-dose viral concentration is used, the route of the infection could be anterograde. Anterograde transport as a possible pathway of viral infection has been demonstrated previously ŽDolivo, 1980; Enquist, 1995.. The spread of PrV via anterograde transport is slower than retrograde transport ŽEnquist, 1995., and a short post-injection time may not be enough for replication in quantities sufficient
Fig. 6. ŽA–D. Representative diagram of coronal sections of the caudal pons ŽA., the pontomedullary junction ŽB., and the rostral medulla ŽC, D, E. ŽSwanson, 1992.. The locations of the virus-infected neurons in the subdivisions of the SSN are indicated by triangles Žlacrimal. or circles Žsubmandibular.. VII s facial nerve; nVII s facial nucleus; PY s pyramidal tract; SOssuperior olive; SVs the nucleus of the spinal trigeminal tract.
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
found medial to the root fibers of the facial nerve. Medially, this subdivision is bordered by the superior olive Žat rostral levels. and the motor facial nucleus Žmore caudally.. The location of these cells is comparable to that of the labelled neurons of the brainstem determined by the injection of PrV-Ba ŽSpencer et al., 1990. or retrograde tracers into the pterygopalatine ganglion ŽSenba et al., 1987; Suzuki et al., 1990; Nemoto et al., 1995. the maxillary nerve ŽInsausiti and Gonzalo, 1980. or the greater superficial petrosal nerve ŽInsausiti and Gonzalo, 1980; Hosoya et al., 1990.. Our double-virus tracer study showed that these cells compose the ventral subdivision of the SSN, while the dorsal part of it is formed by neurons projecting to the submandibular gland. The location of the submandibular subgroup is comparable to the descriptions obtained with tracers injected into the submandibular gland ŽJansen et al., 1992. or the chorda tympani ŽHiura, 1977; Contreras et al., 1980; Mitchell and Templeton, 1981; Nicholson and Severin, 1981., representing the preganglionic parasympathetic neurons projecting to the submandibular ganglion. Insausiti and Gonzalo Ž1980. have pointed out the close topographical connection between the SSN and the A5 catecholaminergic cell group. Although preganglionic cells are partly intermingled with the noradrenergic A5 cells, and some TH-positive neurons are scattered in the SSN ŽHosoya et al., 1990., the two cell populations are clearly separated. None of the preganglionic cells immunostained for TH, and TH-positive A5 cells were not infected by the virus until the late post-inoculation periods. Previous ŽSuzuki et al., 1990; Nemoto et al., 1995. and present double immunostaining revealed that the virus-labelled neurons in the SSN were surrounded by a dense plexus of TH-positive fibers, providing a morphological basis for the catecholaminergic influence of the parasympathetic outflow in the central nervous system. The lacrimal preganglionic neurons receive neuronal inputs from various brain areas, as demonstrated by viral infection of several third-ordered neurons along the entire neuraxis, including the hypothalamus, the limbic system and autonomic brain areas ŽSpencer et al., 1990.. This is in accordance with previous observations from experiments using combined tract-tracing techniques ŽHosoya et al., 1983, 1984, 1990. or immunohistochemistry ŽNemoto et al., 1995. that SSN neurons projecting to the pterygopalatine ganglion receive an abundant synaptic input from the hypothalamus.
5. Conclusion Lacrimal preganglionic parasympathetic neurons occupy the ventral subdivision of the salivatory nucleus. Axons enter the intermediate component of the facial nerve, leave it to enter the greater superficial petrosal nerve, and terminate in the pterygopalatine ganglion. Sympathetic fibers to the lacrimal gland arise in neurons in the
53
IML of the first and second thoracic segments. Additionally, the virus is taken up by sensory trigeminal fibers that innervate the capsule of lacrimal gland and its surrounding tissues. Acknowledgements We are pleased to acknowledge the contributions of Dr G.N. Moskovkin Žremoval of s.c.ggl., Ms Reka ´ Altrichter, Ms Dora Mrs Judit Helfferich, Mrs Zsuzsa Vidra ´ Kezdi, ´ and Mr Jozsef Kiss. We are also pleased to acknowledge ´ the source of the polyclonal antiviral antibody, which was provided through the courtesy of Dr R.R. Miselis. The work was supported by Hungarian Research Grants OTKA No. T-025962, MKM No. 447, and FKFP 0648. References Aujeszky, A., 1902. A contagious disease, not readily distinguishable from rabies, with unknown origin Žin Hungarian.. Veterinarius 25, 387–396. Bartha, A., 1961. Experiments to reduce the virulence of Aujeszky’s virus ´ Žin Hungarian.. Magyar Allatorvosok Lapja 16, 42–45. Boldogkoi, R., Braun, A., Fodor, I., 1996. In ˝ Zs., Medveczky, I., Glavits, ´ vivo studies on Aujeszky’s disease virus mutants. Acta Microbiol. Immunol. Hungarica 43, 307–318. Boldogkoi, ˝ Zs., Braun, A., Medveczky, I., Glavits, B., Gyuro, ´ B., Fodor, I., 1998. Analysis of the equalization of inverted repeats and neurovirulence using a pseudorabies virus mutant strain altered at the UlrIr junction. Virus Genes 17, 89–98. Card, J.P., 1995. Pseudorabies virus replication and assembly in the rodent system. In: Kaplitt, M.G., Loewy, A.D. ŽEds.., Viral Vectors. Gene Therapy and Neuroscience Applications. Academic Press, New York, pp. 319–347. Card, J.P., Rinaman, L., Schwaber, J.S., Miselis, R.R., Whealey, M.E., Robbins, A.K., Enquist, L.W., 1990. Neurotropic properties of pseudorabies virus: uptake and transneuronal passage in the rat central nervous system. J. Neurosci. 10, 1974–1994. Contreras, R.J., Gomez, M.M., Norgren, R., 1980. Central origins of cranial nerve parasympathetic neurons in the rat. J. Comp. Neurol. 190, 373–394. Dolivo, M., 1980. A neurobiological approach to neurotropic viruses. Trends Neurosci. 3, 149–152. Enquist, L.W., 1995. Circuit-specific infection of the mammalian nervous system. Pseudorabies virus, a neurotropic alpha herpesvirus, provides a valuable means for probing cellular interactions in the brain. AMS News 61, 633–638. Hiura, T., 1977. Salivatory neurons innervate the submandibular and sublingual glands in the rat: horseradish peroxidase study. Brain Res. 137, 145–149. Hosoya, Y., Matsushita, M., Sugiura, Y., 1983. A direct hypothalamic projection to the superior salivatory nucleus neurons in the rat. A study using anterograde autoradiographic and retrograde HRP methods. Brain Res. 266, 329–333. Hosoya, Y., Matsushita, M., Sugiura, Y., 1984. Hypothalamic descending afferents to cells of origin of the greater petrosal nerve in the rat, as revealed by a combination of retrograde HRP and anterograde autoradiographic techniques. Brain Res. 290, 141–145. Hosoya, Y., Sugiura, Y., Ito, R.R., Kohno, K., 1990. Descending projections from the hypothalamic paraventricular nucleus to the A5 area, including the superior salivatory nucleus, in the rat. Exp. Brain Res. 82, 513–518.
54
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
Insausiti, R., Gonzalo, L.M., 1980. New data on the precise location of the lacrimo-muconasal nucleus of the rat. Experientia 36, 977–978. Jansen, A.S.P., Ter Horst, G.J., Mettenleiter, T.C., Loewy, A.D., 1992. CNS cell groups projecting to the submandibular parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body-labeled study. Brain Res. 572, 253–260. Jasmin, L., Burkey, A.R., Card, J.P., Basbaum, A.I., 1997. Transneuronal labeling of a nociceptive pathway, the spinoŽtrigemino.parabrachioamygdaloid, in the rat. J. Neurosci. 17, 3751–3765. Mitchell, J., Templeton, D., 1981. The origin of the preganglionic parasympathetic fibers to the mandibular and sublingual salivary glands in the rat: a horseradish peroxidase study. J. Anat. 132, 513–518. Nemoto, T., Konno, A., Chiba, T., 1995. Synaptic contact of neuropeptide- and amine-containing axons on parasympathetic preganglionic neurons in the superior salivatory nucleus of the rat. Brain Res. 685, 33–45. Nicholson, J.E., Severin, C.M., 1981. The superior and inferior salivatory nuclei in the rat. Neuroscience Letters 21, 133–138. Nikkinen, A., Lehtosalo, J.I., Uusitalo, H., Palkama, A., Panula, P., 1984. The lacrimal lands of the rat and the guinea pig are innervated by nerve fibers containing immunoreactivities for substance P and vasoactive intestinal polypeptide. Histochemistry 81, 23–27. Sams, J.M., Jansen, A.S.P., Mettenleiter, T.C., Loewy, A.D., 1995.
Pseudorabies virus mutants as transneuronal markers. Brain Res. 687, 182–190. Senba, E., Daddona, P.E., Nagy, J.I., 1987. A subpopulation of preganglionic parasympathetic neurons in the rat containing adenosine deaminase. Neuroscience 20, 487–502. Spencer, S.E., Sawyer, W.B., Wada, H., Platt, K.B., Loewy, A.D., 1990. CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study. Brain Res. 534, 149–169. Sun, M.K., 1995. Central neural organization and control of sympathetic nervous system in mammals. Prog. Neurobiol. 47, 157–233. Suzuki, N., Hardebo, J.E., Skagerberg, G., Owman, C., 1990. Central origins of preganglionic fibers to the sphenopalatine ganglion in the rat. A fluorescent retrograde tracer study with special reference to its relation to central catecholaminergic system. J. Auton. Nerv. Syst. 30, 101–110. Swanson, L.W., 1992. Brain Maps. Structure of the Rat Brain. Elsevier, Amsterdam. Toth, ´ I.E., Palkovits, M., 1997. Viral labelling of synaptically connected neurons. Neurobiology 5, 17–41. Ugolini, G., 1995. Transneuronal tracing with alpha-herpesviruses: a review of methodology. In: Kaplitt, M.G., Loewy, A.D. ŽEds.., Viral Vectors. Gene Therapy and Neuroscience Applications, Academic Press, New York, pp. 293–317.
Lacrimal preganglionic neurons form a subdivision of the superior salivatory nucleus of rat: transneuronal labelling by pseudorabies virus Ida E. Toth ´ a
a,)
, Zsolt Boldogkoi ˝ a, Istvan ´ Medveczky b, Miklos ´ Palkovits
a
Joint Research Laboratory of Neuromorphology, Semmelweis UniÕersity of Medicine and Hungarian Academy of Sciences, Tuzolto ˝ ´ utca 58, Budapest H-1450, Hungary b Department of Microbiology and Infectious Diseases, UniÕersity of Veterinary Science, Hungaria krt. 23-25, Budapest H-1143, Hungary Received 10 August 1998; received in revised form 23 March 1999; accepted 5 May 1999
Abstract Transneuronal viral tracing was applied to localize preganglionic parasympathetic neurons in the brainstem which innervate the extraorbital lacrimal gland in the rat. The Bartha strain of pseudorabies virus was injected into the lacrimal gland, and after different survival times, the superior cervical and Gasserian ganglia, the upper thoracic spinal cords and the brainstems were immunostained by antiviral antiserum. Virus-labelled neurons appeared in the ganglia and in the ventrolateral part of the ipsilateral brainstem at the pontomedullary junction 45 h after inoculation. The virus-labelled brainstem neurons comprised a subgroup of the superior salivatory nucleus ŽSSN. located between the root fibers of the facial nerve and the nuclei of the superior olive, and were clearly distinguished from the tyrosine hydroxylase ŽTH.-immunopositive, A5 catecholaminergic neurons by double immunostaining. The number of infected cells in the ipsilateral SSN was increased by 72 h, and labelled neurons appeared in the intermediolateral cell column ŽIML. of the ipsilateral thoracic spinal cord. In rats with cervical ganglionectomy prior to the virus injection in the lacrimal gland, virus-infected cells appeared in the SSN, but not in the thoracic spinal cord, indicating that preganglionic SSN cells were infected via parasympathetic axons of the facial nerve. A double-virus tracer labelling technique was applied to determine the topographical relationship between the preganglionic parasympathetic neurons of the lacrimal gland and those of the submandibular gland within the SSN. Simultaneous injection of Bartha strain of pseudorabies virus into the submandibular gland, and a lacZ gene-containing Bartha-derived virus strain into the lacrimal gland Žand vice versa. demarcated a ventral lacrimal and a dorsal submandibular subgroup in the SSN. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Transneuronal viral tracing; Lacrimal gland; Immunohistochemistry
1. Introduction The superior salivatory nucleus ŽSSN. is a group of parasympathetic preganglionic neurons innervating the lacrimal, submandibular and sublingual glands, in addition to the nasal and palatine mucosa, through the pterygopalatine Žalso called sphenopalatine. and submandibular ganglia. In the rat, in contrast with humans, primates and many other mammalian species, early studies involving anatomical Žretrograde degeneration. and electrophysiolog-
AbbreÕiations: Ba-NeutLac, b-galactosidase expressing Bartha strain of pseudorabies virus; IML, intermediolateral cell column; PB, phosphate buffer; pfu, plaque-forming unit; PrV-Ba, Bartha strain of pseudorabies virus; SSN, superior salivatory nucleus; TH, tyrosine hydroxylase ) Corresponding author. Tel.: q36-1-215-6920r3660; fax: q36-1218-1612; e-mail: [email protected]
ical approaches led to controversial results concerning the exact topographical localization of the SSN in the lower brainstem. Recent studies with retrograde tracers resulted in descriptions and names for different subdivisions of the SSN depending on the approach applied. Axons of preganglionic neurons innervating the lacrimal gland and the nasal and palatine mucosa contribute to the greater superficial petrosal nerve, and terminate in the pterygopalatine ganglion. Other preganglionic neurons in the SSN which innervate the submandibular and sublingual glands contribute axons to the submandibular ganglion through the chorda tympani. Retrograde viral transneuronal tracer injected into the submandibular gland ŽJansen et al., 1992., or horseradish peroxidase injected into the chorda tympani ŽHiura, 1977; Contreras et al., 1980; Mitchell and Templeton, 1981; Nicholson and Severin, 1981. revealed neurons in the dorsomedial part of the SSN. Injections of retro-
0165-1838r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 9 . 0 0 0 3 2 - 6
46
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
kept under standard laboratory conditions Žroom temperature, 22 " 18C, with a 12-h light–dark cycle, with lights on at 0600 h.. Regular rat chow and tap water were provided ad libitum. The experimental procedures were approved by the local Animal Care and Use Committee. 2.2. Viruses
Fig. 1. Infection of the extraorbital lacrimal gland. Ž1. Parasympathetic fibers from the SSN ŽS., with synaptic transfer in the pterygopalatine ŽSP. ganglion. Ž2. Sympathetic fibers from the IML in the thoracic spinal cord, with synaptic transfer in the superior cervical ganglion ŽCS.. Ž3. Sensory fibers through the trigeminal nerve ŽV. and ganglion ŽG. to the nucleus of the spinal trigeminal tract ŽSV..
grade tracers ŽInsausiti and Gonzalo, 1980; Senba et al., 1987; Suzuki et al., 1990; Nemoto et al., 1995. or virus ŽSpencer et al., 1990. into the pterygopalatine ganglion or horseradish peroxidase into the greater petrosal nerve ŽHosoya et al., 1984, 1990. resulted in neuronal labelling in the ventrolateral part of the SSN. Reflecting the function of these cells, this subdivision of the SSN is also referred to as the lacrimo-nasopalatine ŽSenba et al., 1987., lacrimo-muconasal ŽInsausiti and Gonzalo, 1980. or lacrimal ŽContreras et al., 1980. nucleus. In the present study, we set out to localize the preganglionic neurons innervating the lacrimal gland by using a viral tracer technique. This technique is capable of revealing multisynaptic connections via specific transynaptic propagation of the virus Žfor details, see Card, 1995; Ugolini, 1995; Toth ´ and Palkovits, 1997.. The neurotropic viruses infect all types of neurons, e.g. parasympathetic, sympathetic and sensory ones, and thus, we studied these three possible pathways through which viruses may enter the brain ŽFig. 1.. Through the application of the Bartha strain of pseudorabies virus ŽPrV-Ba, Aujeszky, 1902; Bartha, 1961. and a genetically engineered b-galactosidase expressing Bartha-derived strain as transneuronal tracer, a subdivision of the SSN projecting exclusively to the extraorbital lacrimal gland of rat was distinguished. We determined the topographical relationship between the lacrimal and submandibular subdivisions of SSN by application of a double virus tracer system, e.g. by injection of immunohistochemically separable virus tracers into these two glands.
2. Experimental procedures 2.1. Animals Twenty-four adult rats of the Sprague–Dawley and Wistar strains were utilized in this study. The animals were
Two pseudorabies viruses: Ži. PrV-Ba ŽAujeszky, 1902; Bartha, 1961. and Žii. a genetically engineered, Bartha-derived strain, expressing Escherichia coli b-galactosidase ŽBa-NeutLac., were used in this study. Ba-NeutLac was constructed by the insertion of the lacZ gene to a ‘neutral’ segment Žplaying no role in the viral virulence. of the Bartha genome ŽBoldogkoi ˝ et al., 1998.. The aim of the insertion of the lacZ gene to the viral DNA was to obtain a marker for discrimination of viruses inoculated into two different peripheral organ. In order not to alter the spreading properties of the lacZ gene-containing virus compared with that of PrV-Ba, the reporter gene was inserted to a genomic location of the parental virus ŽPrV-Ba. which was demonstrated to be neutral with respect to the virulence of PrV ŽBoldogkoi ˝ et al., 1998.. The details on the construction of this virus will be described elsewhere. The viruses were propagated in monolayers of porcine kidney ŽPK-15. cells, and assayed by a standard plaque assay Žfor details, see Boldogkoi ˝ et al., 1996.. The virus suspension was stored at y708C, and before inoculation, it was quickly thawed to 378C in a water bath and kept on ice until used. Aliquots containing 10 8 pfu Žplaque-forming unit.rml, 5 = 10 8 pfurml or 10 9 pfurml were used in the experiments. Immediately, after each inoculation, the titer of the virus suspension was determined. ŽWe observed no decrease in the titer of the virus stock during the storage for several months.. In two rats receiving the highest dose Ž10 9 pfurml., a compact nucleus was observed in the ipsilateral ventrolateral medulla at the pontomedullary junction at a survival time of 72 h. One of the animals receiving the lowest dose of the virus injection was not infected at all, and only 2–3 neuronsrsection were observed in the brainstem in the other animal, at a survival time of 72 h. Thus, in the course of further experiments, 10 9 pfurml titer was used. However, in the double virus tracer experiment, 5 = 10 8 pfurml titer was used for both virus strains since the concentration of Ba-NeutLac was 5 = 10 8 pfurml. 2.3. Injection of Õirus The animals were anesthetized with ketamine Ž80 mgrkg. and xylazine Ž15 mgrkg.. The right extraorbital lacrimal gland was exposed and 10 ml Ž2 = 5 ml. of the virus suspension was injected with a Hamilton syringe Ž5 ml or 10 ml.. The needle was slowly advanced superficially into the gland and kept in situ for 10 min after completion of the injection. The surface of the gland was
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
then rinsed with physiological saline twice, and the incisure was closed. A long-acting penicillin ŽTardomyocell w . was given i.m. to prevent bacterial infection. The rats were sacrificed 20 h Ž n s 2., 45 h Ž n s 2., or 72–76 h Ž n s 4. after the viral injection. In a second experiment, the left superior cervical ganglion was removed under a dissecting microscope, and the next day, the virus was injected into the ipsilateral extraorbital lacrimal gland. These animals were anesthetized, and the brains and the spinal cords were processed for immunohistochemistry following a survival period of 72 h Ž n s 2.. In double-virus tracer experiment, 2 = 5 ml Ba-NeutLac was injected into the right lacrimal gland, and 2 = 5 ml PrV-Ba was injected into the right submandibular gland, and vice versa Ž n s 4 rats.. The titer of both virus stocks was 5 = 10 8 pfurml. The rats were anesthetized and perfused 3 days later, and the brain was then processed for immunohistochemistry. In order to investigate whether the two virus strains can reach the same target neurons, the following experiment was performed. A mixture Ž1:1. of the two strains Ž2 = 5 ml. was injected into the submandibular gland, then 3 days later, the rats Ž n s 2. were perfused and the brainstems were processed for immunohistochemical detection of the virus-infected neurons. The consecu-
47
tive coronal sections were stained for b-galactosidase, for the viral protein, or for double-immunostaining of both epitopes. No significant differences were observed in the number of the infected neurons stained with different manner, suggesting that the two different viruses can reach the same target neurons. In the control experiment, the skin above the lacrimal gland was incised and 20 ml of the PrV-Ba Ž10 9 pfurml. virus suspension was injected onto the surface of gland without opening its connective tissue capsule Ž n s 5 rats.. The incisure was closed 10 min later. Three animals Žtwo with single-virus and one with double-virus injection. were anesthetized 3 days later, and their brains were processed for immunohistochemistry. Virus-labelled neurons were not detected in the brain. The remaining two animals were observed for 12 days. They did not show any sign of illness during this period, except anxiety and mild aggressiveness by day 12, and thus they were sacrificed. The virus-infected animals were kept separated, and all other precautions necessary in work with infective agents were applied. The rats were inspected twice a day. We observed transient sneezing Ždays 2 and 3. when the injection with the highest titer of the virus was made into the lacrimal or submandibular glands. No other symptoms were observed during the 3-day survival period. The rats
Fig. 2. ŽA–D. Virus-labelled ganglion cells Žarrows. in the ipsilateral Gasserian ŽA, C. and superior cervical ŽB, D. ganglia, 45 ŽA, B. and 72 ŽC, D. h after viral injection into the lacrimal gland. Scale bars s 100 mm.
48
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
took food and water and exhibited normal activity and behavior. 2.4. Immunohistochemistry After various survival periods, the rats were anesthetized again and perfused transcardially with 0.9% NaCl Ž50 ml., followed by fixative solution Žabout 350 ml. containing 4% paraformaldehyde and 15% Žvrv. saturated picric acid in 0.1 M phosphate buffer ŽPB. at pH 7.4. Heparin Ž0.5 ml. was given intracardially before the start of the perfusion. The virus-infected cells were visualized by immunoperoxidase staining of cryosections Ž40–50 mm thick. through the brainstem Žcoronal sections., spinal cord Žfrontal sections, thoracic segments 1–4. and both ipsiand contralateral superior cervical and Gasserian ganglia Žsagittal and horizontal sections, respectively.. The tissue samples were pretreated with 30% sucrose, and the superior cervical ganglia were embedded in agar–agar before sectioning. After washing three times in PB for 10 min, the sections were preincubated in 3% H 2 O 2 in methanol for 20 min, and then, after several washings in PB, treated with 10% normal goat serum in PB for 1 h. After rinsing with PB, the sections were incubated with the primary antiserum for 24–48 h at 48C. The polyclonal antiviral antibody Žraised in rabbit, code number Rb134, courtesy of R.R. Miselis, Philadelphia, see Card et al., 1990. was used in a dilution of 1:5000 in 0.1 M PB containing 0.1% bovine serum albumin and 0.05% sodium azide. For double immunostaining, anti-tyrosine hydroxylase ŽTH. antibody Žmonoclonal, INCSTAR. in a dilution of 1:1000, or anti-b-galactosidase Žmonoclonal, Boehringer Mannheim. in a dilution of 1:2500, with a protein content of 4 ngrml. was combined with antiviral antibody staining. The antibodies were visualized with avidinrbiotin peroxidase staining by the ABC technique ŽVectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA.. The immunoperoxidase reaction was developed with 0.02% H 2 O 2 , using 0.05% 3,3X-diaminobenzidine as a chromogen, or intensified with 0.02% 3,3X-diaminobenzidine in 0.5% ammonium nickel sulfate in 0.05 M Tris–HCl, pH 7.6. This intensification was applied to stain the first primary antibody in the double labelling procedure. The sections were washed several times in Tris–HCl buffer, mounted on gelatin-coated slides, air-dried, rinsed in xylene and coverslipped with depex. Occasionally, the sections were coun-
Fig. 3. ŽA–B. Virus-labelled preganglionic neurons in the thoracic IML ipsilateral to the injection site in the lacrimal gland. Post-inoculation time: 72 h. The same cells are indicated by arrows in ŽA. and ŽB. at low and high magnification. Scale barss100 mm ŽA. and 600 mm ŽB..
terstained with Cresyl violet or Kernechtrot before covering the slides.
3. Results 3.1. Kinetics of labelling No virus-infected cells were seen either in the brain or in the ganglia 20 h after inoculation. A few labelled neurons Ž1–3rsection. appeared in the ipsilateral Gasserian ŽFig. 2A. and superior cervical ŽFig. 2B. ganglia 45 h after the injection. Their number was slightly increased by 72 h ŽFig. 2C,D.. No labelled neurons were observed in any contralateral ganglia. In the brain, infected neurons became visible first in the pontomedullary junction of the brainstem at 45 h ŽFig. 4A.. Progressive labelling was observed in the preganglionic parasympathetic and sympathetic neurons in the survival period between 45 and 72 h
Fig. 4. ŽA–C. Virus-labelled cells in the SSN Žarrows.. ŽA. Forty-five h after infection: a few labelled neurons in the SSN, only ipsilateral to the injection. ŽB. Seventy-two h after injection: extended neuronal labelling, but only in the ipsilateral SSN. ŽC. Virus-labelled cells in the ipsilateral SSN 72 h after inoculation in a rat which had undergone prior ipsilateral superior cervical ganglionectomy. Ganglionectomy did not influence the viral infection of the SSN neurons Žarrow.. Inserts show parts of the SSN at higher magnification. The arrows denote the same neurons in the figures. VII s facial nerve; PY s pyramidal tract; nerve; nVII s facial nucleus; SV s nucleus of the spinal trigeminal tract; T s spinal tract of the trigeminal nerve. Scale bars s 220 mm.
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
ŽFig. 4B.. By 72 h after inoculation, few labelled cells Ž0–3rsection. were seen in the oral part of the trigeminal
49
sensory nucleus. These second-order neurons appeared only ipsilateral to the site of the injection.
50
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
No label was noted in the intermediolateral cell column ŽIML. at a survival time of 45 h, although some postganglionic cells had been infected in the ipsilateral superior cervical ganglion ŽFig. 2B.. Virus-labelled cells appeared first in the IML at a survival time of 72 h, when 3–5 infected cellsrsection were seen in the first and second thoracic segments ŽFig. 3A,B.. Infected neurons were not detected on the contralateral side, or in other thoracic segments.
By 72 h after inoculation, a few surrounding glial cells were also labelled around the preganglionic parasympathetic neurons in the brainstem. 3.2. Topography of lacrimal preganglionic neurons in the SSN A few labelled neurons Ž2–5rsection. were observed in the brainstem, ipsilateral to the site of the injection, at a
Fig. 5. ŽA–B. A double-virus tracing. Ba-NeutLac was injected into the lacrimal, and PrV-Ba into the submandibular glands. b-Galactosidase ŽA. and polyclonal antiviral antiserum Žcode no. Rb134. immunostaining of coronal consecutive sections. Cresyl violet counterstaining. The Ba-NeutLac-infected neurons occupy the ventromedial part of the superior salivatory nucleus Žarrows., while the PrV-Ba-labelled cells Žarrowheads. are located dorsomedially. VII s facial nerve; g s genu of the facial nerve; nVII s facial nucleus; PY s pyramidal tract. Scale bars s 220 mm.
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
survival time of 45 h ŽFig. 4A.. These neurons were located in the ventrolateral part of the caudal pons and rostral medulla oblongata, medial to the root fibers of the facial nerve and lateral to the superior olive, or more caudally, to the motor facial nucleus. The number of labelled neurons increased to a maximum of about 35– 40rsection at 72 h ŽFig. 4B., and extended both in dorsoventral and in rostrocaudal directions. The maximal extension of the labelled neurons was about 1.1–1.2 mm rostrocaudally Ž10.1–11.2 mm posterior to the level of the bregma.. The density of labelled cells was slightly variable, depending on the degree of the infection of the animal, but their location was identical in all rats. The area Ža subdivision of the SSN. of virus-labelled lacrimal preganglionic neurons is immediately adjacent to the A5 catecholaminergic cell group. By double immunostaining, involving virus vs. TH, these two groups of cells could be easily distinguished. The smaller, fusiform cells of the virus-labelled preganglionic neurons were always TH-negative, and were situated immediately dorsal to the TH-positive larger, polygonal catecholaminergic cells. No strict borderline could be drawn between the two cell groups since virus-labelled cells were intermingled with TH-positive cells. It is noteworthy that TH-immunopositive fibers occasionally formed a nest around the viruslabelled cells. 3.3. Superior cerÕical ganglionectomy No virus-labelled cells were found in the upper thoracic spinal cord in rats which underwent ipsilateral cervical ganglionectomy prior to virus inoculation into the lacrimal gland. This surgery did not alter the topography and density of the virus-labelled cells in the SSN ŽFig. 4C.. 3.4. Topographical relationship between the lacrimal and submandibular preganglionic neurons in the SSN In order to visualize lacrimal and submandibular preganglionic neurons separately in the SSN, Ba-NeutLac was injected into the lacrimal and PrV-Ba into the submandibular glands of the same animal. The two populations of infected neurons constituted an uninterrupted cell group, the SSN. Ba-NeutLac-infected neurons appeared in the ventrolateral one-third of the SSN, as shown in the previous Žsingle-virus application. experiment in this study. The densest cell population was located 10.60–10.85 mm posterior to the level of the bregma ŽFig. 5A.. PrV-Ba-labelled neurons occupied a more dorsal and somewhat medial position ŽFig. 5B.. The dorsal two-thirds of the SSN contained exclusively PrV-Ba-infected neurons. Thus, cells in the ventral third of the nucleus project to the lacrimal, while others in the dorsal two-thirds project to the submandibular glands. Neurons projecting to the submandibular gland extended more caudally than the lacrimal neurons Žabout 11.75 mm caudal to the level of the bregma.
51
and completely occupied the most caudal portion of the SSN. Although the two subdivisions of the SSN can be clearly separated, their neurons were intermingled at the border of the subdivisions; 3–5 neuronsrsection occurred in the other region. A similar subdivisional distribution was found when the two viruses were injected in the opposite manner.
4. Discussion 4.1. Methodological considerations The retrograde transneuronal labelling technique has already been proved to be a powerful tool for investigations of neuronal connections in the central and peripheral nervous systems ŽDolivo, 1980; Card et al., 1990; Spencer et al., 1990; Jansen et al., 1992; Card, 1995; Enquist, 1995; Sams et al., 1995; Ugolini, 1995; Jasmin et al., 1997; Toth ´ and Palkovits, 1997.. There are two approaches as concerns the titer and dose of the injected herpes viruses to achieve specific and significant transneuronal transfer of the virus from a peripheral organ to the brain. Lower titers Že.g. 2 = 10 5 pfurml. and small doses of injection units Že.g. 10 3 pfu. result in a specific but low rate of injection ŽSams et al., 1995.. When such a low dose and titer are used, the first-order neurons may not be labelled and, certainly, there will be an underrepresentation of labelling in second- and third-order neurons ŽUgolini, 1995.. A high titer Že.g. 5 = 10 8 pfurml. and a higher dose Že.g. 10 6 pfu. per injection are required to achieve extensive labelling and full infectivity with pseudorabies virus ŽCard, 1995; Ugolini, 1995; Toth ´ and Palkovits, 1997.. In the present study, a high dose and a high titer were applied since preliminary experiments showed that significant and effective labelling could be obtained only with a high dose and a high titer of PrV-Ba. The disadvantage of using a high-dose viral concentration is the possible lysis of the infected neurons. In that case, the viruses could be dispersed in the neuropil and could result in nonspecific nonviral labelling of the second- and third-order neurons ŽSams et al., 1995.. In the present study, neuronal chains were followed along to the first-order neurons, i.e. to the synaptical localization of preganglionic neurons which might not be influenced by high-dose-induced cell lysis. 4.2. Retrograde and anterograde labelling Following inoculation into the lacrimal gland, the viruses enter nerve endings and are transported retrogradely to the ipsilateral parasympathetic Žpterygopalatine., sympathetic Žsuperior cervical. and sensory ŽGasserian. ganglia. After replication in these ganglia, the virus travels to the next hierarchical level at the neuronal chain, e.g. it enters the spinal cord and the lower brainstem. It has been observed that the virus may not be equally transported in all neu-
52
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
ronal systems. The interval that is required for the replication, release and transneuronal transfer of viruses may be highly variable, depending on the type of neurons. In the present study, the virus may use three different avenues. 4.2.1. Retrograde tracing of the Õirus in parasympathetic ganglionic and preganglionic neurons Virus injected into the lacrimal gland enters the terminal of the lacrimal nerve which arises in the pterygopalatine ganglion after transfer by the zygomatic nerve. The virus is replicated in the ganglion cells and transsynaptically infects the facial preganglionic neurons having axons in the greater superficial petrosal nerve. Retrograde cell body labelling was then detected in the ipsilateral ventrolateral medulla oblongata in a region that has been termed the SSN Žsee below..
for visualization in sensory neurons. In the present study, the virus was taken up by the trigeminal nerve terminals around the capsule of the lacrimal gland or in the surrounding tissue, infected the cells in the Gasserian ganglion, and subsequently transported to the sensory trigeminal nucleus. 4.3. Lacrimal preganglionic parasympathetic neurons in the SSN The preganglionic parasympathetic neurons which control the activity of the lacrimal gland are located in the ventrolateral brainstem, at a level corresponding to the caudal part of the superior olive down to the level of the rostral third of the motor facial nucleus in the medulla oblongata ŽFig. 6.. The majority of these neurons are
4.2.2. The second route used by the Õirus to enter the brain is the sympathetic chain Neurons in the superior cervical ganglion are infected retrogradely through axons innervating the lacrimal gland. The related preganglionic sympathetic neurons are located in the upper thoracic spinal cord in the IML at the first and second thoracic segments. These preganglionic neurons receive fairly rich neuronal inputs from different brain areas, including cells in the ventrolateral part of the medulla oblongata ŽSuzuki et al., 1990; Sun, 1995.. Theoretically, these brainstem neurons, including those in the SSN, might be infected retrogradely by spinal cord neurons. Cervical ganglionectomy performed before inoculation prevents the labelling through this avenue: in these animals, there was an absence of label in the IML, whereas the labelling in the SSN was like that in the control rats. This finding, together with the observation that infected cells appear in the SSN prior to the IML, clearly indicate that PrV-Ba travels from the lacrimal gland to the SSN through the parasympathetic pathway. 4.2.3. Anterograde transneuronal transfer through sensory trigeminal neurons Despite some suggestive histological and immunohistochemical evidences ŽNikkinen et al., 1984., the existence of the sensory innervation of the lacrimal gland in the rat has not yet been completely established ŽJansen et al., 1992.. Although it has been suggested that pseudorabies virus travels only by retrograde axonal transport in the central nervous system ŽJasmin et al., 1997., viral labelling in the Gasserian ganglionic cells and sensory trigeminal neurons indicates that, under certain conditions, when a high-dose viral concentration is used, the route of the infection could be anterograde. Anterograde transport as a possible pathway of viral infection has been demonstrated previously ŽDolivo, 1980; Enquist, 1995.. The spread of PrV via anterograde transport is slower than retrograde transport ŽEnquist, 1995., and a short post-injection time may not be enough for replication in quantities sufficient
Fig. 6. ŽA–D. Representative diagram of coronal sections of the caudal pons ŽA., the pontomedullary junction ŽB., and the rostral medulla ŽC, D, E. ŽSwanson, 1992.. The locations of the virus-infected neurons in the subdivisions of the SSN are indicated by triangles Žlacrimal. or circles Žsubmandibular.. VII s facial nerve; nVII s facial nucleus; PY s pyramidal tract; SOssuperior olive; SVs the nucleus of the spinal trigeminal tract.
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
found medial to the root fibers of the facial nerve. Medially, this subdivision is bordered by the superior olive Žat rostral levels. and the motor facial nucleus Žmore caudally.. The location of these cells is comparable to that of the labelled neurons of the brainstem determined by the injection of PrV-Ba ŽSpencer et al., 1990. or retrograde tracers into the pterygopalatine ganglion ŽSenba et al., 1987; Suzuki et al., 1990; Nemoto et al., 1995. the maxillary nerve ŽInsausiti and Gonzalo, 1980. or the greater superficial petrosal nerve ŽInsausiti and Gonzalo, 1980; Hosoya et al., 1990.. Our double-virus tracer study showed that these cells compose the ventral subdivision of the SSN, while the dorsal part of it is formed by neurons projecting to the submandibular gland. The location of the submandibular subgroup is comparable to the descriptions obtained with tracers injected into the submandibular gland ŽJansen et al., 1992. or the chorda tympani ŽHiura, 1977; Contreras et al., 1980; Mitchell and Templeton, 1981; Nicholson and Severin, 1981., representing the preganglionic parasympathetic neurons projecting to the submandibular ganglion. Insausiti and Gonzalo Ž1980. have pointed out the close topographical connection between the SSN and the A5 catecholaminergic cell group. Although preganglionic cells are partly intermingled with the noradrenergic A5 cells, and some TH-positive neurons are scattered in the SSN ŽHosoya et al., 1990., the two cell populations are clearly separated. None of the preganglionic cells immunostained for TH, and TH-positive A5 cells were not infected by the virus until the late post-inoculation periods. Previous ŽSuzuki et al., 1990; Nemoto et al., 1995. and present double immunostaining revealed that the virus-labelled neurons in the SSN were surrounded by a dense plexus of TH-positive fibers, providing a morphological basis for the catecholaminergic influence of the parasympathetic outflow in the central nervous system. The lacrimal preganglionic neurons receive neuronal inputs from various brain areas, as demonstrated by viral infection of several third-ordered neurons along the entire neuraxis, including the hypothalamus, the limbic system and autonomic brain areas ŽSpencer et al., 1990.. This is in accordance with previous observations from experiments using combined tract-tracing techniques ŽHosoya et al., 1983, 1984, 1990. or immunohistochemistry ŽNemoto et al., 1995. that SSN neurons projecting to the pterygopalatine ganglion receive an abundant synaptic input from the hypothalamus.
5. Conclusion Lacrimal preganglionic parasympathetic neurons occupy the ventral subdivision of the salivatory nucleus. Axons enter the intermediate component of the facial nerve, leave it to enter the greater superficial petrosal nerve, and terminate in the pterygopalatine ganglion. Sympathetic fibers to the lacrimal gland arise in neurons in the
53
IML of the first and second thoracic segments. Additionally, the virus is taken up by sensory trigeminal fibers that innervate the capsule of lacrimal gland and its surrounding tissues. Acknowledgements We are pleased to acknowledge the contributions of Dr G.N. Moskovkin Žremoval of s.c.ggl., Ms Reka ´ Altrichter, Ms Dora Mrs Judit Helfferich, Mrs Zsuzsa Vidra ´ Kezdi, ´ and Mr Jozsef Kiss. We are also pleased to acknowledge ´ the source of the polyclonal antiviral antibody, which was provided through the courtesy of Dr R.R. Miselis. The work was supported by Hungarian Research Grants OTKA No. T-025962, MKM No. 447, and FKFP 0648. References Aujeszky, A., 1902. A contagious disease, not readily distinguishable from rabies, with unknown origin Žin Hungarian.. Veterinarius 25, 387–396. Bartha, A., 1961. Experiments to reduce the virulence of Aujeszky’s virus ´ Žin Hungarian.. Magyar Allatorvosok Lapja 16, 42–45. Boldogkoi, R., Braun, A., Fodor, I., 1996. In ˝ Zs., Medveczky, I., Glavits, ´ vivo studies on Aujeszky’s disease virus mutants. Acta Microbiol. Immunol. Hungarica 43, 307–318. Boldogkoi, ˝ Zs., Braun, A., Medveczky, I., Glavits, B., Gyuro, ´ B., Fodor, I., 1998. Analysis of the equalization of inverted repeats and neurovirulence using a pseudorabies virus mutant strain altered at the UlrIr junction. Virus Genes 17, 89–98. Card, J.P., 1995. Pseudorabies virus replication and assembly in the rodent system. In: Kaplitt, M.G., Loewy, A.D. ŽEds.., Viral Vectors. Gene Therapy and Neuroscience Applications. Academic Press, New York, pp. 319–347. Card, J.P., Rinaman, L., Schwaber, J.S., Miselis, R.R., Whealey, M.E., Robbins, A.K., Enquist, L.W., 1990. Neurotropic properties of pseudorabies virus: uptake and transneuronal passage in the rat central nervous system. J. Neurosci. 10, 1974–1994. Contreras, R.J., Gomez, M.M., Norgren, R., 1980. Central origins of cranial nerve parasympathetic neurons in the rat. J. Comp. Neurol. 190, 373–394. Dolivo, M., 1980. A neurobiological approach to neurotropic viruses. Trends Neurosci. 3, 149–152. Enquist, L.W., 1995. Circuit-specific infection of the mammalian nervous system. Pseudorabies virus, a neurotropic alpha herpesvirus, provides a valuable means for probing cellular interactions in the brain. AMS News 61, 633–638. Hiura, T., 1977. Salivatory neurons innervate the submandibular and sublingual glands in the rat: horseradish peroxidase study. Brain Res. 137, 145–149. Hosoya, Y., Matsushita, M., Sugiura, Y., 1983. A direct hypothalamic projection to the superior salivatory nucleus neurons in the rat. A study using anterograde autoradiographic and retrograde HRP methods. Brain Res. 266, 329–333. Hosoya, Y., Matsushita, M., Sugiura, Y., 1984. Hypothalamic descending afferents to cells of origin of the greater petrosal nerve in the rat, as revealed by a combination of retrograde HRP and anterograde autoradiographic techniques. Brain Res. 290, 141–145. Hosoya, Y., Sugiura, Y., Ito, R.R., Kohno, K., 1990. Descending projections from the hypothalamic paraventricular nucleus to the A5 area, including the superior salivatory nucleus, in the rat. Exp. Brain Res. 82, 513–518.
54
I.E. Toth ´ et al.r Journal of the Autonomic NerÕous System 77 (1999) 45–54
Insausiti, R., Gonzalo, L.M., 1980. New data on the precise location of the lacrimo-muconasal nucleus of the rat. Experientia 36, 977–978. Jansen, A.S.P., Ter Horst, G.J., Mettenleiter, T.C., Loewy, A.D., 1992. CNS cell groups projecting to the submandibular parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body-labeled study. Brain Res. 572, 253–260. Jasmin, L., Burkey, A.R., Card, J.P., Basbaum, A.I., 1997. Transneuronal labeling of a nociceptive pathway, the spinoŽtrigemino.parabrachioamygdaloid, in the rat. J. Neurosci. 17, 3751–3765. Mitchell, J., Templeton, D., 1981. The origin of the preganglionic parasympathetic fibers to the mandibular and sublingual salivary glands in the rat: a horseradish peroxidase study. J. Anat. 132, 513–518. Nemoto, T., Konno, A., Chiba, T., 1995. Synaptic contact of neuropeptide- and amine-containing axons on parasympathetic preganglionic neurons in the superior salivatory nucleus of the rat. Brain Res. 685, 33–45. Nicholson, J.E., Severin, C.M., 1981. The superior and inferior salivatory nuclei in the rat. Neuroscience Letters 21, 133–138. Nikkinen, A., Lehtosalo, J.I., Uusitalo, H., Palkama, A., Panula, P., 1984. The lacrimal lands of the rat and the guinea pig are innervated by nerve fibers containing immunoreactivities for substance P and vasoactive intestinal polypeptide. Histochemistry 81, 23–27. Sams, J.M., Jansen, A.S.P., Mettenleiter, T.C., Loewy, A.D., 1995.
Pseudorabies virus mutants as transneuronal markers. Brain Res. 687, 182–190. Senba, E., Daddona, P.E., Nagy, J.I., 1987. A subpopulation of preganglionic parasympathetic neurons in the rat containing adenosine deaminase. Neuroscience 20, 487–502. Spencer, S.E., Sawyer, W.B., Wada, H., Platt, K.B., Loewy, A.D., 1990. CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study. Brain Res. 534, 149–169. Sun, M.K., 1995. Central neural organization and control of sympathetic nervous system in mammals. Prog. Neurobiol. 47, 157–233. Suzuki, N., Hardebo, J.E., Skagerberg, G., Owman, C., 1990. Central origins of preganglionic fibers to the sphenopalatine ganglion in the rat. A fluorescent retrograde tracer study with special reference to its relation to central catecholaminergic system. J. Auton. Nerv. Syst. 30, 101–110. Swanson, L.W., 1992. Brain Maps. Structure of the Rat Brain. Elsevier, Amsterdam. Toth, ´ I.E., Palkovits, M., 1997. Viral labelling of synaptically connected neurons. Neurobiology 5, 17–41. Ugolini, G., 1995. Transneuronal tracing with alpha-herpesviruses: a review of methodology. In: Kaplitt, M.G., Loewy, A.D. ŽEds.., Viral Vectors. Gene Therapy and Neuroscience Applications, Academic Press, New York, pp. 293–317.