THE JOURNAL OF COMPARATIVE NEUROLOGY 313~213-226(1991)
Access to Gastric Tissue Promotes the Survival of Axotomized Neurons in the Dorsal Motor Nucleus of the Vagus in Neonatal Rats LINDA RINAMAN AND PAT LEVITT Department of Anatomy and Neurobiology, The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129
ABSTRACT Lesioning the vagus nerve in the neck (cervical vagotomy) results in a rapid and virtually complete loss of motoneurons in the dorsal motor nucleus of the vagus in neonatal rats. The present study sought to determine whether access to gastric target tissue will promote the survival of these motoneurons after axotomy. Quantitative analysis demonstrates that subdiaphragmatic vagotomy, which leaves the cut vagal axons in close proximity to their normal gastric targets, results in significantly less motoneuron loss than cervical vagotomy. Furthermore, the loss of motoneurons after cervical vagotomy can be significantly reduced by transplanting embryonic gastric tissue to the neck of vagotomized neonatal host rats, in the vicinity of the cut axons. The survival effect of transplanted gastric tissue appears specific because control transplants of embryonic bladder tissue fail to reduce motoneuron death after cervical vagotomy. Injections of the neural tracers Fluoro-Gold and cholera toxin-horseradish peroxidase into gastric transplants labeled surviving motoneurons in cervically vagotomized rats, whereas tracer injections into bladder transplants or into host cervical tissues did not. These results indicate that neonatal vagal motoneurons are capable of making the adjustments necessary to survive axotomy if they have access to gastric target cells. The apparent dependence of injured neonatal vagal motoneurons on gastric tissue offers a new system in which to examine in vivo the trophic interactions between neurons and their targets. Key words: vagotomy, gastrointestinal, degeneration,transplantation
Many factors influence neuronal survival during embryonic and early postnatal development, including the establishment and maintenance of connections between neurons and their targets (Landmesser and Pilar, '78;Cunningham, '82; Cowan et al., '84). It is a common observation that both somatic and autonomic motoneurons die when they are separated from their peripheral targets by axotomy during the early postnatal period (LaVelle, '73; Schmalbruch, '84; Jones and LaVelle, '86; Comans et al., '88; Snider and Thanedar, '89; Crews and Wigston, '90; Rinaman and Levitt, '90; Rinaman et al., in press). For example, an extremely rapid and virtually complete loss of the preganglionic parasympathetic motoneurons in the dorsal motor nucleus of the vagus (DMV) follows cervical vagotomy (lesion of the vagus nerve in the neck) in newborn rats (Rinaman and Levitt, '90; Rinaman et al., '91). Anatomical studies in rat indicate that the axons of DMV motoneurons project to the gastrointestinal enteric nervous system (ENS) without collateralizing or leaving the main trunk of the vagus nerve prior to reaching the gastroesophageal junction O
1991 WILEY-LISS, INC.
(Fox and Powley, '85; Bieger and Hopkins, '87; Altschuler et al., '89, '91; Miselis et al., '91). DMV motoneurons may die subsequent to cervical vagotomy because they are deprived of neurotrophic substances manufactured by neuronal and/or nonneuronal cells in the gut. The regressive response of immature DMV motoneurons to axotomy may reflect the continuation of an embryonic dependence on specific target derived trophic factors, as described in other systems (Cowan, '73; Oppenheim, '81, '85; Cunningham, '82; Cowan et al., '84; Hill, '85; Haun and Cunningham, '87;Oppenheim et al., '88; Crews and Wigston, '90). In addition to the detrimental effects of target deprivation, it has also been suggested that neonatal motoneurons are especially vulnerable to axotomy because they are already synthesizing proteins at peak capacity and may not Accepted August 5,1991. Address reprint requests to Dr. Linda Rinaman, Dept. of Anatomy and Neurobiology, The Medical College of Pennsylvania, EPPl Division, 3200 Henry Ave., Philadelphia, PA 19129.
L. RINAMAN AND P. LEVITT
214 be able to mount an adequate regenerative response to injury (LaVelle, '83; Jones and LaVelle, '86). For example, cervical vagotomy amputates a relatively large portion of each DMV motoneuron's axon, a factor that has been shown in other systems to contribute to the rapidity and severity of the lesion response (for reviews see Cragg, '70; Lieberman, '71; Grafstein, '75). Axotomized neonatal DMV neurons may die because they are simply incapable of making the metabolic adjustments necessary to survive the lesion. The present study was designed to test the hypothesis that neonatal DMV motoneurons can survive axotomy provided they have access to an appropriate peripheral target. We first compare the effects of cervical vagotomy and subdiaphragmatic vagotomy (in the abdomen) on the loss of DMV motoneurons to determine whether they can survive a lesion made further from the neuronal cell body and closer to their normal gastric target cells. We then examine whether DMV motoneurons can survive the more severe cervical lesion if their injured axons have access to gastric tissue transplanted to the site of nerve damage.
MATERIALS AND METHODS Animals and anesthesia The embryonic or neonatal offspring of Sprague-Dawley rats (Harlan) were used in all experiments. Rats were housed in an AAALAC licensed animal facility, and their care and treatment adhered to NIH guidelines and the Animal Welfare Act. All rats were deeply anesthetized prior to surgery and sacrifice. Newborn rats [postnatal day 1 (Pl), where PO = day of birth] were ice anesthetized (rapid hypothermia) prior to surgery and were revived by slow warming under a heating lamp. Rats aged P7 to P30 at sacrifice were anesthetized with ketaminelxylazine [five parts ketamine (100 mgsiml) plus one part xylazine (20 mgs/ml); 200 pl/lOO g body weight, ipl. Rats older than P30 at sacrifice were anesthetized with Nembutal (300 p1/ 100 g body weight, ip).
Abbreviations
AChE AP cc
CNS
CT-HRP Cu DMV E ENS eP EPSP FG gP
Gr
H&E HYP IP
lU me NST P
PT se sm SPT te Tr
acetylcholinesterase area postrema central canal central nervous system cholera toxin-horseradishperoxidase cuneate nucleus dorsal motor nucleus of the vagus embryonic day enteric nervous system enteric plexus excitatory postsynapticpotential Fluoro-Gold gastric pits gracile nucleus hemotoxylin and eosin hypoglossal nucleus lamina propria lumen muscularisexterna nucleus of the solitary tract postnatal day pyramidal tract surface epithelium submucosa spinal nucleus of the trigeminal nerve transitional epithelium solitary tract
Fig. 1. Schematic diagram of a typical cross section through the rat medulla oblongata at the level of the area postrema (caudal to the opening of the 4th ventricle). The micrographs in Figures 2,3, and 6-8 are of tissue sections from approximately the same rostrocaudal level and include the area indicated by the hatched box in this schematic. Left and right are as depicted. Arrowheads point out the left DMV, which is axotomized in Figures 2, 3, and 6-8. AP, area postrema; cc, central canal; Cu, cuneate nucleus; DMV, dorsal motor nucleus of the vagus; Gr, gracile nucleus; H Y P , hypoglossal nucleus; NA, nucleus ambiguus; NST, nucleus of the solitary tract; PT, pyramidal tract; SpT, spinal nucleus of the trigeminal nerve; Tr, solitary tract (adapted from Paxinos and Watson, '86). Scale bar = 500 Fm.
Newborn (Pl) rats were placed in one of four experimental groups: (1)cervical vagotomy; no transplant (n = 13); (2) subdiaphragmatic vagotomy; no transplant (n = 8); (3) cervical vagotomy; gastric transplant (n = 18),(4)cervical vagotomy; bladder transplant (n = 11).
Transplant procedures Transplantation of fetal gastric tissue was partially based on a preliminary report demonstrating the survival and development of embryonic rat gastric tissue transplanted to the forehead of host rats (Horgan and Van der Kooy, '89). In the present study, rat fetuses at 17 days of gestation (E17) were removed from the uterine horns, placed in sterile ice cold F-10 Nutrient Mixture (Gibco), and decapitated. Tissue samples (approximately one mm3)from donor embryos were collected from the antral region of the ventral stomach wall or from the bladder wall and were placed in separate containers of fresh ice cold F-10. The antral region of the ventral stomach wall contains a concentrated population of enteric neurons that receive direct innervation specifically by motoneurons in the left DMV in adult rats (Shapiro and Miselis, '85; Kirchgessner and Gershon, '89). Enteric neurons and well defined nerve plexuses are already present in the stomach wall by E l 7 (Rothman and Gershon, '82; Rothman et al., '84). The bladder does not receive vagal innervation; the bladder's postganglionic parasympathetic
Fig. 2. Nissl stained sections through the medullas of four vagotomized rats, none of which received tissue implants. Arrowheads point out the axotomized DMV. A: Two weeks after left cervical vagotomy, there is virtually complete loss of DMV motoneurons. B: Four weeks after left cervical vagotomy, motoneuronal loss is similar to that seen (in A) after two weeks. C: Two weeks after ventral subdiaphragmatic vagotomy, a large number of DMV motoneurons remain on the lesioned side, although their number is reduced compared to the intact side. D: Four weeks after ventral subdiaphragmatic vagotomy, motoneuronal loss on the lesioned side remains incomplete. cc, central canal. Scale har in A = 500 ym, applies also to B-D.
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS
Figure 2
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neurons are innervated by preganglionic parasympathetic motoneurons in the sacral spinal cord (Nyberg-Hansen,'66). In the P1 rats to be implanted with gastric or bladder tissue, the ventral surface of the neck was swabbed with 70% ethanol and a short midline incision made. With the aid of a dissecting microscope, the skin was retracted and the left carotid artery was exposed at the level of the omohyoid muscle by blunt dissection. One piece of embryonic gastric or bladder tissue was implanted medial to the carotid artery, and the left cervical vagus nerve was then sectioned (see below). No more than 10 minutes elapsed between dissection of the embryonic tissue samples and implantation into P 1 hosts.
Vagotomy procedures For cervical vagotomy, the left vagus nerve was exposed at the level of the omohyoid muscle, gently separated from the carotid artery, and severed. In rats that received a fetal gastric or bladder tissue transplant, the proximal end of the severed vagus nerve was placed directly onto the surface of the transplant. In some of the P1 rats that were cervically vagotomized with no transplant, the left vagus nerve was first thoroughly crushed with fine forceps and 2-4 p1 of Fluorogold (FG) neural tracer (Fluorochrome International; 0.5% in sterile saline) was injected into the nerve at the crush site to label axotomized DMV motoneurons retrogradely. Assurance of complete axotomy was achieved by subsequently cutting the vagus nerve just distal to the FG injection site. FG injections were made with a pulled glass micropipette (20 pm tip diameter) affixed to the tip of a 10 p1 Hamilton syringe. Neck incisions were sutured with 7-0 silk (Ethilon). For subdiaphragmatic vagotomy, the abdomen was swabbed with 70% ethanol and a ventral midline incision was made through the skin and abdominal musculature. The gastroesophageal junction was exposed by careful retraction of the liver. With the aid of a dissecting microscope, the ventral vagus nerve (the distal extension of the left cervical vagus nerve) was identified in its course along the esophagus and was cut at the gastroesophageal junction, just above the point where gastric vagal branches ramify. Abdominal incisions were sutured in two layers with 7-0 silk (Ethilon).
Tracer injection into tissue transplants In some rats that received a gastric or bladder transplant, FG or cholera toxin-horseradish peroxidase (CT-HRP; donated by R.R. Miselis) was injected into the transplant two days prior to sacrifice. As a control, some of the rats that were cervically vagotomized with no transplant received FG or CT-HRP tracer injections into the muscles and other host tissues surrounding the site of cervical vagotomy. In each rat, 10-15 ~1 of CT-HRP (0.2% in sterile saline) or FG (0.5% in sterile saline) was injected in multiple 1-2 p1 aliquots. Injections were made through a pulled glass micropipette (tip diameter 25-40 pm) secured to a 10 p1 Hamilton syringe with sealing wax. Although the membrane binding properties of CT-HRP limit its diffusion from sites of injection (discussed in Rinaman and Miselis, '87), the following measures were also taken to further ensure specificity of CT-HRP labeling: the injecting micropipette was held in place for approximately one minute at each injection site, and each site was then swabbed with bits of gauze soaked in sterile saline. Following the final CT-HRP injection, the entire cervical area was flooded three times with warm sterile saline and then blotted with gauzes prior
TABLE 1. Effect of Vagotomy and Transplant Procedures on Loss of Neonatally Axotomized DMV Motoneurons Fourteen Days Postlesion'
Group
Total number of neurons in lesioned DMV
Total number of neurons in intact DMV
1. Cervical Vagotomy, no Transplant (rat i.d.#) 89-275 7 9047 13 90-68 27 90-69 14
% Loss of axotomized DMV neurons [(intact-lesioned) x loo%] intact
390 492 570 515 AverageLoss 2. Subdiaphragmatic Vagotomy, no Transplant (rat i.d.#) 90-76 193 439 90-77 360 481 90-78 136 551 90-79 101 463 Average Loss 2 3. Cervical Vagotomy, Gastric Transplant (rat i.d.#) 90-16 304 670 90-17 112 384 90-19 203 556 90-106 66 551 Average Loss z 4. Cervical Vagotomy, Bladder Transplant (rat id.#) 90-95 25 367 90-100 16 526 90-101 7 485 90-102 16 502 AverageLoss 2
*
98.26 loss 97.46 95.3% 97.37" S.D.: 97.1 i 1.2% 56.0%loss 25.2% 75.3% 78.2% S.D.:58.7 t 24 4% 54.6%loss 71.0% 63.56 88.0% S.D.: 69.3 ? 14.2% 93.28 loss 97.08
99.0% 96.88 S.D.: 96.5 2 2.496
'Statistical comparisons were made between groups using the Mann-Whitney U Test. Significant differences (p s 0 05) in DMV montoneuronal loss were found between Groups 1and 2 , Groups 1and 3, Groups 2 and 4, and Groups 3 and 4.
to suturing the cervical incision. No special precautions were taken to limit spread of FG, which is known to diffuse from peripheral injection sites (Yoshida et al., '88) and to be picked up by intact peripheral fibers in neonatal rats (Leong and Ling, '90; Rinaman et al., in press; see Discussion).
Histological preparation One to eight weeks after vagotomy, rats were anesthetized and sacrificed by transcardiac perfusion fixation. In rats injected with CT-HRP, perfusates consisted of an initial saline rinse followed by infusion of ice cold phosphate buffer containing 1% paraformaldehyde and 2.5%glutaraldehyde (pH 7.41, followed by infusion of 10% buffered sucrose. Brainstems were removed and cryoprotected in 30% buffered sucrose. For all other rats, the initial saline rinse was followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). These brainstems and transplant tissues were postfixed in situ for 24 hours at 4"C, then removed and cryoprotected in 30%buffered sucrose. Medullas were sectioned in the coronal plane with a sliding microtome at 40 pm intervals throughout the rostrocaudal extent of the DMV, which in rats extends from the inferior olive to C2-C3 of the spinal cord. Tissue sections used for Nissl analysis were mounted on gelatin coated slides, air dried, and stained with cresyl violet. Tissue sections used for localization of transported FG were mounted, air dried in the dark, and coverslipped using GelMount (Biomeda). Some sections containing FG were temporarily coverslipped with buffer and photographed for FG localization prior to staining with cresyl violet. Tissue sections used for localization of transported CT-HRP were enzymatically treated using a variation of Mesulam's ('78) tetramethyl benzidine procedure (Rinaman and Miselis, '87), mounted on gelatin coated slides, rapidly dehydrated
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Fig. 3. Tissue section from a cervically vagotomized rat in which the left vagus nerve was injected with FG at the time of vagotomy (two week survival). Arrowheads delineate the axotomized DMV. In A, the section is stained for Nissl substance to reveal an apparently complete loss of DMV motoneurons on the side of the lesion. In B, the same tissue section is viewed with fluorescence (prior to Nissl staining) to demonstrate loss of FG labeled neurons in the left DMV. The small,
very bright cells in the axotomized DMV are non-neuronal macrophages, including microglia (see text). As no attempt was made to restrict peripheral diffusion of the FG from the injection site, almost all of the Nissl stained motoneurons in the intact (right) DMV are also lightly labeled with FG. This makes evident the high correlation between neurons identified with FG labeling and with Nissl staining (compare A and B). cc, central canal. Scale bar in A = 500 pm, applies also to B.
and coverslipped with DPX. Some HRP processed tissue sections were stained with cresyl violet prior to coverslipping. Gastric and bladder transplant tissues were cryostat sectioned at 16 km intervals. Cryostat sections through the tissue transplants were stained with hemotoxylin and eosin (H&E) according to routine procedures, or were processed for acetylcholinesterase (AChE) localization (Lewis, '61).
analysis of the number of Nissl stained DMV motoneurons was restricted to rats surviving two weeks after vagotomy. A two week survival period was chosen because cell loss in the lesioned DMVs of cervically vagotomized rats, including those receiving gastric and bladder tissue transplants, reached a plateau by this time that was maintained for up to eight weeks (see Results). Longer survival times were not examined. Medullary tissue sections from the rostrocaudal level of the DMV containing the midregion of the area postrema (four 40 krn thick sections per rat) were used for DMV cell counts, as the cytoarchitectural boundaries of the DMV tend to become obscure at more rostral and caudal levels of the nucleus. At the level of the area postrema, the DMV is
Quantitative analysis of DMV motoneuronal survival Tissue sections were examined from rats in all four groups surviving between one and eight weeks after vagotomy. For statistical comparisons, however, quantitative
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Fig. 4. H&E stained sections through a typical gastric transplant (two week survival) demonstrating the healthy, histological differentiation of the tissue and its incorporation into the host cervical area. In A, a low magnification micrograph reveals the general organization of the transplant. Note the well developed gastric pits (gp), lamina propria (lp) and muscularis externa (me). The large arrow (boxed B) indicates an
area which is enlarged in B. At this magnification, well differentiated mucous secreting cells are evident in the gastric pits and gastric glands (gg). C is from another area of the same transplant showing gastric glands and pits in cross section, surrounded by the lamina propria. lu, lumen; se, surface epithelium of gg; sm, submucosa. Scale bar in A = 500 km; B and C X 4.
bounded ventrally by a thin plate of glial cells (visible in Nissl stained tissue) separating it from the underlying hypoglossal nucleus, dorsally by small cells of the medial subnucleus and the subnucleus centralis of the NST, laterally by an imaginary line drawn ventrally from the solitary tract, and medially by the central canal. Tissue ~ sections were analyzed microscopically with a 1 0 objective. DMV motoneurons were identified as large cells with a distinct Nissl stained cytoplasm and proximal dendritic projections. Well defined nuclear profiles of these cells were counted, as the average nuclear diameter of surviving axotomized neurons in the lesioned DMV did not differ significantly from the average nuclear diameter of motoneurons in the intact DMV. For each rat, cell counts from the intact and vagotomized sides of each section were totaled and the percent reduction of DMV neurons on the lesioned side compared to the unlesioned side was calculated {[(intactlesioned)/intact] x loo%]. Statistical differences between DMV motoneuron loss in the four experimental groups were determined using the Mann-Whitney U Test, with p values I 0.05 considered significant.
RESULTS Cervical vagotomy, no transplant Severing the left cervical vagus nerve in P1 rats consistently resulted in an almost complete loss of Nissl stained motoneurons in the ipsilateral DMV by two weeks postlesion (Fig. 2A,B; see Fig. 1 for histological orientation). Quantitative analysis revealed an average 97.1% reduction in the number of DMV neurons on the lesioned vs. intact side in each rat (Table 1). Examination of FG labeling and Nissl staining in the same tissue sections revealed that counts of Nissl stained motoneurons in the intact and lesioned DMV match well with counts of FG labeled neurons (Figs. 3, 8; see Discussion). This verified that the apparent loss of DMV motoneurons was an actual loss and was not due to morphological alterations of the axotomized DMV neurons that would prevent their identification in Nissl stained tissue. The small, very bright FG labeled cells visible in the lesioned DMV in Figure 3 have been immunocytochemically identified as nonneuronal macrophages, including microglia, that phagocytose the FG when the
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Fig. 5. AChE stained tissue section through a typical gastric transplant (two week survival). Many AChE positive nerve fibers and enteric neurons are present in the transplant. The large arrow in A (boxed B) indicates an area that is enlarged in B. ep, enteric plexus; n, neurons in plexus. Scale bar in A = 500 pm;
B x 4.
labeled motoneurons die (Rinaman et al., '91; see also Crews and Wigston, '90). Control injections of FG or CT-HRP into the internal neck muscles and other host tissues surrounding the site of cervical vagotomy in these rats never resulted in labeled neurons in the axotomized DMV. Light FG labeling of motoneurons in the intact (right) DMV and in both hypoglossal nuclei was always observed following FG injection of the lesioned vagus nerve (Fig. 3) or injection of host cervical
tissues, consistent with FG's propensity to spread from peripheral injection sites (see Methods). In contrast, CTHRP labeling of neurons in the intact DMV or hypoglossal nuclei was never seen following injection into host cervical tissues, consistent with the lack of peripheral spread of CT-HRP or its uptake by intact nerve fibers (see Methods). Positive, within animal controls for the neuronal uptake and transport of both tracers was provided by the presence of labeled motoneurons in the ipsilateral nucleus ambiguus
Figure 6
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS
Fig. 7. Two alternate tissue sections from a cervically vagotomized rat with gastric tissue transplant (four week survival) that was injected with CT-HRP two days prior to sacrifice. Arrowheads point out the axotomized DMV. A is HRP-treated and Nissl-stained; B is HRPtreated only. The motoneurons that survived cervical vagotomy are
221
retrogradely labeled following CT-HRP injection into the gastric transplant tissue. Anterogradely labeled vagal sensory axons and terminals are also evident in the dorsally situated NST. cc, central canal. Scale bar in A = 500 pm, applies also to B.
and ventral horn of the cervical spinal cord which are known to innervate muscles in the cervical region.
demonstrated many positive nerve fibers and neurons in well defined enteric plexuses (Fig. 5). Rats with gastric transplants consistently had many Subdiaphragmatic vagotomy, no Nissl stained motoneurons remaining in the axotomized Severing the ventral subdiaphragmatic vagus nerve at DMV two weeks after cervical vagotomy (Fig. 6A; Fig. BA). the gastroesophagealjunction resulted in an average 58.7% Quantitative analysis revealed an average 69.3% reduction reduction of Nissl stained motoneurons in the axotomized of neurons in the lesioned DMV as compared to the intact DMV as compared to the intact DMV at two weeks postle- DMV in these rats (Table l),a significantly smaller cell loss sion (Table 1; Fig. 2C). Although DMV cell loss in subdia- than in rats that were cervically vagotomized but received phragmatically vagotomized rats was more variable than in no transplants. Qualitatively, DMV cell loss in rats with the other three groups (see Table 11, it was significantlyless gastric transplants appeared somewhat greater than DMV than DMV cell loss in cervically vagotomized rats that cell loss in subdiaphragmatically vagotomized rats, but the received no transplant. Examination of tissue sections from difference was not statistically significant. The effect of the subdiaphragmatically vagotomized rats surviving for longer gastric tissue transplant on DMV cell survival remained periods revealed a similar between animal variability in evident in rats surviving for longer periods (Fig. 6B; Fig. 7), with many Nissl stained motoneurons remaining in the DMV cell loss (Fig. 2D). axotomized DMV as long as eight weeks after vagotomy. Cervical vagotomy, gastric transplant Injection of CT-HRP into the gastric transplants rouAs early as one week after transplantation into P1 host tinely resulted in retrograde labeling of surviving DMV rats, gastric transplant tissues were vascularized, had motoneurons and their dendritic processes on the lesioned grown in size, and had well delineated luminal cavities. side, as well as anterograde (transganglionic) labeling of Cervically vagotomized rats with gastric transplants devel- vagal sensory fibers in the nucleus of the solitary tract oped externally visible enlargements on the left ventrolat- (NST; Fig. 7). CT-HRP labeled neurons were never oberal side of the neck caused by growth of the transplant served in the contralateral (intact) DMV, but labeled motissue. Examination of H&E stained cryostat sections toneurons were occasionally observed in the ipsilateral through the transplants revealed gastric rugae and other nucleus ambiguus and ventral horn of the cervical spinal morphological features typical of gastric tissue (Fig. 4). cord, likely resulting from tracer injection into the host Examination of AChE stained gastric transplant sections muscles underlying the transplant tissue. As with CT-HRP injection, injection of FG into the gastric transplants labeled surviving motoneurons in the axotomized DMV (Fig. 8 ) as well as motoneurons in the nucleus ambiguus and spinal cord. Unlike CT-HRP, FG also retrogradely Fig. 6. Nissl stained tissue sections through the medullas of four cervically vagotomized rats. Arrowheads denote the axotomized DMV. labeled motoneurons in the intact DMV due to tracer spread. A With; gastric tissue implant, two week survival, a moderate number of DMV motoneurons remain on the lesioned side. B: DMV motoneurons are also evident on the lesioned side after four weeks in a rat that received a gastric tissue implant. C: With a bladder tissue implant, two week survival, loss of DMV motoneurons on the lesioned side appears complete. D: Loss of axotomized DMV neurons is evident in another rat with a bladder transplant, four weeks post lesion. cc, central canal. Scale bar in A = 500 i m , applies also to B-D.
Cervical vagotomy, bladder transplant Examination of H&E stained sections through bladder growth, vascularization, and morphology typical of bladder tissue (Fig. 9). AChE positive nerve fibers were present in and around the smooth muscle
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Fig. 8. Alternate tissue sections from a cervically vagotomized rat that received a gastric tissue implant (two week survival). The gastric tissue implant was injected with FG two days prior to sacrifice. Arrowheads point out the axotomized DMV. A: Nissl stained section
reveals surviving DMV motoneurons on the side of the lesion. B: Fluorescence view shows motoneurons retrogradely labeled with FG in the lesioned DMV. cc, central canal. Scale bar in A = 500 pn, applies also to B.
bundles in the transplant tissue, and a small number of AChE positive neurons were observed in clusters of 3 or 4 in the smooth muscle and adventitia. In marked contrast to the increased DMV cell survival seen in cervically vagotomized rats with gastric transplants, rats with bladder transplants exhibited almost complete loss of cervically axotomized DMV neurons (Fig. 6C,D). Quantitative analysis revealed an average 96.5% reduction in Nissl stained DMV motoneurons on the lesioned side (Table l), statistically no different than the DMV cell loss seen in rats that were cervically vagotomized with no transplant tissue. Injection of FG or CT-HRP into bladder tissue transplants never labeled neurons in the axotomized DMV. However, both tracers labeled neurons in the other locations described above following injections into host cervical tissues and gastric tissue transplants, providing a positive control for tracer transport.
DISCUSSION This study demonstrates that neonatal rat DMV motoneurons can survive axotomy if they have access to gastric target tissue. The survival effect of gastric tissue lasts for at least two months, by which time the neonatally lesioned rats have reached maturity. As longer survival times were not examined, it is not known whether the apparent rescue of these axotomized neurons is permanent. In addition, although a significant number of axotomized DMV neurons are “rescued” in the presence of gastric tissue, many of the axotomized DMV neurons in subdiaphragmatically lesioned rats, and in cervically lesioned rats with gastric transplants, do not survive. Survival may be incomplete for a number of reasons, possibly including abberrant regenerative vagal sprouting (see Evans and Murray, ’54; King and Thomas, ’71).
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Fig. 9. H&E stained section through a typical bladder transplant (two week survival) demonstrating the healthy, histological differentiation of the tissue and its incorporation into the host cervical area. In A, a low magnification view depicts well organized tissue, including a
surface epithelium surrounding the lumen (lu), extensive lamina propria (lp), and smooth muscle (sm) bundles. The area depicted by a large arrow and a box is enlarged in B, where transitional epithelium (te) typical of the bladder is seen. Scale bar in A = 500 +m; B x 4.
It is difficult to know whether the survival of neonatal DMV motoneurons following subdiaphragmatic vagotomy is attributable more to enhanced access to gastric targets or to reduced lesion severity. While the distance of the lesion from the neuronal cell body certainly cannot be excluded as a contributing factor (LaVelle, '83; Jones and LaVelle, '86; see also Murphy et al., 'go), the closer proximity of the severed axons to appropriate gastric target cells and presumably to some survival promoting factor(s) associated with
those cells appears to play an important role. This is supported by our demonstration that the normally rapid and virtually complete loss of cervically axotomized neonatal DMV motoneurons can be significantly reduced by transplanting gastric tissue to the site of cervical vagotomy. DMV cell loss in cervically vagotomized rats with gastric transplants does not differ significantly from DMV cell loss in subdiaphragmatically vagotomized rats. The ability of gastric tissue to promote the survival of axotomized DMV
224 motoneurons appears specific, because access to transplanted bladder tissue (another parasympathetically innervated viscus) provides no survival effect.
Possible trophic interactions between DMV motoneurons and gastric target cells In certain respects, DMV motoneurons are more closely akin to intrinsic central neurons than to somatic motoneurons in that their axons terminate not on muscle cells but on other neurons (see Lewis et al., '72). The postsynaptic targets of DMV motoneurons are neurons in the ENS. The ENS is a complex and independent nervous system containing a diversity of intrinsic neuronal types and has many structural and morphological features in common with the CNS (Gabella, '87; Kirchgessner and Gershon, 'go), including a blood-myenteric plexus barrier that may be functionally analogous to the CNS' blood-brain barrier (Gershon and Bursztajn, '78; Gershon et al., '79). The peripheral axons of vagal neurons normally invade the blood-myenteric barrier, allowing access to and possible retrograde transport of unique trophic substances secreted by neuronal and/or non-neuronal gastric cells. It has been repeatedly postulated that trophic substances produced by target cells are critical to the development and survival of the neurons that innervate those targets (Cowan, '73; Landmesser and Pilar, '78; Oppenheim, '81, '85, '88; Cunningham, '82; Gordon, '83; Bijlsma et al., '84; Cowan et al., '84; Hill, '85; Crews and Wigston, '90). The new observation that gastric tissue (but not bladder or host cervical tissues) decreases the loss of axotomized DMV neurons suggests that these motoneurons also depend on specific interactions with their appropriate target cell populations for survival during the early postnatal period. The specificity of the interaction between DMV motoneurons and gastric tissue suggests that more general substances produced by host tissues in the cervical region or by smooth muscle and other cells in the bladder are insufficient in promoting the survival of axotomized vagal motoneurons. CT-HRP and FG tracer injection into gastric transplants labeled motoneurons in the lesioned DMV, whereas tracer injections into bladder transplants or host cervical tissues did not. This indicates that the proximal ends of the severed axons were present and capable of retrograde transport in rats with a gastric transplant, but not in rats with a bladder transplant or with no transplant. We did not determine whether vagal axons actually regenerated to contact neurons or other cells in the gastric transplants, but the presence of AChE positive enteric ganglia allows for the possibility that DMV-ENSinnervation can be established in this situation. It should be kept in mind that the putative acquisition of specific target derived trophic factors may by itself be only partially responsible for producing the observed survival effect on injured DMV motoneurons. Access to gastric target tissue following axotomy may simply be one requisite step in a more complex process that ultimately leads to the reestablishment of normal central synaptic inputs, producing a balance that is itself essential for motoneuronal survival (Cunningham, '82; Hill, '85; Oppenheim, '89; Snider and Thanedar, '89). Further in vivo and in vitro experiments should be able to specifically address this issue.
L. RINAMAN AND P. LEVITT
Speculations on a role for sensory deafferentation in the response of axotomized DMV motoneurons Concurrent damage to vagal sensory neurons necessarily occurs during vagotomy, and may be a key factor in the inability of target deprived DMV motoneurons to recover from the lesion. The availability of a proper peripheral target after subdiaphragmatic vagotomy or after cervical vagotomy with gastric transplants may enhance the survival of axotomized DMV motoneurons partly because these procedures enhance the survival of the axotomized sensory neurons that normally terminate centrally on the motoneurons. Injection of CT-HRP into the gastric tissue transplants anterogradely (transganglionically) labeled vagal sensory axon terminals in the NST, but vagal sensory labeling was not seen after injection of CT-HRP into bladder transplants. One can speculate that target specific factors also promote the survival of vagal sensory neurons after axotomy. This raises an interesting question: in addition to a possible dependence on intact inputs from CNS neurons as mentioned in the preceding paragraph, might injured DMV motoneurons also depend on inputs from vagal sensory neurons? Physiological studies have shown that intact vagal sensory input is necessary for the normal tonic activity of DMV motoneurons. Vagotomy causes the immediate cessation of spontaneous action potentials in DMV motoneurons, although EPSPs can still be recorded (Barber and Burks, '83). Vagal sensory inputs drive DMV motoneurons not only through central disynaptic and polysynaptic circuits, but also through direct monosynaptic contacts on DMV motoneuronal dendrites (Neuhuber and Sandoz, '86; Rinaman et al., '89). Damage to vagal sensory neurons, therefore, alters or removes a significant source of afferent drive and potential anterogradely supplied trophic support to DMV motoneurons. Transsynaptic degeneration of certain populations of deafferented central neurons is a well known phenomenon in developing animals (Cowan, '70; Van der Loos and Woolsey, '73; Smith, '74; Cunningham, '82; Okado and Oppenheim, '84; Furber et al., '87). It will be intriguing to discover whether DMV motoneurons also exhibit a dependence on their vagal sensory innervation for survival.
Speculations on DMV cell loss following vagotomy in mature animals Most mature somatic motoneurons respond to axotomy with chromatolysis and dendritic atrophy, but their axons regenerate and the neurons recover (Cragg, '70; Lieberman, '71; Grafstein, '75; Baker et al., '81; Bijlsma et al., '84; Murphy et al., '90). In fact, at least some populations of mature somatic motoneurons survive even when they are not allowed to reinnervate their normal muscular targets (Acheson et al., '42; Cull, '74; Goldring et al., '80; Snider and Thanedar, '89), suggesting that they no longer require specific target derived factors for survival. In marked contrast, it has been known for more than 100 years that the majority of mature DMV motoneurons ultimately atrophy and disintegrate following cervical vagotomy in a variety of mammalian species (Dees, 1889;Geist, '33; Lewis et al., '70, '72; Sumner and Watson, '71; Sumner, '75; Aldskogius, '78; Aldskogius et al., '80; Szereda-Przestaszewska, '85; Laiwand et al., '87; Ling et al., '87). The uniquely necrobiotic reaction of mature DMV motoneurons to axotomy may reflect a continuation of the developmental target
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS dependence suggested by the present work. Further experiments should be able to demonstrate whether access to gastric tissue reduces the incidence of DMV motoneuronal death following vagotomy in mature rats. In conclusion, we have demonstrated that neonatal DMV motoneurons can survive axotomy if they are provided access to appropriate peripheral target cells, irregardless of the distance of the lesion from the motoneuronal cell body. The vagal visceral circuit is readily accessible for further experimental manipulations aimed at determining the specific contributions of its central and peripheral components on the development and long term survival of its constituent neurons, as well as to define and assay the potential macromolecular candidates that mediate these effects.
ACKNOWLEDGMENTS This work was supported by NIMH grant MH45507, NIH Training Grant lT32NS07287, and NRSA Grant 1F32NS08900. We are grateful to Drs. Mary Barbe, Pat Card, Tim Cunningham, Michael Goldberger,Forrest Haun, Tim Himes, Hazel Murphy, and Alan Tessler for their insightful comments on the manuscript.
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226 Lieberman, A.R. (1971)The axon reaction: a review of the principal features of perikaryal responses to axon injury. Int. Rev. Neurobiol. 14t49-124. Ling,E.A., J.Y. Shieh, C.Y. Wen, T.Y.Yick,and W.C. Wong(1987) Thedorsal motor nucleus of the vagus nerve of the hamster: ultrastructure of vagal neurons and their responses to vagotomy. J. Anat. 152161-172. Mesulam, M.-M. (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: A non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26: 106-117. Miselis, R.R., L. Rinaman, S.M. Altschuler, X. Bao, and R.B. Lynn. (1991) Viscerotopic representation of the alimentary canal innervation. In Y. Tache and D. Wingate (eds): Brain-Gut Interactions. London: CRC Press, Chapter 1. Murphy, E.H., J. Brown, P.G. Iannuzzelli, and R. Baker (1990)Regeneration and soma size changes following axotomy of the trochlear nerve. J. Comp. Neurol. 292524-536. Neuhuber, W.L., and P.A. Sandoz (1986)Vagal primary afferent terminals in the dorsal motor nucleus of the rat: are they making monosynaptic contacts on preganglionic efferent neurons? Neurosci. Lett. 69,126-130. NybergHansen, R. (1966) Innervation and nervous control of the urinary bladder. Acta Neurol. Scand. 42(Suppl. 20):7-24. Okado, N. and R.W. Oppenheim (1984) Cell death of motoneurons in the chick embryo spinal cord. IX. The loss of motoneurons followingremoval of afferent inputs. J. Neurosci. Res. 4:1639-1652. Oppenheim, R.W. (1981) Neuronal cell death and some related regressive phenomena during neurogenesis: a selective review and progress report. In M.W. Cowan (ed): Studies in Developmental Neurobiology: Essays in Honor of Victor Hamburger. New York: Oxford Univ. Press, pp. 74-133. Oppenheim, R.W. (1985) Naturally occurring cell death during neural development. TINS 17:487-493. Oppenheim, R.W., L.J. Haverkamp, D. Prevette, J.L. MeManaman, and S.H. Appel(1988) Reduction of naturally occurring motoneuron death in vivo by a target-derived neurotrophic factor. Science 24Ot919-922. Oppenheim, R.W. (1989) The neurotrophic theory and naturally occurring motoneuron death. TINS 12252-255.
L. RINAMAN AND P. LEVITT Paxinos, G. and C. Watson (1986) The Rat Brain in Stereotaxic Coordinates. Australia: Academic Press. Rinaman, L., and R.R. Miselis (1987)Vagal innervation of rat pancreas using cholera toxin-horseradish peroxidase conjugate. J. Auton. Nerv. Syst. 21:109-125. Rinaman, L., J.P. Card, J.S. Schwaber, and R.R. Miselis (1989) Ultrastructural demonstration of a gastric monosynaptic vagal circuit in the nucleus of the solitary tract in rat. J. Neurosci. 9,1985-1996, Rinaman, L., and P. Levitt (1990) Rapid degeneration of axotomized vagal motoneurons in neonatal rats. Anat. Rec. 226t84-85 (Abstract). Rinaman, L., C.E. Milligan, and P. Levitt. Persistence of Fluoro-Gold following degeneration of labeled motoneurons is due to phagocytosis by microglia and macrophages. Neurosci. 44:765-777. Rothman, T.P., and M.D. Gershon (1982) Phenotypic expression in the developing murine enteric nervous system. J. Neurosci. 2381-393. Rothman, T.P., G. Nilaver, and M.D. Gershon (1984) Colonization of the developing murine enteric nervous system and subsequent phenotypic expression by the precursors of peptidergic neurons. J. Comp. Neurol. 22513-23. Schmalbruch, H. (1984) Motoneuron death after sciatic nerve section in newborn rats. J. Comp. Neurol. 224t252-258. Shapiro, R.E., and R.R. Miselis (1985) The central organization of the vagus nerve innervating the stomach of the rat. J. Comp. Neurol. 238t473-488. Smith, D.E. (1974) The effect of dederentation on the postnatal development of Clarke’s nucleus in the kitten-a Golgi study. Brain Res. 74t119-130. Snider, W.D., and S. Thanedar (1989) Target dependence of hypoglossal motor neurons during development and in maturity. J. Comp. Neurol. 279:489498. Szereda-Przestaszewska, M. (1985) Retrograde degeneration within the dorsal motor vagal nucleus following bilateral vagotomy in rabbits. A d a Anat. 121:133-139. Van der Loos, H., and T.A. Woolsey (1973) Somatosensory cortex: structural alterations following early injury to sense organs. Science 179:395-398. Yoshida, J., E.H. Polley, L.M. Nyhus, and P.E. Donahue (1988) Labeling of nerve cells in the dorsal motor nucleus of the vagus of rats by retrograde transport of Fluoro-Gold. Brain Res. 455: 1-8.
Access to Gastric Tissue Promotes the Survival of Axotomized Neurons in the Dorsal Motor Nucleus of the Vagus in Neonatal Rats LINDA RINAMAN AND PAT LEVITT Department of Anatomy and Neurobiology, The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129
ABSTRACT Lesioning the vagus nerve in the neck (cervical vagotomy) results in a rapid and virtually complete loss of motoneurons in the dorsal motor nucleus of the vagus in neonatal rats. The present study sought to determine whether access to gastric target tissue will promote the survival of these motoneurons after axotomy. Quantitative analysis demonstrates that subdiaphragmatic vagotomy, which leaves the cut vagal axons in close proximity to their normal gastric targets, results in significantly less motoneuron loss than cervical vagotomy. Furthermore, the loss of motoneurons after cervical vagotomy can be significantly reduced by transplanting embryonic gastric tissue to the neck of vagotomized neonatal host rats, in the vicinity of the cut axons. The survival effect of transplanted gastric tissue appears specific because control transplants of embryonic bladder tissue fail to reduce motoneuron death after cervical vagotomy. Injections of the neural tracers Fluoro-Gold and cholera toxin-horseradish peroxidase into gastric transplants labeled surviving motoneurons in cervically vagotomized rats, whereas tracer injections into bladder transplants or into host cervical tissues did not. These results indicate that neonatal vagal motoneurons are capable of making the adjustments necessary to survive axotomy if they have access to gastric target cells. The apparent dependence of injured neonatal vagal motoneurons on gastric tissue offers a new system in which to examine in vivo the trophic interactions between neurons and their targets. Key words: vagotomy, gastrointestinal, degeneration,transplantation
Many factors influence neuronal survival during embryonic and early postnatal development, including the establishment and maintenance of connections between neurons and their targets (Landmesser and Pilar, '78;Cunningham, '82; Cowan et al., '84). It is a common observation that both somatic and autonomic motoneurons die when they are separated from their peripheral targets by axotomy during the early postnatal period (LaVelle, '73; Schmalbruch, '84; Jones and LaVelle, '86; Comans et al., '88; Snider and Thanedar, '89; Crews and Wigston, '90; Rinaman and Levitt, '90; Rinaman et al., in press). For example, an extremely rapid and virtually complete loss of the preganglionic parasympathetic motoneurons in the dorsal motor nucleus of the vagus (DMV) follows cervical vagotomy (lesion of the vagus nerve in the neck) in newborn rats (Rinaman and Levitt, '90; Rinaman et al., '91). Anatomical studies in rat indicate that the axons of DMV motoneurons project to the gastrointestinal enteric nervous system (ENS) without collateralizing or leaving the main trunk of the vagus nerve prior to reaching the gastroesophageal junction O
1991 WILEY-LISS, INC.
(Fox and Powley, '85; Bieger and Hopkins, '87; Altschuler et al., '89, '91; Miselis et al., '91). DMV motoneurons may die subsequent to cervical vagotomy because they are deprived of neurotrophic substances manufactured by neuronal and/or nonneuronal cells in the gut. The regressive response of immature DMV motoneurons to axotomy may reflect the continuation of an embryonic dependence on specific target derived trophic factors, as described in other systems (Cowan, '73; Oppenheim, '81, '85; Cunningham, '82; Cowan et al., '84; Hill, '85; Haun and Cunningham, '87;Oppenheim et al., '88; Crews and Wigston, '90). In addition to the detrimental effects of target deprivation, it has also been suggested that neonatal motoneurons are especially vulnerable to axotomy because they are already synthesizing proteins at peak capacity and may not Accepted August 5,1991. Address reprint requests to Dr. Linda Rinaman, Dept. of Anatomy and Neurobiology, The Medical College of Pennsylvania, EPPl Division, 3200 Henry Ave., Philadelphia, PA 19129.
L. RINAMAN AND P. LEVITT
214 be able to mount an adequate regenerative response to injury (LaVelle, '83; Jones and LaVelle, '86). For example, cervical vagotomy amputates a relatively large portion of each DMV motoneuron's axon, a factor that has been shown in other systems to contribute to the rapidity and severity of the lesion response (for reviews see Cragg, '70; Lieberman, '71; Grafstein, '75). Axotomized neonatal DMV neurons may die because they are simply incapable of making the metabolic adjustments necessary to survive the lesion. The present study was designed to test the hypothesis that neonatal DMV motoneurons can survive axotomy provided they have access to an appropriate peripheral target. We first compare the effects of cervical vagotomy and subdiaphragmatic vagotomy (in the abdomen) on the loss of DMV motoneurons to determine whether they can survive a lesion made further from the neuronal cell body and closer to their normal gastric target cells. We then examine whether DMV motoneurons can survive the more severe cervical lesion if their injured axons have access to gastric tissue transplanted to the site of nerve damage.
MATERIALS AND METHODS Animals and anesthesia The embryonic or neonatal offspring of Sprague-Dawley rats (Harlan) were used in all experiments. Rats were housed in an AAALAC licensed animal facility, and their care and treatment adhered to NIH guidelines and the Animal Welfare Act. All rats were deeply anesthetized prior to surgery and sacrifice. Newborn rats [postnatal day 1 (Pl), where PO = day of birth] were ice anesthetized (rapid hypothermia) prior to surgery and were revived by slow warming under a heating lamp. Rats aged P7 to P30 at sacrifice were anesthetized with ketaminelxylazine [five parts ketamine (100 mgsiml) plus one part xylazine (20 mgs/ml); 200 pl/lOO g body weight, ipl. Rats older than P30 at sacrifice were anesthetized with Nembutal (300 p1/ 100 g body weight, ip).
Abbreviations
AChE AP cc
CNS
CT-HRP Cu DMV E ENS eP EPSP FG gP
Gr
H&E HYP IP
lU me NST P
PT se sm SPT te Tr
acetylcholinesterase area postrema central canal central nervous system cholera toxin-horseradishperoxidase cuneate nucleus dorsal motor nucleus of the vagus embryonic day enteric nervous system enteric plexus excitatory postsynapticpotential Fluoro-Gold gastric pits gracile nucleus hemotoxylin and eosin hypoglossal nucleus lamina propria lumen muscularisexterna nucleus of the solitary tract postnatal day pyramidal tract surface epithelium submucosa spinal nucleus of the trigeminal nerve transitional epithelium solitary tract
Fig. 1. Schematic diagram of a typical cross section through the rat medulla oblongata at the level of the area postrema (caudal to the opening of the 4th ventricle). The micrographs in Figures 2,3, and 6-8 are of tissue sections from approximately the same rostrocaudal level and include the area indicated by the hatched box in this schematic. Left and right are as depicted. Arrowheads point out the left DMV, which is axotomized in Figures 2, 3, and 6-8. AP, area postrema; cc, central canal; Cu, cuneate nucleus; DMV, dorsal motor nucleus of the vagus; Gr, gracile nucleus; H Y P , hypoglossal nucleus; NA, nucleus ambiguus; NST, nucleus of the solitary tract; PT, pyramidal tract; SpT, spinal nucleus of the trigeminal nerve; Tr, solitary tract (adapted from Paxinos and Watson, '86). Scale bar = 500 Fm.
Newborn (Pl) rats were placed in one of four experimental groups: (1)cervical vagotomy; no transplant (n = 13); (2) subdiaphragmatic vagotomy; no transplant (n = 8); (3) cervical vagotomy; gastric transplant (n = 18),(4)cervical vagotomy; bladder transplant (n = 11).
Transplant procedures Transplantation of fetal gastric tissue was partially based on a preliminary report demonstrating the survival and development of embryonic rat gastric tissue transplanted to the forehead of host rats (Horgan and Van der Kooy, '89). In the present study, rat fetuses at 17 days of gestation (E17) were removed from the uterine horns, placed in sterile ice cold F-10 Nutrient Mixture (Gibco), and decapitated. Tissue samples (approximately one mm3)from donor embryos were collected from the antral region of the ventral stomach wall or from the bladder wall and were placed in separate containers of fresh ice cold F-10. The antral region of the ventral stomach wall contains a concentrated population of enteric neurons that receive direct innervation specifically by motoneurons in the left DMV in adult rats (Shapiro and Miselis, '85; Kirchgessner and Gershon, '89). Enteric neurons and well defined nerve plexuses are already present in the stomach wall by E l 7 (Rothman and Gershon, '82; Rothman et al., '84). The bladder does not receive vagal innervation; the bladder's postganglionic parasympathetic
Fig. 2. Nissl stained sections through the medullas of four vagotomized rats, none of which received tissue implants. Arrowheads point out the axotomized DMV. A: Two weeks after left cervical vagotomy, there is virtually complete loss of DMV motoneurons. B: Four weeks after left cervical vagotomy, motoneuronal loss is similar to that seen (in A) after two weeks. C: Two weeks after ventral subdiaphragmatic vagotomy, a large number of DMV motoneurons remain on the lesioned side, although their number is reduced compared to the intact side. D: Four weeks after ventral subdiaphragmatic vagotomy, motoneuronal loss on the lesioned side remains incomplete. cc, central canal. Scale har in A = 500 ym, applies also to B-D.
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS
Figure 2
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neurons are innervated by preganglionic parasympathetic motoneurons in the sacral spinal cord (Nyberg-Hansen,'66). In the P1 rats to be implanted with gastric or bladder tissue, the ventral surface of the neck was swabbed with 70% ethanol and a short midline incision made. With the aid of a dissecting microscope, the skin was retracted and the left carotid artery was exposed at the level of the omohyoid muscle by blunt dissection. One piece of embryonic gastric or bladder tissue was implanted medial to the carotid artery, and the left cervical vagus nerve was then sectioned (see below). No more than 10 minutes elapsed between dissection of the embryonic tissue samples and implantation into P 1 hosts.
Vagotomy procedures For cervical vagotomy, the left vagus nerve was exposed at the level of the omohyoid muscle, gently separated from the carotid artery, and severed. In rats that received a fetal gastric or bladder tissue transplant, the proximal end of the severed vagus nerve was placed directly onto the surface of the transplant. In some of the P1 rats that were cervically vagotomized with no transplant, the left vagus nerve was first thoroughly crushed with fine forceps and 2-4 p1 of Fluorogold (FG) neural tracer (Fluorochrome International; 0.5% in sterile saline) was injected into the nerve at the crush site to label axotomized DMV motoneurons retrogradely. Assurance of complete axotomy was achieved by subsequently cutting the vagus nerve just distal to the FG injection site. FG injections were made with a pulled glass micropipette (20 pm tip diameter) affixed to the tip of a 10 p1 Hamilton syringe. Neck incisions were sutured with 7-0 silk (Ethilon). For subdiaphragmatic vagotomy, the abdomen was swabbed with 70% ethanol and a ventral midline incision was made through the skin and abdominal musculature. The gastroesophageal junction was exposed by careful retraction of the liver. With the aid of a dissecting microscope, the ventral vagus nerve (the distal extension of the left cervical vagus nerve) was identified in its course along the esophagus and was cut at the gastroesophageal junction, just above the point where gastric vagal branches ramify. Abdominal incisions were sutured in two layers with 7-0 silk (Ethilon).
Tracer injection into tissue transplants In some rats that received a gastric or bladder transplant, FG or cholera toxin-horseradish peroxidase (CT-HRP; donated by R.R. Miselis) was injected into the transplant two days prior to sacrifice. As a control, some of the rats that were cervically vagotomized with no transplant received FG or CT-HRP tracer injections into the muscles and other host tissues surrounding the site of cervical vagotomy. In each rat, 10-15 ~1 of CT-HRP (0.2% in sterile saline) or FG (0.5% in sterile saline) was injected in multiple 1-2 p1 aliquots. Injections were made through a pulled glass micropipette (tip diameter 25-40 pm) secured to a 10 p1 Hamilton syringe with sealing wax. Although the membrane binding properties of CT-HRP limit its diffusion from sites of injection (discussed in Rinaman and Miselis, '87), the following measures were also taken to further ensure specificity of CT-HRP labeling: the injecting micropipette was held in place for approximately one minute at each injection site, and each site was then swabbed with bits of gauze soaked in sterile saline. Following the final CT-HRP injection, the entire cervical area was flooded three times with warm sterile saline and then blotted with gauzes prior
TABLE 1. Effect of Vagotomy and Transplant Procedures on Loss of Neonatally Axotomized DMV Motoneurons Fourteen Days Postlesion'
Group
Total number of neurons in lesioned DMV
Total number of neurons in intact DMV
1. Cervical Vagotomy, no Transplant (rat i.d.#) 89-275 7 9047 13 90-68 27 90-69 14
% Loss of axotomized DMV neurons [(intact-lesioned) x loo%] intact
390 492 570 515 AverageLoss 2. Subdiaphragmatic Vagotomy, no Transplant (rat i.d.#) 90-76 193 439 90-77 360 481 90-78 136 551 90-79 101 463 Average Loss 2 3. Cervical Vagotomy, Gastric Transplant (rat i.d.#) 90-16 304 670 90-17 112 384 90-19 203 556 90-106 66 551 Average Loss z 4. Cervical Vagotomy, Bladder Transplant (rat id.#) 90-95 25 367 90-100 16 526 90-101 7 485 90-102 16 502 AverageLoss 2
*
98.26 loss 97.46 95.3% 97.37" S.D.: 97.1 i 1.2% 56.0%loss 25.2% 75.3% 78.2% S.D.:58.7 t 24 4% 54.6%loss 71.0% 63.56 88.0% S.D.: 69.3 ? 14.2% 93.28 loss 97.08
99.0% 96.88 S.D.: 96.5 2 2.496
'Statistical comparisons were made between groups using the Mann-Whitney U Test. Significant differences (p s 0 05) in DMV montoneuronal loss were found between Groups 1and 2 , Groups 1and 3, Groups 2 and 4, and Groups 3 and 4.
to suturing the cervical incision. No special precautions were taken to limit spread of FG, which is known to diffuse from peripheral injection sites (Yoshida et al., '88) and to be picked up by intact peripheral fibers in neonatal rats (Leong and Ling, '90; Rinaman et al., in press; see Discussion).
Histological preparation One to eight weeks after vagotomy, rats were anesthetized and sacrificed by transcardiac perfusion fixation. In rats injected with CT-HRP, perfusates consisted of an initial saline rinse followed by infusion of ice cold phosphate buffer containing 1% paraformaldehyde and 2.5%glutaraldehyde (pH 7.41, followed by infusion of 10% buffered sucrose. Brainstems were removed and cryoprotected in 30% buffered sucrose. For all other rats, the initial saline rinse was followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). These brainstems and transplant tissues were postfixed in situ for 24 hours at 4"C, then removed and cryoprotected in 30%buffered sucrose. Medullas were sectioned in the coronal plane with a sliding microtome at 40 pm intervals throughout the rostrocaudal extent of the DMV, which in rats extends from the inferior olive to C2-C3 of the spinal cord. Tissue sections used for Nissl analysis were mounted on gelatin coated slides, air dried, and stained with cresyl violet. Tissue sections used for localization of transported FG were mounted, air dried in the dark, and coverslipped using GelMount (Biomeda). Some sections containing FG were temporarily coverslipped with buffer and photographed for FG localization prior to staining with cresyl violet. Tissue sections used for localization of transported CT-HRP were enzymatically treated using a variation of Mesulam's ('78) tetramethyl benzidine procedure (Rinaman and Miselis, '87), mounted on gelatin coated slides, rapidly dehydrated
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS
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Fig. 3. Tissue section from a cervically vagotomized rat in which the left vagus nerve was injected with FG at the time of vagotomy (two week survival). Arrowheads delineate the axotomized DMV. In A, the section is stained for Nissl substance to reveal an apparently complete loss of DMV motoneurons on the side of the lesion. In B, the same tissue section is viewed with fluorescence (prior to Nissl staining) to demonstrate loss of FG labeled neurons in the left DMV. The small,
very bright cells in the axotomized DMV are non-neuronal macrophages, including microglia (see text). As no attempt was made to restrict peripheral diffusion of the FG from the injection site, almost all of the Nissl stained motoneurons in the intact (right) DMV are also lightly labeled with FG. This makes evident the high correlation between neurons identified with FG labeling and with Nissl staining (compare A and B). cc, central canal. Scale bar in A = 500 pm, applies also to B.
and coverslipped with DPX. Some HRP processed tissue sections were stained with cresyl violet prior to coverslipping. Gastric and bladder transplant tissues were cryostat sectioned at 16 km intervals. Cryostat sections through the tissue transplants were stained with hemotoxylin and eosin (H&E) according to routine procedures, or were processed for acetylcholinesterase (AChE) localization (Lewis, '61).
analysis of the number of Nissl stained DMV motoneurons was restricted to rats surviving two weeks after vagotomy. A two week survival period was chosen because cell loss in the lesioned DMVs of cervically vagotomized rats, including those receiving gastric and bladder tissue transplants, reached a plateau by this time that was maintained for up to eight weeks (see Results). Longer survival times were not examined. Medullary tissue sections from the rostrocaudal level of the DMV containing the midregion of the area postrema (four 40 krn thick sections per rat) were used for DMV cell counts, as the cytoarchitectural boundaries of the DMV tend to become obscure at more rostral and caudal levels of the nucleus. At the level of the area postrema, the DMV is
Quantitative analysis of DMV motoneuronal survival Tissue sections were examined from rats in all four groups surviving between one and eight weeks after vagotomy. For statistical comparisons, however, quantitative
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Fig. 4. H&E stained sections through a typical gastric transplant (two week survival) demonstrating the healthy, histological differentiation of the tissue and its incorporation into the host cervical area. In A, a low magnification micrograph reveals the general organization of the transplant. Note the well developed gastric pits (gp), lamina propria (lp) and muscularis externa (me). The large arrow (boxed B) indicates an
area which is enlarged in B. At this magnification, well differentiated mucous secreting cells are evident in the gastric pits and gastric glands (gg). C is from another area of the same transplant showing gastric glands and pits in cross section, surrounded by the lamina propria. lu, lumen; se, surface epithelium of gg; sm, submucosa. Scale bar in A = 500 km; B and C X 4.
bounded ventrally by a thin plate of glial cells (visible in Nissl stained tissue) separating it from the underlying hypoglossal nucleus, dorsally by small cells of the medial subnucleus and the subnucleus centralis of the NST, laterally by an imaginary line drawn ventrally from the solitary tract, and medially by the central canal. Tissue ~ sections were analyzed microscopically with a 1 0 objective. DMV motoneurons were identified as large cells with a distinct Nissl stained cytoplasm and proximal dendritic projections. Well defined nuclear profiles of these cells were counted, as the average nuclear diameter of surviving axotomized neurons in the lesioned DMV did not differ significantly from the average nuclear diameter of motoneurons in the intact DMV. For each rat, cell counts from the intact and vagotomized sides of each section were totaled and the percent reduction of DMV neurons on the lesioned side compared to the unlesioned side was calculated {[(intactlesioned)/intact] x loo%]. Statistical differences between DMV motoneuron loss in the four experimental groups were determined using the Mann-Whitney U Test, with p values I 0.05 considered significant.
RESULTS Cervical vagotomy, no transplant Severing the left cervical vagus nerve in P1 rats consistently resulted in an almost complete loss of Nissl stained motoneurons in the ipsilateral DMV by two weeks postlesion (Fig. 2A,B; see Fig. 1 for histological orientation). Quantitative analysis revealed an average 97.1% reduction in the number of DMV neurons on the lesioned vs. intact side in each rat (Table 1). Examination of FG labeling and Nissl staining in the same tissue sections revealed that counts of Nissl stained motoneurons in the intact and lesioned DMV match well with counts of FG labeled neurons (Figs. 3, 8; see Discussion). This verified that the apparent loss of DMV motoneurons was an actual loss and was not due to morphological alterations of the axotomized DMV neurons that would prevent their identification in Nissl stained tissue. The small, very bright FG labeled cells visible in the lesioned DMV in Figure 3 have been immunocytochemically identified as nonneuronal macrophages, including microglia, that phagocytose the FG when the
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS
219
Fig. 5. AChE stained tissue section through a typical gastric transplant (two week survival). Many AChE positive nerve fibers and enteric neurons are present in the transplant. The large arrow in A (boxed B) indicates an area that is enlarged in B. ep, enteric plexus; n, neurons in plexus. Scale bar in A = 500 pm;
B x 4.
labeled motoneurons die (Rinaman et al., '91; see also Crews and Wigston, '90). Control injections of FG or CT-HRP into the internal neck muscles and other host tissues surrounding the site of cervical vagotomy in these rats never resulted in labeled neurons in the axotomized DMV. Light FG labeling of motoneurons in the intact (right) DMV and in both hypoglossal nuclei was always observed following FG injection of the lesioned vagus nerve (Fig. 3) or injection of host cervical
tissues, consistent with FG's propensity to spread from peripheral injection sites (see Methods). In contrast, CTHRP labeling of neurons in the intact DMV or hypoglossal nuclei was never seen following injection into host cervical tissues, consistent with the lack of peripheral spread of CT-HRP or its uptake by intact nerve fibers (see Methods). Positive, within animal controls for the neuronal uptake and transport of both tracers was provided by the presence of labeled motoneurons in the ipsilateral nucleus ambiguus
Figure 6
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS
Fig. 7. Two alternate tissue sections from a cervically vagotomized rat with gastric tissue transplant (four week survival) that was injected with CT-HRP two days prior to sacrifice. Arrowheads point out the axotomized DMV. A is HRP-treated and Nissl-stained; B is HRPtreated only. The motoneurons that survived cervical vagotomy are
221
retrogradely labeled following CT-HRP injection into the gastric transplant tissue. Anterogradely labeled vagal sensory axons and terminals are also evident in the dorsally situated NST. cc, central canal. Scale bar in A = 500 pm, applies also to B.
and ventral horn of the cervical spinal cord which are known to innervate muscles in the cervical region.
demonstrated many positive nerve fibers and neurons in well defined enteric plexuses (Fig. 5). Rats with gastric transplants consistently had many Subdiaphragmatic vagotomy, no Nissl stained motoneurons remaining in the axotomized Severing the ventral subdiaphragmatic vagus nerve at DMV two weeks after cervical vagotomy (Fig. 6A; Fig. BA). the gastroesophagealjunction resulted in an average 58.7% Quantitative analysis revealed an average 69.3% reduction reduction of Nissl stained motoneurons in the axotomized of neurons in the lesioned DMV as compared to the intact DMV as compared to the intact DMV at two weeks postle- DMV in these rats (Table l),a significantly smaller cell loss sion (Table 1; Fig. 2C). Although DMV cell loss in subdia- than in rats that were cervically vagotomized but received phragmatically vagotomized rats was more variable than in no transplants. Qualitatively, DMV cell loss in rats with the other three groups (see Table 11, it was significantlyless gastric transplants appeared somewhat greater than DMV than DMV cell loss in cervically vagotomized rats that cell loss in subdiaphragmatically vagotomized rats, but the received no transplant. Examination of tissue sections from difference was not statistically significant. The effect of the subdiaphragmatically vagotomized rats surviving for longer gastric tissue transplant on DMV cell survival remained periods revealed a similar between animal variability in evident in rats surviving for longer periods (Fig. 6B; Fig. 7), with many Nissl stained motoneurons remaining in the DMV cell loss (Fig. 2D). axotomized DMV as long as eight weeks after vagotomy. Cervical vagotomy, gastric transplant Injection of CT-HRP into the gastric transplants rouAs early as one week after transplantation into P1 host tinely resulted in retrograde labeling of surviving DMV rats, gastric transplant tissues were vascularized, had motoneurons and their dendritic processes on the lesioned grown in size, and had well delineated luminal cavities. side, as well as anterograde (transganglionic) labeling of Cervically vagotomized rats with gastric transplants devel- vagal sensory fibers in the nucleus of the solitary tract oped externally visible enlargements on the left ventrolat- (NST; Fig. 7). CT-HRP labeled neurons were never oberal side of the neck caused by growth of the transplant served in the contralateral (intact) DMV, but labeled motissue. Examination of H&E stained cryostat sections toneurons were occasionally observed in the ipsilateral through the transplants revealed gastric rugae and other nucleus ambiguus and ventral horn of the cervical spinal morphological features typical of gastric tissue (Fig. 4). cord, likely resulting from tracer injection into the host Examination of AChE stained gastric transplant sections muscles underlying the transplant tissue. As with CT-HRP injection, injection of FG into the gastric transplants labeled surviving motoneurons in the axotomized DMV (Fig. 8 ) as well as motoneurons in the nucleus ambiguus and spinal cord. Unlike CT-HRP, FG also retrogradely Fig. 6. Nissl stained tissue sections through the medullas of four cervically vagotomized rats. Arrowheads denote the axotomized DMV. labeled motoneurons in the intact DMV due to tracer spread. A With; gastric tissue implant, two week survival, a moderate number of DMV motoneurons remain on the lesioned side. B: DMV motoneurons are also evident on the lesioned side after four weeks in a rat that received a gastric tissue implant. C: With a bladder tissue implant, two week survival, loss of DMV motoneurons on the lesioned side appears complete. D: Loss of axotomized DMV neurons is evident in another rat with a bladder transplant, four weeks post lesion. cc, central canal. Scale bar in A = 500 i m , applies also to B-D.
Cervical vagotomy, bladder transplant Examination of H&E stained sections through bladder growth, vascularization, and morphology typical of bladder tissue (Fig. 9). AChE positive nerve fibers were present in and around the smooth muscle
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L. RINAMAN AND P. LEVITT
Fig. 8. Alternate tissue sections from a cervically vagotomized rat that received a gastric tissue implant (two week survival). The gastric tissue implant was injected with FG two days prior to sacrifice. Arrowheads point out the axotomized DMV. A: Nissl stained section
reveals surviving DMV motoneurons on the side of the lesion. B: Fluorescence view shows motoneurons retrogradely labeled with FG in the lesioned DMV. cc, central canal. Scale bar in A = 500 pn, applies also to B.
bundles in the transplant tissue, and a small number of AChE positive neurons were observed in clusters of 3 or 4 in the smooth muscle and adventitia. In marked contrast to the increased DMV cell survival seen in cervically vagotomized rats with gastric transplants, rats with bladder transplants exhibited almost complete loss of cervically axotomized DMV neurons (Fig. 6C,D). Quantitative analysis revealed an average 96.5% reduction in Nissl stained DMV motoneurons on the lesioned side (Table l), statistically no different than the DMV cell loss seen in rats that were cervically vagotomized with no transplant tissue. Injection of FG or CT-HRP into bladder tissue transplants never labeled neurons in the axotomized DMV. However, both tracers labeled neurons in the other locations described above following injections into host cervical tissues and gastric tissue transplants, providing a positive control for tracer transport.
DISCUSSION This study demonstrates that neonatal rat DMV motoneurons can survive axotomy if they have access to gastric target tissue. The survival effect of gastric tissue lasts for at least two months, by which time the neonatally lesioned rats have reached maturity. As longer survival times were not examined, it is not known whether the apparent rescue of these axotomized neurons is permanent. In addition, although a significant number of axotomized DMV neurons are “rescued” in the presence of gastric tissue, many of the axotomized DMV neurons in subdiaphragmatically lesioned rats, and in cervically lesioned rats with gastric transplants, do not survive. Survival may be incomplete for a number of reasons, possibly including abberrant regenerative vagal sprouting (see Evans and Murray, ’54; King and Thomas, ’71).
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS
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Fig. 9. H&E stained section through a typical bladder transplant (two week survival) demonstrating the healthy, histological differentiation of the tissue and its incorporation into the host cervical area. In A, a low magnification view depicts well organized tissue, including a
surface epithelium surrounding the lumen (lu), extensive lamina propria (lp), and smooth muscle (sm) bundles. The area depicted by a large arrow and a box is enlarged in B, where transitional epithelium (te) typical of the bladder is seen. Scale bar in A = 500 +m; B x 4.
It is difficult to know whether the survival of neonatal DMV motoneurons following subdiaphragmatic vagotomy is attributable more to enhanced access to gastric targets or to reduced lesion severity. While the distance of the lesion from the neuronal cell body certainly cannot be excluded as a contributing factor (LaVelle, '83; Jones and LaVelle, '86; see also Murphy et al., 'go), the closer proximity of the severed axons to appropriate gastric target cells and presumably to some survival promoting factor(s) associated with
those cells appears to play an important role. This is supported by our demonstration that the normally rapid and virtually complete loss of cervically axotomized neonatal DMV motoneurons can be significantly reduced by transplanting gastric tissue to the site of cervical vagotomy. DMV cell loss in cervically vagotomized rats with gastric transplants does not differ significantly from DMV cell loss in subdiaphragmatically vagotomized rats. The ability of gastric tissue to promote the survival of axotomized DMV
224 motoneurons appears specific, because access to transplanted bladder tissue (another parasympathetically innervated viscus) provides no survival effect.
Possible trophic interactions between DMV motoneurons and gastric target cells In certain respects, DMV motoneurons are more closely akin to intrinsic central neurons than to somatic motoneurons in that their axons terminate not on muscle cells but on other neurons (see Lewis et al., '72). The postsynaptic targets of DMV motoneurons are neurons in the ENS. The ENS is a complex and independent nervous system containing a diversity of intrinsic neuronal types and has many structural and morphological features in common with the CNS (Gabella, '87; Kirchgessner and Gershon, 'go), including a blood-myenteric plexus barrier that may be functionally analogous to the CNS' blood-brain barrier (Gershon and Bursztajn, '78; Gershon et al., '79). The peripheral axons of vagal neurons normally invade the blood-myenteric barrier, allowing access to and possible retrograde transport of unique trophic substances secreted by neuronal and/or non-neuronal gastric cells. It has been repeatedly postulated that trophic substances produced by target cells are critical to the development and survival of the neurons that innervate those targets (Cowan, '73; Landmesser and Pilar, '78; Oppenheim, '81, '85, '88; Cunningham, '82; Gordon, '83; Bijlsma et al., '84; Cowan et al., '84; Hill, '85; Crews and Wigston, '90). The new observation that gastric tissue (but not bladder or host cervical tissues) decreases the loss of axotomized DMV neurons suggests that these motoneurons also depend on specific interactions with their appropriate target cell populations for survival during the early postnatal period. The specificity of the interaction between DMV motoneurons and gastric tissue suggests that more general substances produced by host tissues in the cervical region or by smooth muscle and other cells in the bladder are insufficient in promoting the survival of axotomized vagal motoneurons. CT-HRP and FG tracer injection into gastric transplants labeled motoneurons in the lesioned DMV, whereas tracer injections into bladder transplants or host cervical tissues did not. This indicates that the proximal ends of the severed axons were present and capable of retrograde transport in rats with a gastric transplant, but not in rats with a bladder transplant or with no transplant. We did not determine whether vagal axons actually regenerated to contact neurons or other cells in the gastric transplants, but the presence of AChE positive enteric ganglia allows for the possibility that DMV-ENSinnervation can be established in this situation. It should be kept in mind that the putative acquisition of specific target derived trophic factors may by itself be only partially responsible for producing the observed survival effect on injured DMV motoneurons. Access to gastric target tissue following axotomy may simply be one requisite step in a more complex process that ultimately leads to the reestablishment of normal central synaptic inputs, producing a balance that is itself essential for motoneuronal survival (Cunningham, '82; Hill, '85; Oppenheim, '89; Snider and Thanedar, '89). Further in vivo and in vitro experiments should be able to specifically address this issue.
L. RINAMAN AND P. LEVITT
Speculations on a role for sensory deafferentation in the response of axotomized DMV motoneurons Concurrent damage to vagal sensory neurons necessarily occurs during vagotomy, and may be a key factor in the inability of target deprived DMV motoneurons to recover from the lesion. The availability of a proper peripheral target after subdiaphragmatic vagotomy or after cervical vagotomy with gastric transplants may enhance the survival of axotomized DMV motoneurons partly because these procedures enhance the survival of the axotomized sensory neurons that normally terminate centrally on the motoneurons. Injection of CT-HRP into the gastric tissue transplants anterogradely (transganglionically) labeled vagal sensory axon terminals in the NST, but vagal sensory labeling was not seen after injection of CT-HRP into bladder transplants. One can speculate that target specific factors also promote the survival of vagal sensory neurons after axotomy. This raises an interesting question: in addition to a possible dependence on intact inputs from CNS neurons as mentioned in the preceding paragraph, might injured DMV motoneurons also depend on inputs from vagal sensory neurons? Physiological studies have shown that intact vagal sensory input is necessary for the normal tonic activity of DMV motoneurons. Vagotomy causes the immediate cessation of spontaneous action potentials in DMV motoneurons, although EPSPs can still be recorded (Barber and Burks, '83). Vagal sensory inputs drive DMV motoneurons not only through central disynaptic and polysynaptic circuits, but also through direct monosynaptic contacts on DMV motoneuronal dendrites (Neuhuber and Sandoz, '86; Rinaman et al., '89). Damage to vagal sensory neurons, therefore, alters or removes a significant source of afferent drive and potential anterogradely supplied trophic support to DMV motoneurons. Transsynaptic degeneration of certain populations of deafferented central neurons is a well known phenomenon in developing animals (Cowan, '70; Van der Loos and Woolsey, '73; Smith, '74; Cunningham, '82; Okado and Oppenheim, '84; Furber et al., '87). It will be intriguing to discover whether DMV motoneurons also exhibit a dependence on their vagal sensory innervation for survival.
Speculations on DMV cell loss following vagotomy in mature animals Most mature somatic motoneurons respond to axotomy with chromatolysis and dendritic atrophy, but their axons regenerate and the neurons recover (Cragg, '70; Lieberman, '71; Grafstein, '75; Baker et al., '81; Bijlsma et al., '84; Murphy et al., '90). In fact, at least some populations of mature somatic motoneurons survive even when they are not allowed to reinnervate their normal muscular targets (Acheson et al., '42; Cull, '74; Goldring et al., '80; Snider and Thanedar, '89), suggesting that they no longer require specific target derived factors for survival. In marked contrast, it has been known for more than 100 years that the majority of mature DMV motoneurons ultimately atrophy and disintegrate following cervical vagotomy in a variety of mammalian species (Dees, 1889;Geist, '33; Lewis et al., '70, '72; Sumner and Watson, '71; Sumner, '75; Aldskogius, '78; Aldskogius et al., '80; Szereda-Przestaszewska, '85; Laiwand et al., '87; Ling et al., '87). The uniquely necrobiotic reaction of mature DMV motoneurons to axotomy may reflect a continuation of the developmental target
GASTRIC TISSUE RESCUES AXOTOMIZED DMV NEURONS dependence suggested by the present work. Further experiments should be able to demonstrate whether access to gastric tissue reduces the incidence of DMV motoneuronal death following vagotomy in mature rats. In conclusion, we have demonstrated that neonatal DMV motoneurons can survive axotomy if they are provided access to appropriate peripheral target cells, irregardless of the distance of the lesion from the motoneuronal cell body. The vagal visceral circuit is readily accessible for further experimental manipulations aimed at determining the specific contributions of its central and peripheral components on the development and long term survival of its constituent neurons, as well as to define and assay the potential macromolecular candidates that mediate these effects.
ACKNOWLEDGMENTS This work was supported by NIMH grant MH45507, NIH Training Grant lT32NS07287, and NRSA Grant 1F32NS08900. We are grateful to Drs. Mary Barbe, Pat Card, Tim Cunningham, Michael Goldberger,Forrest Haun, Tim Himes, Hazel Murphy, and Alan Tessler for their insightful comments on the manuscript.
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