THE ANATOMICAL RECORD 248:129–136 (1997) Ultrastructural Changes in the Nerve Fiber Population of Anastomosed Vagal and Spinal Accessory Nerves in the Sheep PIERRE DELORME,1* ANTOINETTE ROUSSEAU,2 JACQUELINE BERNARD,1 BARRY F. LEEK,3 AND JEAN-PAUL ROUSSEAU2 1Laboratoire de Neurobiologie Fonctionnelle, UST Lille, Villeneuve d’Ascq Cedex, France 2Laboratoire de Physiologie de la Reproduction, C.N.R.S./I.N.R.A., Université Pierre et Marie Curie, Paris Cedex, France 3Department of Veterinary Physiology and Biochemistry, University College of Dublin, Dublin, Ireland ABSTRACT Background: The ultrastructure of the vagal and spinal accessory nerves was studied 1) in normal sheep and 2) in sheep in which an experimental crossed-nerve anastomosis had been made by sectioning the supranodose vagal and spinal accessory nerves, then suturing the distal end of the vagal nerve to the distal end of the spinal accessory nerve, and allowing time for regeneration to occur. This study was carried out in order to analyze the modifications liable to occur when this technique is used and to specify the origin and the nature of the fibers that colonize the spinal accessory nerve. Methods: The study was performed in 4- to 5-month-old sheep. After the surgical procedure, the animals were housed indoors during 1 year until their sacrifice by fixative perfusion. Then, nerve samples were dissected out, processed for electron microscopy, examined, and systematically photographed. After printing, the diameters of the nerve fibers were determined. Results: In sheep, the ratios of nonmyelinated to myelinated fibers (NF/MF) in the infranodose and supranodose vagal nerve and accessory spinal nerve were 1.21, 1.67, and 3.21, respectively. In both parts of the vagal nerve, the myelinated fibers had a unimodal diameter distribution around a peak of 4 mm; whereas, in the spinal accessory nerve, they were distributed bimodally, and 53% had values of 15–18 mm. After making the above anastomosis, the centrifugal vagal fibers degenerated, and the NF/MF ratios increased in the centripetal infranodose vagal nerve, in the reinnervating supranodose vagal nerve, and in the reinnervated spinal accessory nerve (approximately 1.87, 1.72, and 6.04, respectively). In all of these nerves, the myelinated fibers had a unimodal distribution with a peak at 4 mm, as in the vagal nerve of normal sheep. Conclusions: These results reveal the large part taken by the nonmyelinated fibers in the nerve fiber population of the vagal nerve and support the vagal origin of the fibers reinnervating the spinal accessory nerve. Anat. Rec. 248:129–136, 1997. r 1997 Wiley-Liss, Inc. Key words: heterogeneous nerve anastomosis; vagal nerve; spinal accessory nerve; ultrastructure; nerve fiber populations To study vagal receptor activity in conscious sheep, Rousseau (1970, 1973) conceived and successfully developed a method of heterogeneous nerve anastomosis between the peripheral stump of a cut supranodose vagal nerve (SVN) and the peripheral stump of the ipsilateral spinal accessory nerve (SAN). Allowing time for nerve regeneration and muscle reinnervation enables centripetal vagal receptor activation to be reflected in activation of motor units of a superficial skeletal muscle in the neck. With this technique (Fig. 1A,B), the peripheral stump of the SVN end, sectioned r 1997 WILEY-LISS, INC. as cranially as possible, is sutured to the peripheral stump of the SAN, the SAN being the motor nerve of the mastoidohumeral muscle in sheep. After an interval of several months, there is partial reinnervation of this denervated muscle by certain centripetal vagal axons. Received January 9, 1995; accepted October 28, 1996. *Correspondence to: Professor Pierre Delorme, Laboratoire de Neurobiologie Fonctionnelle, UST Lille, F.59655 Villeneuve d’Ascq Cedex, France. Contract grant sponsor: M.E.N., and CNRS; Contract grant no: V.A. 308. 130 P. DELORME ET AL. Fig. 1. Vagal and spinal accessory nerves located in the neck of the sheep. Schematic illustrations of the normal anatomical structure (A), the heterogeneous anastomosed nerve after 1 year (B), and the section of the supranodose vagal nerve without anastomosis shown after 40 days (C). The numbers show the nerve samples taken. Their analyses are reported in this order in Table 1. IVN, infranodose vagal nerve; NG, nodose ganglion; SVN, supranodose vagal nerve; SAN, spinal The term ‘‘centripetal vagal axons’’ is used to include afferent axons that constitute the majority of centripetal axons as well as a small number of recurrent contralateral vagal motor axons whose existence was demonstrated by Dalal et al. (1988). Electrophysiological studies have confirmed the functional reality of reinnervation (Coget and Rousseau, 1983; Falempin and Rousseau, 1983). Of the 26,000– 36,000 neurons located in the nodose ganglion, only one afferent vagal axon out of every 600 is able to reestablish neuromuscular junctions (Rousseau and Falempin, 1984). Before determining the ultrastructure of the vagal fibers that reinnervate the SAN, fiber populations of the SVN and the infranodose vagal nerve (IVN) were analyzed. Previously, the vagal nerve of sheep has been the object of light microscopic studies that were unable to reveal the contribution of the nonmyelinated fraction. For this reason, we have analyzed first the normal nerves (IVN, SVN, SAN; Fig. 1A) and, then, the anastomosed nerves [reinnervating centripetal IVN (RCIVN), reinnervated SAN (RSAN); Fig. 1B] and the vagus nerve after the degeneration of the efferent fibers [centripetal IVN (CIVN); Fig. 1C]. accessory nerve; MHM, mastoidohumeral muscle; RCIVN, reinnervating centripetal infranodose vagal nerve; RSAN, reinnervated spinal accessory nerve; CIVN, centripetal infranodose vagal nerve. Long dashed line, vagal centripetal (motor) fibers; Dashed-and-dotted lines, spinal accessory motor fibers; solid lines, vagal centrifugal (sensory) fibers; short dashed line, degenerated fibers. MATERIALS AND METHODS Nerve anastomoses (Fig. 1B) were performed on the left side in 4- to 5-month-old sheep of the ‘‘Préalpes du Sud’’ breed following the procedure described by Falempin and Rousseau (1983). After the surgical procedure, the sheep were housed indoors, allowed free access to water, and fed a diet of meadow hay ad libitum plus 300 g/day of pellets. One year after the anastomosis, the reinnervation of the mastoidohumeral muscle was demonstrated in each sheep by recording the electrical activity of reinnervated motor units, which was evoked by stimulating the anastomosed vagal nerve in its cervical. The three animals were killed immediately afterward. The vagus nerve in its infranodose part up to the suture site and to the peripheral SAN stump were dissected. We checked that the suture site was entirely disconnected from the surrounding tissues, namely, from the proximal ends of both nerves, which could still be easily located by the presence of clips. To determine the population of the centripetal vagal contingent, the left vagal nerve was sectioned above the nodose ganglion (Fig. 1C) in a fourth sheep. This sheep was 15 months old at the time of the vagal section and NERVE FIBER POPULATIONS AFTER HETEROGENEOUS NERVE ANASTOMOSIS 131 Fig. 2. A demonstration of the extent to which the nervous tissue sections may be masked by the wires of a support grid. A: Semithin section from a spinal nerve observed under light microscopy. B: The same section as it appears when it is displayed on a grid. was born in the same breeding and during the same period as the three other sheep. It was killed 40 days after the operation. All experiments were carried out in compliance with the principles of laboratory animal care and the French laws on the protection of animals. In particular, operative techniques were performed under general anesthesia. An intravenous injection (200 mg of sodium pentobarbitone and 500 mg of sodium thiopentone dissolved in 25 ml of physiological saline) induced a brief anesthesia, which allowed the insertion of an endotracheal tube. Then, anesthesia was maintained by inhalation of 0.5–2.0% halothane in air. The tissues of the neck and the head were fixed by perfusion through the left common carotid artery of 4 liters of a glutaraldehyde (2.5%) paraformaldehyde (0.5%) solution in 0.2 M sodium cacodylate buffer, pH 7.4. The nerves were dissected out, fixed for another 2 hours by immersion in the same fixative, and then cut up to a size compatible with processing for electron microscopy. The fragments were washed with cacodylate buffer, postfixed for 1 hour in 1% buffered osmium tetroxide, then dehydrated in graded alcohols and propylene oxide, and finally embedded in Epon 812. Semithin or ultrathin sections were cut on a Reichert OM U3 Ultrotome. Semithin sections were stained with toluidine blue to allow location of the fiber bundles (Fig. 2A). Ultrathin sections collected on 75 mesh grids coated with Formvar film were contrasted with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 1A electron microscope. The unavoidable presence of the grid wires (Fig. 2B) masking areas of greater or lesser importance did not allow the complete study of the nerves: Only the bundles showing a sufficiently free area were analyzed. The analysis was completed by observations of the adjacent sections. Therefore, the choice of the analyzed bundles was purely arbitrary. The visible parts of the bundles were first carefully drawn and were then systematically photographed at an initial magnification of 32,000, the photographs partly overlapping in order to facilitate the reconstruction of the nerve section. After printing, the bundles were reconstructed, and the diameters of the nonmyelinated fibers (NFs) and the myelinated fibers (MFs) were determined, taking into account the final magnification of 34,000. Because the section of a fiber was rarely round, we decided to determine its diameter by taking the average of a long and short axis, including the myelin sheath, to make our results more comparable with those obtained from light microscopic observations (Tiao and Blakemore, 1976). Nerve samples of the three sheep with nerve anastomosis were observed in the electron microscope, but, in consideration of the vast amount of work, morphometric studies were only performed from IVN, SVN, SAN, RCIVN, and RSAN samples of one of the sheep; the samples from the other two sheep were used only for 132 P. DELORME ET AL. qualitative observations. Morphometric studies were also carried out from the CIVN sample of the fourth sheep, including the supranodose section of the vagus nerve, to compare its composition and distribution of the centripetal fibers with those of the RCIVN sample in the infranodose level. RESULTS Qualitative Aspects The distribution of the nerve fibers in the normal IVN or SVN (Fig. 3A) is not homogeneous. This heterogeneity is due not only to the differences between the fiber diameters but also to the repartition of these various fibers among the different nerve bundles and inside each bundle. The relatively small diameter of most fibers that compose this nerve results in a high-density fiber population. On the contrary, in the normal SAN (Figs. 2A, 3B), the distribution appears much more homogeneous. Density of fibers is relatively low, as the fibers of larger diameter are the most numerous. This explains why, for nearly equivalent nerve areas analyzed, the fiber count was very different between samples of normal vagus nerve and normal SAN (Table 1). Observation of the sectioned then anastomosed SAN (Fig. 1B) reveals a very collagenous endoneurium divided into small fascicles by the long, thin processes of endoneurial cells (Fig. 3D). Inside these thin-walled muffs, small- and middle-diameter vagal fibers, both MFs and NFs, alone or in groups of two or three, colonize the peripheral stump of the SAN, which is now emptied of its own axons. Similitude of observations in the samples of the three sheep, including reinnervation of vagal fibers into the SAN, led us to carry out the morphometric studies on the same animal. Quantitative Aspects Normal vagal nerve The normal vagal nerve was sampled central to the nodose ganglion (i.e., SVN) and peripheral to the ganglion (i.e., infranodose ganglion; IVN). Analysis of the IVN indicated that, of the 7,589 fibers measured, 3,443 were myelinated (i.e., 45% of the total; Table 1). The MF diameter distribution was unimodal (Fig. 4A). This fiber population was particularly concentrated between 2 µm and 7 µm. The NF distribution was also unimodal: The diameters of the NF fibers ranged from 0.25 to 2 µm, with a peak close to 1.5 µm (Fig. 4A). In the SVN (Fig. 4A), the proportion of MFs was slightly lower than in the IVN, because these fibers represented only 37% of the total population (3,720 of 9,935; Table 1). The diameter ranges over which both fiber types were distributed were nearly the same as in the infranodose part, although the MF peak was again located at 4 µm, whereas the NF peak had shifted to 0.75 µm. Sectioned SVN The vagal nerves were sectioned central to the nodose ganglion with and without creating an anastomosis. In both cases, the centrifugal efferent fibers degenerated, so that the peripheral stump sampled distal to the nodose ganglion would contain only centripetal fibers. In the CIVN (Fig. 4C), the MFs represented 35% of the fiber population. It was slightly lower than the 37% of the IVN population. Their distribution was always unimodal, but the peak shifted to 3 µm. On the other hand, the NF population displayed a greater range (0.25–4.0 µm), and its peak was shifted toward slightly greater diameters (1.5–1.75 µm). In the RCIVN (Fig. 4B), the MF diameter distribution was like that in the IVN, but the most important component of the MF population, although it was still centered at 4 µm, fell from 32% to 24% of the total. Moreover, the NF/MF proportion was increased from 1.2 to 1.72 (2,020 of 5,514 RCIVN fibers analyzed; Table 1). The NF distribution ranged from 0.25 to 2.5 µm, but it did not have a well-defined peak. Normal SAN Analysis of the SAN demonstrated that the MF component was smaller than the NF component (Fig. 5A): Indeed, it represented only one-fourth (24%) of the fibers measured (Table 1). The total fiber population of the SAN can be estimated as about 9,600, because Falempin (1981) estimated the total number of MFs to be about 2,300. The MF diameter spectrum was bimodal, with a lower peak (16% of the MFs) of 6–8 µm and a higher peak (53% of the MFs) of 15–18 µm. The NFs were distributed between 0.25 and 1.25 µm, with a peak of 0.75 µm. RSAN After anastomosing the distal stump of the sectioned vagal nerve to the distal stump of the sectioned SAN, the regenerating axons of the vagal centripetal neurons invaded the empty endoneurial fascicles of the degenerated spinal accessory motor nerve fibers (RSAN; Fig. 5B). In the RSAN, the NF spectrum was a little modified; its peak of 0.5 µm had slightly shifted compared with the NF peak in the normal SAN. The MF spectrum had changed substantially. The bimodal MF distribution in the normal SAN became unimodal in the RSAN, with a peak at 4 µm. This peak had the same location as that of the SVN, IVN, and CIVN. Comparison of the MF sizes in the different photomicrographs of Figure 3 illustrates this fact. The modal MF percentage in the normal SAN (24%) is even lower in the RSAN and corresponds to only 14% of the total fiber population (Table 1). DISCUSSION The aim of this study was to analyze and to compare the populations of MFs and NFs in the normal vagal nerve and SAN with nerves in which a peripheral vagoaccessory nerve anastomosis had been made (Fig. 1B) as a means of recording afferent vagal activity in conscious sheep (Rousseau, 1973). The relatively heterogeneous distribution of the various nerve fibers in the Fig. 3. Ultrastructural aspect of the myelinated (MFs) and nonmyelinated (NFs) nerve fibers in bundles from the normal vagal nerve (A), the normal spinal accessory nerve (B), the reinnervating centripetal infranodose vagal nerve (C), and the reinnervated spinal accessory nerve (D). Sometimes, Schwann cells (S) are observed near nerve fibers that are surrounded by abundant collagen (Co). The arrow in D indicates the thin processes of the endoneurial cells that form the small endoneurial fascicles. The MF diameters of the RSAN (D) are like those of the SVN and RCIVN myelinated fibers (A,C). Scale bars 5 2 µm. NERVE FIBER POPULATIONS AFTER HETEROGENEOUS NERVE ANASTOMOSIS Fig. 3. 133 134 P. DELORME ET AL. TABLE 1. Populations of nerve fibers in the various nerves studied No. of sample in Figure 1 Analyzed nerves 1 2 3 4 5 6 Normal infranodose vagus nerve (IVN) Normal supranodose vagus nerve (SVN) Normal spinal accessory nerve (SAN) Reinnervating centripetal IVN (RCIVN) Reinnervated SAN (RSAN) Centripetal infranodose vagus nerve (CIVN) Nonmyelinated Myelinated No. of fibers (NF) fibers (MF) analyzed Total no. (TN) of bundles measured fibers Number % of TN Number % of TN NF/MF 5 5 3 4 5 16 7,589 9,935 1,386 5,514 3,799 16,613 4,146 6,215 1,057 3,494 3,260 10,842 55 63 76 63 86 65 3,443 3,720 329 2,020 539 5,771 45 37 24 37 14 35 1.2 1.67 3.21 1.72 6.04 1.87 Fig. 4. Comparisons of the distributions (expressed as percentages of the total fibers) of the myelinated (MFs) or nonmyelinated (NFs) nerve fibers between the normal infranodose (IVN) and supranodose (SVN) vagal nerves (A), between the IVN and the reinnervating centripetal infranodose vagal nerve (RCIVN; B), between the IVN and the centripetal infranodose vagus nerve (CIVN; C), and between the RCIVN and the CIVN (D). Each pie chart indicates the number and the percentage of the analyzed MFs and NFs for each nerve. The numbers above the pie charts summarize the total number of fibers analyzed in each nerve studied. vagal nerves induced us to analyze more bundles in this nerve than in the SAN, the fiber distribution of which seemed more homogeneous. To obtain a reasonably true fiber representation, all the workable bundles that were not masked by the grid wires were photographed and were then reconstituted before analysis. bution of vagal MFs is not exceptional, because it is also found in other species, such as the cat (Burgh-Daly et al., 1953; Mei et al., 1980) and the ferret (Asala and Brower, 1966), whereas Bronson et al. (1978) have described an MF bimodal distribution in the rat vagal nerve. The originality of this study lies mostly in the fact that the NF component in normal vagal nerves of sheep was estimated. The NFs represent 55% of the fibers in the infranodose part and represent 63% in the supranodose part (Table 1). These proportions are considerably greater than the 10–30% of the NFs estimated in thoracic vagal nerve by Habel (1956). The only previous studies (Iggo, 1956; Dussardier, 1960) on vagal nerves in sheep used light microscopy and led to an underestimation of the NF component of these nerves. However, this component in sheep is less important than Composition of the Normal Vagus Nerve Estimation of the total numbers The MF distributions in the SVN and the IVN were near enough to those that were described by Iggo (1956) or Dussardier (1960) from light microscopy observations. These authors found that the great majority of the MFs (about 80%) had diameters between 2 and 4 µm, and, in our study, a similar percentage pertained to the fibers, with a peak of 4 µm. Such a unimodal distri- NERVE FIBER POPULATIONS AFTER HETEROGENEOUS NERVE ANASTOMOSIS 135 Supranodose vs. infranodose Fig. 5. Comparisons of the distributions of the myelinated (MFs) and nonmyelinated (NFs) nerve fibers between the normal spinal accessory nerve (SAN) and the reinnervated spinal accessory nerve (RSAN; A) and between the RSAN and the reinnervating centripetal infranodose vagal nerve (RCIVN; B). For details, see the legend to Figure 4. that observed by electron microscopy in the cat vagal nerves, where it represents 68–85% of the infranodose fibers and 86–90% of the supranodose fibers (Mei et al., 1980). The relative wealth of MFs in the vagal nerves of sheep could be accounted for by the large innervation of the complex stomach of ruminants, the sequential motility of which is triggered once every 45 seconds by the medullary gastric centers through the vagal nerves. From our observations, the total number of fibers in the cervical vagal nerve could be determined only approximately. The number of bundles that we have analyzed corresponded to about one-sixth of the whole area of the IVN section. Therefore, we can estimate the total nerve fiber population of the IVN to be about 45,000 fibers. Dussardier (1960) estimated that there were 10,000–12,000 MFs present in each dorsal thoracic vagal nerve and 8,000–10,000 in the common ventral thoracic vagal trunk. Taking into account the 45% of MFs that we found in the IVN and the observation that the IVN contains the fibers from one of the dorsal thoracic vagal nerves and one-half of those from the ventral thoracic common vagal trunk, we can calculate from Dussardier’s results the total population of the IVN to be 31,000–38,000 fibers. Consequently, the results of Dussardier (1960) represent an underestimation, probably because of the difficulty in counting the NFs by light microscopy. The different composition of the SVN and the IVN must be emphasized. The SVN contains more NFs than the IVN, the NF/MF ratio being 1.67 against 1.2 (Table 1). The difference between these two levels is plainly less marked than in the cat, in which the NF/MF ratios are 7 to 9 and 4 to 6 for the supranodose and infranodose parts of the vagal nerves, respectively (Mei et al., 1980). This comparison confirms the more important myelinization of cervical vagal nerve fibers in sheep. Similar to cat vagal nerves, the unimodal distribution of the NFs presents different maxima when we consider the whole SVN (0.75 µm) on the one hand and the whole IVN or its centripetal contingent (1.25–1.5 µm) on the other hand. In the infranodose region, the CIVN remaining after supranodose nerve section and the degeneration of the centrifugal (motor) fibers contains relatively fewer MFs than the normal IVN (35% vs. 45%). The presence of the centripetal fibers in the normal IVN, therefore, is responsible for the higher percentage of MFs, suggesting that efferent vagal fibers are proportionally more myelinated than afferent vagal fibers. If the 16,613 centripetal fibers (Table 1) that we counted in the CIVN represented about two-thirds of the area of this nerve, then we can estimate the infranodose afferent component to be 25,000 fibers, i.e., about 55% of the estimated total IVN population of 45,000 fibers. Taking into account the whole population, if 45% of all fibers are MFs and 35% of all centripetal fibers are myelinated, then the calculation shows 60% of the infranodose efferent component to be NFs, i.e., 27% of the total population in which there are 45% efferent fibers. These estimations are in line with those given by Prechtl and Powley (1990) for the rat. Those authors consider that the efferent fiber component of the vagal nerves is more important than it has been proposed previously, namely, 10% (Agostini et al., 1957; Evans and Murray, 1954; Kemp, 1973). Compared with the afferent MF spectrum of the CIVN, the IVN MF spectrum is shifted to the right (Fig. 4C), suggesting that the myelinated centrifugal fibers generally have a greater diameter that the centripetal fibers. Reinnervation of the SAN The new population, which consists principally of small-diameter MFs, differs markedly from the population of the normal SAN (compare Fig. 3D with Fig. 3B) and rather resembles the population observed in the normal vagus nerve (Fig. 3A) or in the reinnervating IVN (Fig. 3C). The relative similarity of the fiber distribution (Fig. 4D) and of the MF percentages, 35% and 37%, respectively (Table 1), in the CIVN and in the RCIVN, is not surprising, because, in both cases, the nerves are deprived of their efferent component. The MF component changes from being bimodal in the intact SAN to becoming unimodal in the RSAN. Distributed around a peak at 4 µm, the spectrum of their diameters is identical to that observed in the RCIVN. On the other hand, the NF diameter is smaller in the RSAN than in the RCIVN (Fig. 5B). In other respects, they are plainly more numerous, because, in the RSAN, the NF/MF ratio is 3.5 times greater than in the RCIVN. This situation could be due to the fact that, in the RSAN, many axons from vagal sensory neurons, 136 P. DELORME ET AL. which colonize the endoneurial fascicles of the recipient nerve, are in the process of maturation and have not yet reached their final size. It is possible to compare the diameter spectrum of the RCIVN fibers with the distribution of the conduction velocities measured between two points of stimulation on this nerve (Falempin and Rousseau, 1983). In sheep, the maximum of the evoked responses (44%) is obtained by the stimulation of fibers with conduction velocities of 6–12 meters/second corresponding to fiber diameters of about 1–2 µm. Eighteen percent of the responses are evoked by stimulation of fibers of diameters less than 1 µm, and 25% are evoked by stimulation of fibers of 2-4 µm in diameter. In other words, the diameter spectrum, which is deduced from the distribution of conduction velocities, is shifted to the left compared with those established by direct histological observation. This means that, among the vagal centripetal fibers running below the nodose ganglion, about 62% of those that had reinnervated the neck muscle are either slightly myelinated or nonmyelinated, with diameters equal or less than 2 µm. They would correspond to the peripheral processes of sensory neurons, because retrograde labeling of nerve cells was evidenced in the ipsilateral nodose ganglion following successful reinnervations (Falempin and Rousseau, 1983). They could also be centrally directed recurrent collateral branches of preganglionic efferent gastric fibers from the contralateral vagus nerve, as suggested by subsequent results (Dalal et al., 1988). ACKNOWLEDGMENTS The authors thank Yolande Dodey and Sophie Duclos for their secretarial help and Claudine Guichard for her photographic assistance. This research was supported by grants from M.E.N. and from C.N.R.S. (U.A. 308). LITERATURE CITED Agostini, E., J.E. Chinnock, M.D. Burgh-Daly, and J.G. Murray 1957 Functional and histological studies on the vagus nerve and its branches to the heart lungs and abdominal viscera in the cat. J. Physiol., 135:182–205. Asala, S.A., and A.J. Brower 1986 An electron microscope study of vagus nerve composition in the ferret. Anat. Embryol., 175:247– 253. Bronson, R.T., H. Bishop, and E.T. Hedley-White 1978 A contribution to the electron microscopic morphometric analysis of peripheral nerves. J. Comp. Neurol., 178:177–186. Burgh-Daly, M.D., and D.H.L. Evans 1953 Functional and histological changes in the vagus nerve of the cat after degenerative section at various levels. J. Physiol., 120:579–595. Coget, J., and J.-P. Rousseau 1983 Reinnervation of striated muscle by peripheral vagal fibers cut above or below the nodose ganglion in the cat and rabbit. J. Physiol., 335:481–493. Dalal, A., B.F. Leek, and J.-P. Rousseau 1988 Gastric centripetal vagal unitary activity in conscious sheep. J. Physiol., 406:137P. Dussardier, M. 1960 Recherches sur le contrôle bulbaire de la motricité gastrique chez les Ruminants. Thèse Doct. Sci., Paris, 199 pp. Evans, D.H.L., and J.G. Murray 1954 Histological and functional studies on the fiber composition of the vagus nerve of the Rabbit. J. Anat., 88:320–337. Falempin, M. 1981 Contribution à l’étude des afférences vagales digestives chez l’animal éveillé. Thèse Doct. Sci., Lille, 211 pp., 79 illustrations. Falempin, M., and J.-P. Rousseau 1983 Reinnervation of skeletal muscles by vagal sensory fibers in the sheep, cat and rabbit. J. Physiol., 335:467–479. Falempin, M., and J.-P. Rousseau 1984 Activity of lingual, laryngeal and oesophageal receptors in conscious sheep. J. Physiol., 347: 47–58. Habel, R.E. 1956 A study of the innervation of the ruminant stomach. Cornell Vet., 46:555–628. Iggo, A. 1956 Central nervous control of gastric movements in sheep and goats. J. Physiol., 131:248–256. Kemp, D.R. 1973 A histological and functional study of the gastric mucosal innervation in the dog. I. The quantification of the fiber content of the normal subdiaphragmatic vagal trunks and their abdominal branches. Austral. NZ J. Surg., 43:288–294. Mei, N., M. Condamin, and A. Boyer 1980 The composition of the vagus nerve of the cat. Cell Tissue Res., 209:423–431. Prechtl, J.C., and T.L. Powley 1990 The fiber composition of the abdominal vagus of the rat. Anat. Embryol., 181:101–115. Rousseau, J.-P. 1970 Contribution à l’étude de la réinnervation et de l’éructation chez le mouton. Thèse Doct. Sci., Aix-Marseille, 156 pp. Rousseau. J.-P. 1973 Réinnervation du muscle mastoido-huméral par les axones des neurones sensitifs vagaux. J. Physiol. (Paris), 67:308A. Rousseau, J.-P., and M. Falempin 1984 Reinnervation of a striated muscle by vagal sensory axons. J. Autonom. Nervous Syst., 10:217–224. Tiao, Y.C., and C. Blakemore 1976 Regional specialization in golden hamster’s retina. J. Comp. Neurol., 168:439–458.