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Origin of the parasympathetic preganglionic fibers to the distal colon of the rabbit as demonstrated by the horseradish peroxidase method
Neuroscience Letters, 63 ( t 986) 17- 22 Elsevier Scicntitic Publishers Ireland Ltd.
17
NSL 03698
ORIGIN OF THE P A R A S Y M P A T H E T I C PREGANGLIONIC FIBERS TO THE DISTAL C O L O N OF THE RABBIT AS D E M O N S T R A T E D BY THE HORSERADISH PEROXIDASE METHOD
A.R.D. BESSANT and J. ROBERTSON-R1NTOUL
Department ~I' Anatom)" and E.~perimental Pathology, University o/'SI. Andrews, St. Andrew.~, Fili"( ~ . K. ,, (Received July 23rd. 1985: Revised version received September l lth, 1985; Accepted September 16th. 1985)
Key ~ordv.
colon
horseradish peroxidase technique
preganglionic parasympalhelic ncuron
rabbit
The extrinsic innerwttion of the rabbit distal colon was studied by the use of the horseradish peroxidase (ttRP) tracing technique. After injection of HRP, labelled cells were observed in sacral spinal cord segments $2 $5, primarily in the lateral intermediate grey matter. Labelled neurons were also observed m the lateral funiculus and sometimes in the veniral horn. No labelled cells were found in the vagal nuclei, contrary to the reports of earlier investigators. Anterograde transport of HRP resulted in labelling of visceral afferent neurons in dorsal root ganglia, whilst the central processes of afferent neurons cntercd kissauer's tract and formed a collateral pathway along the lateral edge of the dorsal horn which terminaled m close apposition to labelled preganglionic efferent neurons.
The central origin of preganglionic parasympathetic neurons innervating the gastrointestinal tract has been studied using various techniques including cytoarchitectonics [13], chromatolysis [11, 12], electrophysiology [15] and retrograde axonal transport of horseradish peroxidase (HRP) [5, 6, 9, 10]. It is generally held that the parasympathetic preganglionic fibers which pass to the myenteric and submucous plexuses in the intestinal wall above the splenic ttcxurc originate in the dorsal m o t o r nucleus of the vagus nerve (dmnX) and in the sacral parasympathetic nucleus (SPN) below that. Kalia [6] further claims that all those abdominal viscera below the diaphragm which receive a vagal supply are innervated by effcrents from the nucleus ambiguus as well as the dmnX. Recent studies have cast doubt, however, on the accepted description, suggesting instead that the dmnX may contain the cell bodies of neurons supplying the whole length of the gastrointestinal tract. Satomi et al. [14] observed labelled somata in the dmnX of cats after injection of H R P into the distal colon and rectum, whilst Conners et al. [3] describe labelling of the myenteric plexus of the distal colon of rats after injection of an autoradiographic tracer into the dmnX. This study was carried out with two main objectives: ( 1) to provide a detailed dcscription of the location of cells forming the SPN and to determine their relationship to visceral afferents we felt that this was necessary because the majority of previous investigators [5, 8 10] have not injected H R P into the gut wall directly, where it t l l a V (1~,(i4-~'~40 S65 0*~.S0 ~ 1986 Elsevier Scientific Publishers Ireland l.ld.
18 be endocytosed by nerve termini, but relied on applying it to the severed end of the pelvic nerve; and (2) to verify or refute the findings of Satomi et al. [14] and Connors et al. [3]. Ten New Zealand White rabbits of both sexes were used in this study. The animals were given 0.44 ml/kg of Sagatal anesthetic (pentobarbitone sodium BP; May & Baker, Dagenham, U.K.) i.v., and a midline incision was made in the lower abdominal wall. Skin, fascia and fibro muscular layers were incised and reflected laterally. Fat, bladder and overlying bowel were eased aside and held in place with warm gauze soaked in sterile water to permit access to the lower end of the large intestine. Between 60 and 130/A of 40~o H R P (Sigma type VI) were injected under the peritoneal coat of the rectum or distal colon at various distances above the colorectal junction. Precautions were taken to minimize leakage of H R P since this can lead to uptake by adjacent structures and result in false labelling. Small (5-10/~I) injections were made at several discrete locations into a segment of the bowel, isolated by gauze swabs, and the needle withdrawn gradually, with absorbent Gelfoam (Sterispor, The Boots, Nottingham, U.K.) applied over the injection site. After a 48-72-h recovery period the animals were given 1.5 mg of Largactil (May & Baker) i.m., to act as a vasodilator, and 25 mg heparin (BDH) i.v. Thirty minutes later they were killed by an overdose of diethyl ether. The thoracic cavity was opened, and the animals were then perfused transcardially with Ringer-Lock solution followed by 1~o paraformaldehyde-2.5~o glutaraldehyde in 0.1 M phosphate buffer. The medulla oblongata and sacral spinal cord were removed, postfixed for 1 h and transferred to a phosphate-buffered sucrose solution for at least 15 h. The fixed tissue was then sectioned at 50/~m on a Leitz 1310 freezing microtome (dorsal root ganglia were sectioned at 30 #m) and processed for H R P according to the method of Mesulam [7]. Sections were mounted on glass slides and counterstained with red neutral. Cell dimensions were measured with a MOP-I analyser (Reichert-Jung) in conjunction with an Apple II microcomputer. Diagrams o f cell distributions were prepared with a camera lucida drawing table connected to a BBC microcomputer. After HRP injection into the distal colon, retrogradely labelled neurons were seen bilaterally, forming two unbroken columns, in the sacral spinal cord. At least 80,0~,~ of the labelled cells in each animal was located in $3 and $4 spinal segments, with variable numbers in $2 and $5. The greatest number of labelled cells on one side of the spinal cord was 1006 (corrected by a factor of 0.74 for cells appearing twice [1]), and they extended through approximately 9.t mm of the spinal cord (corrected by a factor of 1.15 for shrinkage of the cord during fixation [9]). Labelled somata tended to lie in a fairly well demarcated group along the lateral border of the dorsal horn and in the intermediate grey matter. This corresponds to the position of the intermediolateral cell column and comprises part of the SPN (Fig. 1). A distinct lateral horn, comparable to that seen in the thoracolumbar region [2], was not seen at this level of the spinal cord, but up to 10~o of labelled cells lay at varying depths within the lateral funiculus. These correspond to the 'pars funicularis' of the SPN as described by Petras and Cummings [12]. At the most rostral end of the SPN, labelled cells were also found to extend along the lateral margin of the
19
Fig. 1. Diagram of the sacral parasympathetic nucleus (spn) of the rabbit. The preganglionic parasympathetic neurons tend to lie along the lateral border of the dorsal column of grey matter with their long axes parallel to that border and also within the lateral funiculus of white matter. ventral horn, lateral to the s o m a t i c m o t o r nuclei. A n a l m o s t identical d i s t r i b u t i o n has been o b s e r v e d in the rat [5]. The p r e g a n g l i o n i c p a r a s y m p a t h e t i c n e u r o n s c o n s t i t u t i n g the S P N were small to m e d i u m sized (mean d i a m e t e r , 12.7-23.4/~m) and typically oval or spindle shaped. Dendrites e x t e n d e d dorsally, m e d i a l l y a n d laterally, whilst axons were seen to course ventrally a r o u n d the lateral b o r d e r o f the ventral horn and enter the ventral roots. N o labelled cells were ever o b s e r v e d in any o f the vagal nuclei following injection o f H R P into the distal colon. Visceral afferent n e u r o n s in sacral d o r s a l r o o t ganglia ( D R G ) were labelled by a n t e r o g r a d e t r a n s p o r t o f H R P . These cells o u t n u m b e r e d p r e g a n g l i o n i c efferents by a ratio o f 3:1 to 4:1, but their respective segmental d i s t r i b u t i o n s c o r r e l a t e d well. Labelled D R G cells were typically ovoid, p s e u d o - u n i p o l a r cells, a n d they tended to be a m o n g s t the smaller cells o f the ganglia (averaging 23.8 32.7/~m). Within the spi-
20
w
Fig. 2. Bright-field photomicrograph of a transverse section from $4 of rabbit 6. Reaction product granules are present in Lissauer's tract (LT) and form a discrete band along the lateral edge of the dorsal horn. This band is the lateral collateral pathway (LCP), and it can be observed terminating in relation to cells of the SPN in the bottom right of the picture. In some sections the appearance of reaction product in LT and LCP is markedly different and suggests that LT may contain longitudinally orienled fibers, whilst the LCP is formed by fibers coursing ventrally in the transverse plane and their terminal fields.
21 hal cord primary afferent fibers were revealed as trails of reaction product granules in the dorsal root entry zone and Lissauer's tract. They also formed a prominent collateral fiber bundle along the lateral edge of the dorsal horn. This bundle, which corresponds to the qateral collateral pathway' seen in cats [8], terminated in relation to cells of the SPN (Fig. 2). Our study has confirmed many aspects of the description of the SPN provided by the earlier investigators who utilized less direct approaches [5, 8 10], but some discrepancies were found. We were unable to demonstrate a viscerotopic organization within the SPN since we only studied the colonic innervation, but it may be worthy of note that the location of labelled somata in our rabbit species compared more closely with that seen in monkeys [10] than with that of the cat [9], which is the only animal in which a clear viscerotopic subdivision of the SPN has been claimed to exist. A 'medial collateral pathway" along the medial edge of the dorsal horn, which has been described tk)r the cat [8], was not observed in our studies. This may indicate that afferent Iibers other than those passing from the colon were present in the branches of the pelvic nerve which these workers chose to sever and bathe in H R P rather than injecting the marker into the viscus being studied, illustrating the possible drawbacks of" this approach. The considerable overlap between incoming afferent fibers and the dendrites of preganglionic neurons of the SPN was remarkably demonstrated and suggests that perhaps a monosynaptic reflex arc might operate in the control of colonic motility. The considerable central delay (45 6(} ms) in the colonic reflex, as demonstrated by clectrophysiological studies in the cat [4], seems to preclude this, however, and may imply the presence of interneurons in the region of the SPN. The absence of labelled somata in the dmnX in our experiments contradicts the findings of Satomi et al. [14] and Connors et al. [3]. The earlier H R P study [14] may have suffered from artefactual labelling due to leakage of H R P to adjacent structures, notably the overlying small intestine which is vagally innervated. We suggest this because the authors also observed labelled cells in several apparently aberrant sites in the sacral spinal cord, implying leakage of tracer to pelvic wall musculature. In our own studies several precautions were taken to prevent such leakage. Doubts regarding the autoradiographic study [3] arise from the authors" description of granules overlying somata in the myenteric plexus of the distal colon, where the mycntcric neurons actually appear to be fully labelled. This seems to indicate a transneuronal passage of the tracer (tritiated leucine), which could invalidate their results. Despite this, we feel that the findings of these studies cannot be dismissed. It is interesting to note that Wiesendanger and Wiesendanger [16] provide evidence that lectin wheat germ agglutinin (tritiated leucine) conjugated to the marker H R P , or to the radioactive iodine, may be transported across cell boundaries. Since it is possible that the caudal extent of the territory of the vagus nerve may vary between species, we propose that further studies be carried out to determine more precisely the point at which the vagal innervation of the gut ends in various experimental animals. This study is now underway.
22 1 Abercrombie, M., Estimation of nuclear population from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 Chung, J.M., Chung, K. and Wurster, R.D., Sympathetic preganglionic neurons of the cat spinal cord: horseradish peroxidase study, Brain Res., 91 (1975) 126-131. 3 Connors, N.A., Sullivan, J.M. and Kubb, K.S., An autoradiographic study of the distribution of fibres from the dorsal motor nucleus to the digestive tube in the rat, Acta Anal., 115 (1983) 266-271. 4 De Groat, W.C. and Krier, J., The sacral parasympathetic reflex pathway regulating colonic motility and defaecation in the cat, J. Physiol. (London), 276 (1978) 481--500. 5 Hancock, M.B. and Peveto, C.A., Pre-ganglionic neurons in the sacral spinal cord of the rat: a horseradish peroxidase study, Neurosci. Lett, 11 (1979) 1-5. 6 Kalia, M., Brainstem localization of vagal preganglionic neurons, J. Auton. Nerv. Syst.. 3 (1981) 451 481. 7 Mesulam, M.-M., Tetramethyl benzidine for HRP neuro-histochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualising neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 8 Morgan, C., Nadelhaft. 1. and De Groat, W.C., The distribution of visceral primary afferents from the pelvic nerve to Lissauers Tract and the spinal grey matter and its relationship to the sacral parasympathetic nucleus, J. Comp. Neurol., 201 (1981) 415440. 9 Nadelhaft, I., De Groat, W.C. and Morgan, C., Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat as revealed by the retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 193 (1980) 265-281. 10 Nadelhaft, 1., Roppolo, J., Morgan, C. and De Groat, W.C., Parasympathetic pre-ganglionic neurons and visceral primary afferents in monkey sacral spinal cord revealed following application of horseradish peroxidase to the pelvic nerve, J. Comp. Neurol., 216 (1983) 36-52. 11 Oliver, J.E., Bradley, W.E. and Fletcher, T.F., Identification of pre-ganglionic parasympathetic neurons in the sacral spinal cord of the cat, J. Comp. Neurol., 137 (1969) 387-412. 12 Petras, J.M. and Cummings, J.F., Autonomic neurons in the spinal cord of the rhesus monkey: a correlation of the findings of cytoarchitectonics and sympathectomy with fibre degeneration after dorsal rhizotomy, J. Comp. Neurol., 146 (1972) 189--218. 13 Rexed, B., A cytoarchitectonic atlas of the spinal cord of the cat, J. Comp. Neurol., 100 (1954) 297379. 14 Satomi, H., Yamamoto, T., Ise, H. and Takatama, H., Origins of the preganglionic fibres to the cat intestine as demonstrated by the horseradish peroxidase method, Brain Res., 151 (1978) 571 578. 15 Schnitzlein, H.M., Hoffman, H.H., Hamlett, D.H. and Howell, E., A study of the sacral parasympathetic nucleus, J. Comp. Neurol., 120 (1963) 477-493. 16 Wiesendanger, R. and Wiesendanger, M., Cerebello-cortical linkage in the monkey as revealed by transcellular labeling with the lectin wheat germ agglutinin conjugated to the marker horseradish peroxidase, Exp. Brain Res., 59 (1985) 105- 117.
17
NSL 03698
ORIGIN OF THE P A R A S Y M P A T H E T I C PREGANGLIONIC FIBERS TO THE DISTAL C O L O N OF THE RABBIT AS D E M O N S T R A T E D BY THE HORSERADISH PEROXIDASE METHOD
A.R.D. BESSANT and J. ROBERTSON-R1NTOUL
Department ~I' Anatom)" and E.~perimental Pathology, University o/'SI. Andrews, St. Andrew.~, Fili"( ~ . K. ,, (Received July 23rd. 1985: Revised version received September l lth, 1985; Accepted September 16th. 1985)
Key ~ordv.
colon
horseradish peroxidase technique
preganglionic parasympalhelic ncuron
rabbit
The extrinsic innerwttion of the rabbit distal colon was studied by the use of the horseradish peroxidase (ttRP) tracing technique. After injection of HRP, labelled cells were observed in sacral spinal cord segments $2 $5, primarily in the lateral intermediate grey matter. Labelled neurons were also observed m the lateral funiculus and sometimes in the veniral horn. No labelled cells were found in the vagal nuclei, contrary to the reports of earlier investigators. Anterograde transport of HRP resulted in labelling of visceral afferent neurons in dorsal root ganglia, whilst the central processes of afferent neurons cntercd kissauer's tract and formed a collateral pathway along the lateral edge of the dorsal horn which terminaled m close apposition to labelled preganglionic efferent neurons.
The central origin of preganglionic parasympathetic neurons innervating the gastrointestinal tract has been studied using various techniques including cytoarchitectonics [13], chromatolysis [11, 12], electrophysiology [15] and retrograde axonal transport of horseradish peroxidase (HRP) [5, 6, 9, 10]. It is generally held that the parasympathetic preganglionic fibers which pass to the myenteric and submucous plexuses in the intestinal wall above the splenic ttcxurc originate in the dorsal m o t o r nucleus of the vagus nerve (dmnX) and in the sacral parasympathetic nucleus (SPN) below that. Kalia [6] further claims that all those abdominal viscera below the diaphragm which receive a vagal supply are innervated by effcrents from the nucleus ambiguus as well as the dmnX. Recent studies have cast doubt, however, on the accepted description, suggesting instead that the dmnX may contain the cell bodies of neurons supplying the whole length of the gastrointestinal tract. Satomi et al. [14] observed labelled somata in the dmnX of cats after injection of H R P into the distal colon and rectum, whilst Conners et al. [3] describe labelling of the myenteric plexus of the distal colon of rats after injection of an autoradiographic tracer into the dmnX. This study was carried out with two main objectives: ( 1) to provide a detailed dcscription of the location of cells forming the SPN and to determine their relationship to visceral afferents we felt that this was necessary because the majority of previous investigators [5, 8 10] have not injected H R P into the gut wall directly, where it t l l a V (1~,(i4-~'~40 S65 0*~.S0 ~ 1986 Elsevier Scientific Publishers Ireland l.ld.
18 be endocytosed by nerve termini, but relied on applying it to the severed end of the pelvic nerve; and (2) to verify or refute the findings of Satomi et al. [14] and Connors et al. [3]. Ten New Zealand White rabbits of both sexes were used in this study. The animals were given 0.44 ml/kg of Sagatal anesthetic (pentobarbitone sodium BP; May & Baker, Dagenham, U.K.) i.v., and a midline incision was made in the lower abdominal wall. Skin, fascia and fibro muscular layers were incised and reflected laterally. Fat, bladder and overlying bowel were eased aside and held in place with warm gauze soaked in sterile water to permit access to the lower end of the large intestine. Between 60 and 130/A of 40~o H R P (Sigma type VI) were injected under the peritoneal coat of the rectum or distal colon at various distances above the colorectal junction. Precautions were taken to minimize leakage of H R P since this can lead to uptake by adjacent structures and result in false labelling. Small (5-10/~I) injections were made at several discrete locations into a segment of the bowel, isolated by gauze swabs, and the needle withdrawn gradually, with absorbent Gelfoam (Sterispor, The Boots, Nottingham, U.K.) applied over the injection site. After a 48-72-h recovery period the animals were given 1.5 mg of Largactil (May & Baker) i.m., to act as a vasodilator, and 25 mg heparin (BDH) i.v. Thirty minutes later they were killed by an overdose of diethyl ether. The thoracic cavity was opened, and the animals were then perfused transcardially with Ringer-Lock solution followed by 1~o paraformaldehyde-2.5~o glutaraldehyde in 0.1 M phosphate buffer. The medulla oblongata and sacral spinal cord were removed, postfixed for 1 h and transferred to a phosphate-buffered sucrose solution for at least 15 h. The fixed tissue was then sectioned at 50/~m on a Leitz 1310 freezing microtome (dorsal root ganglia were sectioned at 30 #m) and processed for H R P according to the method of Mesulam [7]. Sections were mounted on glass slides and counterstained with red neutral. Cell dimensions were measured with a MOP-I analyser (Reichert-Jung) in conjunction with an Apple II microcomputer. Diagrams o f cell distributions were prepared with a camera lucida drawing table connected to a BBC microcomputer. After HRP injection into the distal colon, retrogradely labelled neurons were seen bilaterally, forming two unbroken columns, in the sacral spinal cord. At least 80,0~,~ of the labelled cells in each animal was located in $3 and $4 spinal segments, with variable numbers in $2 and $5. The greatest number of labelled cells on one side of the spinal cord was 1006 (corrected by a factor of 0.74 for cells appearing twice [1]), and they extended through approximately 9.t mm of the spinal cord (corrected by a factor of 1.15 for shrinkage of the cord during fixation [9]). Labelled somata tended to lie in a fairly well demarcated group along the lateral border of the dorsal horn and in the intermediate grey matter. This corresponds to the position of the intermediolateral cell column and comprises part of the SPN (Fig. 1). A distinct lateral horn, comparable to that seen in the thoracolumbar region [2], was not seen at this level of the spinal cord, but up to 10~o of labelled cells lay at varying depths within the lateral funiculus. These correspond to the 'pars funicularis' of the SPN as described by Petras and Cummings [12]. At the most rostral end of the SPN, labelled cells were also found to extend along the lateral margin of the
19
Fig. 1. Diagram of the sacral parasympathetic nucleus (spn) of the rabbit. The preganglionic parasympathetic neurons tend to lie along the lateral border of the dorsal column of grey matter with their long axes parallel to that border and also within the lateral funiculus of white matter. ventral horn, lateral to the s o m a t i c m o t o r nuclei. A n a l m o s t identical d i s t r i b u t i o n has been o b s e r v e d in the rat [5]. The p r e g a n g l i o n i c p a r a s y m p a t h e t i c n e u r o n s c o n s t i t u t i n g the S P N were small to m e d i u m sized (mean d i a m e t e r , 12.7-23.4/~m) and typically oval or spindle shaped. Dendrites e x t e n d e d dorsally, m e d i a l l y a n d laterally, whilst axons were seen to course ventrally a r o u n d the lateral b o r d e r o f the ventral horn and enter the ventral roots. N o labelled cells were ever o b s e r v e d in any o f the vagal nuclei following injection o f H R P into the distal colon. Visceral afferent n e u r o n s in sacral d o r s a l r o o t ganglia ( D R G ) were labelled by a n t e r o g r a d e t r a n s p o r t o f H R P . These cells o u t n u m b e r e d p r e g a n g l i o n i c efferents by a ratio o f 3:1 to 4:1, but their respective segmental d i s t r i b u t i o n s c o r r e l a t e d well. Labelled D R G cells were typically ovoid, p s e u d o - u n i p o l a r cells, a n d they tended to be a m o n g s t the smaller cells o f the ganglia (averaging 23.8 32.7/~m). Within the spi-
20
w
Fig. 2. Bright-field photomicrograph of a transverse section from $4 of rabbit 6. Reaction product granules are present in Lissauer's tract (LT) and form a discrete band along the lateral edge of the dorsal horn. This band is the lateral collateral pathway (LCP), and it can be observed terminating in relation to cells of the SPN in the bottom right of the picture. In some sections the appearance of reaction product in LT and LCP is markedly different and suggests that LT may contain longitudinally orienled fibers, whilst the LCP is formed by fibers coursing ventrally in the transverse plane and their terminal fields.
21 hal cord primary afferent fibers were revealed as trails of reaction product granules in the dorsal root entry zone and Lissauer's tract. They also formed a prominent collateral fiber bundle along the lateral edge of the dorsal horn. This bundle, which corresponds to the qateral collateral pathway' seen in cats [8], terminated in relation to cells of the SPN (Fig. 2). Our study has confirmed many aspects of the description of the SPN provided by the earlier investigators who utilized less direct approaches [5, 8 10], but some discrepancies were found. We were unable to demonstrate a viscerotopic organization within the SPN since we only studied the colonic innervation, but it may be worthy of note that the location of labelled somata in our rabbit species compared more closely with that seen in monkeys [10] than with that of the cat [9], which is the only animal in which a clear viscerotopic subdivision of the SPN has been claimed to exist. A 'medial collateral pathway" along the medial edge of the dorsal horn, which has been described tk)r the cat [8], was not observed in our studies. This may indicate that afferent Iibers other than those passing from the colon were present in the branches of the pelvic nerve which these workers chose to sever and bathe in H R P rather than injecting the marker into the viscus being studied, illustrating the possible drawbacks of" this approach. The considerable overlap between incoming afferent fibers and the dendrites of preganglionic neurons of the SPN was remarkably demonstrated and suggests that perhaps a monosynaptic reflex arc might operate in the control of colonic motility. The considerable central delay (45 6(} ms) in the colonic reflex, as demonstrated by clectrophysiological studies in the cat [4], seems to preclude this, however, and may imply the presence of interneurons in the region of the SPN. The absence of labelled somata in the dmnX in our experiments contradicts the findings of Satomi et al. [14] and Connors et al. [3]. The earlier H R P study [14] may have suffered from artefactual labelling due to leakage of H R P to adjacent structures, notably the overlying small intestine which is vagally innervated. We suggest this because the authors also observed labelled cells in several apparently aberrant sites in the sacral spinal cord, implying leakage of tracer to pelvic wall musculature. In our own studies several precautions were taken to prevent such leakage. Doubts regarding the autoradiographic study [3] arise from the authors" description of granules overlying somata in the myenteric plexus of the distal colon, where the mycntcric neurons actually appear to be fully labelled. This seems to indicate a transneuronal passage of the tracer (tritiated leucine), which could invalidate their results. Despite this, we feel that the findings of these studies cannot be dismissed. It is interesting to note that Wiesendanger and Wiesendanger [16] provide evidence that lectin wheat germ agglutinin (tritiated leucine) conjugated to the marker H R P , or to the radioactive iodine, may be transported across cell boundaries. Since it is possible that the caudal extent of the territory of the vagus nerve may vary between species, we propose that further studies be carried out to determine more precisely the point at which the vagal innervation of the gut ends in various experimental animals. This study is now underway.
22 1 Abercrombie, M., Estimation of nuclear population from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 Chung, J.M., Chung, K. and Wurster, R.D., Sympathetic preganglionic neurons of the cat spinal cord: horseradish peroxidase study, Brain Res., 91 (1975) 126-131. 3 Connors, N.A., Sullivan, J.M. and Kubb, K.S., An autoradiographic study of the distribution of fibres from the dorsal motor nucleus to the digestive tube in the rat, Acta Anal., 115 (1983) 266-271. 4 De Groat, W.C. and Krier, J., The sacral parasympathetic reflex pathway regulating colonic motility and defaecation in the cat, J. Physiol. (London), 276 (1978) 481--500. 5 Hancock, M.B. and Peveto, C.A., Pre-ganglionic neurons in the sacral spinal cord of the rat: a horseradish peroxidase study, Neurosci. Lett, 11 (1979) 1-5. 6 Kalia, M., Brainstem localization of vagal preganglionic neurons, J. Auton. Nerv. Syst.. 3 (1981) 451 481. 7 Mesulam, M.-M., Tetramethyl benzidine for HRP neuro-histochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualising neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 8 Morgan, C., Nadelhaft. 1. and De Groat, W.C., The distribution of visceral primary afferents from the pelvic nerve to Lissauers Tract and the spinal grey matter and its relationship to the sacral parasympathetic nucleus, J. Comp. Neurol., 201 (1981) 415440. 9 Nadelhaft, I., De Groat, W.C. and Morgan, C., Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat as revealed by the retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 193 (1980) 265-281. 10 Nadelhaft, 1., Roppolo, J., Morgan, C. and De Groat, W.C., Parasympathetic pre-ganglionic neurons and visceral primary afferents in monkey sacral spinal cord revealed following application of horseradish peroxidase to the pelvic nerve, J. Comp. Neurol., 216 (1983) 36-52. 11 Oliver, J.E., Bradley, W.E. and Fletcher, T.F., Identification of pre-ganglionic parasympathetic neurons in the sacral spinal cord of the cat, J. Comp. Neurol., 137 (1969) 387-412. 12 Petras, J.M. and Cummings, J.F., Autonomic neurons in the spinal cord of the rhesus monkey: a correlation of the findings of cytoarchitectonics and sympathectomy with fibre degeneration after dorsal rhizotomy, J. Comp. Neurol., 146 (1972) 189--218. 13 Rexed, B., A cytoarchitectonic atlas of the spinal cord of the cat, J. Comp. Neurol., 100 (1954) 297379. 14 Satomi, H., Yamamoto, T., Ise, H. and Takatama, H., Origins of the preganglionic fibres to the cat intestine as demonstrated by the horseradish peroxidase method, Brain Res., 151 (1978) 571 578. 15 Schnitzlein, H.M., Hoffman, H.H., Hamlett, D.H. and Howell, E., A study of the sacral parasympathetic nucleus, J. Comp. Neurol., 120 (1963) 477-493. 16 Wiesendanger, R. and Wiesendanger, M., Cerebello-cortical linkage in the monkey as revealed by transcellular labeling with the lectin wheat germ agglutinin conjugated to the marker horseradish peroxidase, Exp. Brain Res., 59 (1985) 105- 117.