Neuroseience Letters, 11 (1979) 1--5 © Elsevier/North-Holland Scientific Publishers Ltd.
PREGANGLIONICNEURONS IN THE SACRAL SPINAL CORD OF THE RAT:, AN HRPSTUDY M.B. HANCOCK and C.A. PEVETO The Department Of Anatomy, The University of Texas Medical Branch, Galveston, TX 77550(U.S.A.) (Received October 2nd, 19~78) (Revised version received October 19th, 1978) (Accepted November 1st, 1978)
SUMMARY
The pelvic nerve in the rat was exposed to H R P in order to localizethe cells of origin of preganglionic fibers coursing in this nerve. Labeled cells were located in spinal segments Ls-SI, primarily in the lateral intermediate gray. Occasionally, an HRP.filled cellwas observed in the ventral horn in association with somatic motoneurons.
The location in the sacral spinal cord of preganglionic neurons contributing fibers to the pelvic nerve in the cat has been determined e~ectrophysiologicaUy [4,12] and with retrograde chromatolysis [10]. Retrograde transport of horseradish peroxidase (HRP) in the pelwc nerve of the c~t and monkey was employed to localize preganglionic neurons in the sacral spinal cord [9]. In each of these studies, the cells sending fibers into the pelvic nerve were localized in a nucleus in the lateral intermediate gray of the sacral spinal cord. The longitudinal distribution of the neurons was variable; generally the cells extended over two segments, primarily $2 and $3. Coggeshall et al. [ 2] observed that the L6-$1 ventral roots in t h e m t contained comparatively large numbers of unmyelinated 8rid small myelinated fibers and postulated that ~he autonomic outfloWOf the pelvis takes origin in these spinal segments in the rat. They were stvrprised at the,relatively more rostral level of this outflow compared to that in other mammal~,:a finding which requires confirmation. Recently, :the authors [5] observed that the majority of preganglionic neur0nsWith axons c o ~ g in thehypogastric nerve of the mt are located al0ng t h e / ~ d ~ e ; in thedorsal gray commissure 0f spinal segments LrL2, a finding: ~ c t l y c o n l ~ 7 t o the results of previous studies in which sympathetic p ~ g h ' 0 n i C neur6~i!~ze~ ~l ~ e d ~ p ~ a r i l y t? the lateralintermediate gmy~[l~3~i~i~13];iThe ~ U e l o c a t i o n of the majority of preganglionic neurons innel~t~gii~thepelvic,--vis~m,in the rat ~ the hypogastric nen,e suggests the
possibility that the sacral preganglionic cells may too have an unpredictable distribution in the spinal gray. The r~:trograde transport of HRP was used in the present experiments in order to confirm the level of autonomic outflow to the pelvic viscera in the rat and to determine the cross.sectional distribution of preganglionic neurons contributing fibers to the pelvic nerve. Experiments were performed on adult male rats (Sprague--Dawley). The animals were anesthetized with an intraperitoneal injection of a solution of urethane (500 mg/kg) and nembutal (30 mg/kg) and the pelvic viscera were exposed. The pelvic nerve was i~entified as is coursed medially to the major pelvic g~glion where it was cut and dissected laterally. The nerve was then immersed in a molten low melting point wax which was allowed to solidify. The soft wax was gently packed down around the exposed end of the nerve in order to. form a seal and to provide a depression in which a drop of 30% solution of horseradish peroxidase (HRP, Sigma type VI) in saline would be placed. The cut end of the pelvic nerve was exposed to the HRP solution for three hc,urs. Following a survival period of.three days, the animals were anesthetized and perfused transcardially with a solution of 0.4% paraformaldehyde and 2.4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The lower lumbar and sacral spinal cord was immediately removed and placed in fixative for about two hours and then transferred to a 30% sucrose solution in which it remained overnight. Frozen sections (35 ~m) were cut and reacted with tetramethylbenzidine [6]. The sections were mounted and counterstained with neutral red. Pregsnglionic neurons, labeled by the retrograde transport of H R P in the pelvic nerve, were observed in three animals. Negative results obtained in several other animals may have been due to either inadequate perfusion and fixation or excessive trauma to the pelvic nerve as it was dissected. HRP-filled preganglionic neurons were present in spinal segments L~-S~;the majority of the cel~ were located in segment Sz. In transverse sections,the cellswere most frequently observed in tight clustersin the lateralintermediate gray (Fig. 1). These ctustersoften extended laterally,ventral to the zona reticularis,~ving the appearance of a distinct'lateralhorn'. In other sections,labeled cellswere aggrega~l along the lateralborder of the intermediate gray and were generally aligned with the border (Fig. 2), Occasionally, labeled cellswere seen in the middle of the intermediate gray; these cellswere generally oriented horizontally. Nc, labeled cellswere observed near the central canal or in the contralateral[~ay matter. In severalsections, HRP.filled neurons were located in the dorsal part of the ventral horn in association with somatic motor nuclei. Labeled preganglionic neurons were polygonal to fusiform in shape with a range'o:~mean diameters of 13--32 ~ m and an average mean diameter of 20 ~m. Approximately 3501 labeled cellswer e present in each of two mdmals in which cell counts were made. Labeled axons were not observed. The results ofi~epresent experiments ~have shownthat the cellsof originof pregan~!ionic fibers coursing in the pelvic nerve in the rat are located in spinal ,,.
,
.,
~
.
.
.
•
.
-
segments L6-Sz, a finding which confirms the observations of Coggeshall e t a ] . [2] who expressed concern over the comparatively more rostra] preganglionic outflow from this region of the cord in the rat as determined from their s t u d y of ventral r o o t fiber composition.
Fig. 1. Transverse section of the Ss spinal segment containing HRP filled preganglionie neurons in the right lateral intermediate gray. The calibration bar repre lents 200 ~m.
Fig. 2. Transverse sect/on o f L 6 spinal cord contalninglsb~led ~eIls along the right border of the intermediate gray. A small HRP-fdledneuron (indicated by arrow) is present medial to a somatic. :motor nucleus in the dorsal pa~t of the:,~entral horn.,The,.ealibration, bar represents I00 ~m.
The present authors have recently shown that the majority of the cells of origin of preganglionic fibers coursing in the hypogastric nerve of the rat are situated along the midline of spinal segments LI-L2, in the dorsal gray comm!~ure [ 5 ] . The transverse distribution of sympathetic preganglionic neurons at more rostral levels of the rat cord appears to be similar to that observed in other mammals; the majority of these cells are. located in the lateral intermediate gray matter. The location of cells sending fibers into the hypogastric nerve in the rat is comparable to that of preganglionic neurons in the pigeon which form a column, dorsal to the central canal [ 7,8]. This column (column of Terni) has both thoraco-lumbsr and sacral components; the latter is thought to represent the sacral componen't of the parasy mpathetic division of the autonomic nervous system [7]. The present experiments were thus prompted to determine whether a midline preganglionic cell column, with fiber projections in the pelvic nerve, also exists in the sacral cord of the rat. The results of this study provide no evidence of such a midline column, and show that the transverse distribution of sacral preganglionic neurons in the rat spinal cord is similar to that described for other mammals. A few labeled cells were seen in the dorsal part of the ventral horn in proximity to somatic motoneurons. Oliver et al. [10] reported observing an occasional chromatolytic neuron in this region of the ventral horn in the cat following repeated pelvic nerve sections. REFERENCES
1 Chung, J.M., Chung, K. and Wurster, R., Sympathetic preganglionic neurons of the cat spinal cord: horseradish peroxidase study~ Brain Res., 91 (1975) 126--131. 2 Coggeshall, R.E., Emery, D.G., Ito, H. and Maynard, C.W., Unmyelinated and small myelinated axons in rat ventral roots, J. comp. Neurol., 173 (1977) 175--184. 3 Cummings, J.F., Thoracolumbar preganglionic neurons and adrenal innervation in the dog, Acta Anat., 73 (1969) 27--37. 4 DeGroat, W.C. and Ryall, R.W., The identification and characteristics of sacral parasympathetic preganglionic neurones, J. Physiol. (Lond.), 196 (1968) 563--577. 5 Hancock, M.B. and Peveto, C.A., A preganglionic autonomic nucleus in the dorsal gray commissure of the lumbar spinal cord in the rat, J. comp. Neurol., in press. 6 Hardy, H. and Helmet, L., A safer and more sensitive substitute for diaminobenzidine in the light microscopic demonstration of retrograde and anterograde axonal transport of HRP, Neurosei. Le~t., 5 (1977) 235--240. 7 Huber, J.F., Nerve roots and nuclear groups in the spinal cord in the pigeon, J. comp. Neurol., 65 (1936) 43---91. 8 MacDonald, R.L. and Cohen, D.H., Cells of origin of sympathetic pre- and postganglionic cardioaceeleratory fibers in the pigeon, J. comp. Neurol., 140 (1970) 343--357. 9 Nadelhaft, I., Morgan, C., Schauble, T. and DeGroat, W.C., Localization of the sacral autonomic (parasympathetic) nucleus in the spinal cord of the cat and monkey by the horseradish peroxidase technique, Neurosci. A~str., 3 (1977) 24. 10 Oliver, J.E., Jr., Bradley, W.E. and Fletcher, T.F., Identification of preganglionic parasympathetic neurons in the sacral spinal cord of the cat, J. comp. Neurt~l., 137 (1969) 321--328. 11 Petras, J.M. and Fadert, A.I., The origin of sympathetic preganglionic neurons in the
dog, Brain. Res., 144 (1978) 353--357. 12 Sehnitzlein, H.M., Hoffman, H.H:, Hamlett, D.M. and Howell, E.M., A study of the sacral parasympathetic nucleus, J. comp. Neurol., 137 (1969) 329--346. 13 Schramm, L , Adair, J.R., Stribling, J. and Gray, L., Preganglionic innervation of the adrenal gland of the rat: a study using horseradish peroxidase, Exp. Ne,rol., 49 (1975) 540--553.
PREGANGLIONICNEURONS IN THE SACRAL SPINAL CORD OF THE RAT:, AN HRPSTUDY M.B. HANCOCK and C.A. PEVETO The Department Of Anatomy, The University of Texas Medical Branch, Galveston, TX 77550(U.S.A.) (Received October 2nd, 19~78) (Revised version received October 19th, 1978) (Accepted November 1st, 1978)
SUMMARY
The pelvic nerve in the rat was exposed to H R P in order to localizethe cells of origin of preganglionic fibers coursing in this nerve. Labeled cells were located in spinal segments Ls-SI, primarily in the lateral intermediate gray. Occasionally, an HRP.filled cellwas observed in the ventral horn in association with somatic motoneurons.
The location in the sacral spinal cord of preganglionic neurons contributing fibers to the pelvic nerve in the cat has been determined e~ectrophysiologicaUy [4,12] and with retrograde chromatolysis [10]. Retrograde transport of horseradish peroxidase (HRP) in the pelwc nerve of the c~t and monkey was employed to localize preganglionic neurons in the sacral spinal cord [9]. In each of these studies, the cells sending fibers into the pelvic nerve were localized in a nucleus in the lateral intermediate gray of the sacral spinal cord. The longitudinal distribution of the neurons was variable; generally the cells extended over two segments, primarily $2 and $3. Coggeshall et al. [ 2] observed that the L6-$1 ventral roots in t h e m t contained comparatively large numbers of unmyelinated 8rid small myelinated fibers and postulated that ~he autonomic outfloWOf the pelvis takes origin in these spinal segments in the rat. They were stvrprised at the,relatively more rostral level of this outflow compared to that in other mammal~,:a finding which requires confirmation. Recently, :the authors [5] observed that the majority of preganglionic neur0nsWith axons c o ~ g in thehypogastric nerve of the mt are located al0ng t h e / ~ d ~ e ; in thedorsal gray commissure 0f spinal segments LrL2, a finding: ~ c t l y c o n l ~ 7 t o the results of previous studies in which sympathetic p ~ g h ' 0 n i C neur6~i!~ze~ ~l ~ e d ~ p ~ a r i l y t? the lateralintermediate gmy~[l~3~i~i~13];iThe ~ U e l o c a t i o n of the majority of preganglionic neurons innel~t~gii~thepelvic,--vis~m,in the rat ~ the hypogastric nen,e suggests the
possibility that the sacral preganglionic cells may too have an unpredictable distribution in the spinal gray. The r~:trograde transport of HRP was used in the present experiments in order to confirm the level of autonomic outflow to the pelvic viscera in the rat and to determine the cross.sectional distribution of preganglionic neurons contributing fibers to the pelvic nerve. Experiments were performed on adult male rats (Sprague--Dawley). The animals were anesthetized with an intraperitoneal injection of a solution of urethane (500 mg/kg) and nembutal (30 mg/kg) and the pelvic viscera were exposed. The pelvic nerve was i~entified as is coursed medially to the major pelvic g~glion where it was cut and dissected laterally. The nerve was then immersed in a molten low melting point wax which was allowed to solidify. The soft wax was gently packed down around the exposed end of the nerve in order to. form a seal and to provide a depression in which a drop of 30% solution of horseradish peroxidase (HRP, Sigma type VI) in saline would be placed. The cut end of the pelvic nerve was exposed to the HRP solution for three hc,urs. Following a survival period of.three days, the animals were anesthetized and perfused transcardially with a solution of 0.4% paraformaldehyde and 2.4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The lower lumbar and sacral spinal cord was immediately removed and placed in fixative for about two hours and then transferred to a 30% sucrose solution in which it remained overnight. Frozen sections (35 ~m) were cut and reacted with tetramethylbenzidine [6]. The sections were mounted and counterstained with neutral red. Pregsnglionic neurons, labeled by the retrograde transport of H R P in the pelvic nerve, were observed in three animals. Negative results obtained in several other animals may have been due to either inadequate perfusion and fixation or excessive trauma to the pelvic nerve as it was dissected. HRP-filled preganglionic neurons were present in spinal segments L~-S~;the majority of the cel~ were located in segment Sz. In transverse sections,the cellswere most frequently observed in tight clustersin the lateralintermediate gray (Fig. 1). These ctustersoften extended laterally,ventral to the zona reticularis,~ving the appearance of a distinct'lateralhorn'. In other sections,labeled cellswere aggrega~l along the lateralborder of the intermediate gray and were generally aligned with the border (Fig. 2), Occasionally, labeled cellswere seen in the middle of the intermediate gray; these cellswere generally oriented horizontally. Nc, labeled cellswere observed near the central canal or in the contralateral[~ay matter. In severalsections, HRP.filled neurons were located in the dorsal part of the ventral horn in association with somatic motor nuclei. Labeled preganglionic neurons were polygonal to fusiform in shape with a range'o:~mean diameters of 13--32 ~ m and an average mean diameter of 20 ~m. Approximately 3501 labeled cellswer e present in each of two mdmals in which cell counts were made. Labeled axons were not observed. The results ofi~epresent experiments ~have shownthat the cellsof originof pregan~!ionic fibers coursing in the pelvic nerve in the rat are located in spinal ,,.
,
.,
~
.
.
.
•
.
-
segments L6-Sz, a finding which confirms the observations of Coggeshall e t a ] . [2] who expressed concern over the comparatively more rostra] preganglionic outflow from this region of the cord in the rat as determined from their s t u d y of ventral r o o t fiber composition.
Fig. 1. Transverse section of the Ss spinal segment containing HRP filled preganglionie neurons in the right lateral intermediate gray. The calibration bar repre lents 200 ~m.
Fig. 2. Transverse sect/on o f L 6 spinal cord contalninglsb~led ~eIls along the right border of the intermediate gray. A small HRP-fdledneuron (indicated by arrow) is present medial to a somatic. :motor nucleus in the dorsal pa~t of the:,~entral horn.,The,.ealibration, bar represents I00 ~m.
The present authors have recently shown that the majority of the cells of origin of preganglionic fibers coursing in the hypogastric nerve of the rat are situated along the midline of spinal segments LI-L2, in the dorsal gray comm!~ure [ 5 ] . The transverse distribution of sympathetic preganglionic neurons at more rostral levels of the rat cord appears to be similar to that observed in other mammals; the majority of these cells are. located in the lateral intermediate gray matter. The location of cells sending fibers into the hypogastric nerve in the rat is comparable to that of preganglionic neurons in the pigeon which form a column, dorsal to the central canal [ 7,8]. This column (column of Terni) has both thoraco-lumbsr and sacral components; the latter is thought to represent the sacral componen't of the parasy mpathetic division of the autonomic nervous system [7]. The present experiments were thus prompted to determine whether a midline preganglionic cell column, with fiber projections in the pelvic nerve, also exists in the sacral cord of the rat. The results of this study provide no evidence of such a midline column, and show that the transverse distribution of sacral preganglionic neurons in the rat spinal cord is similar to that described for other mammals. A few labeled cells were seen in the dorsal part of the ventral horn in proximity to somatic motoneurons. Oliver et al. [10] reported observing an occasional chromatolytic neuron in this region of the ventral horn in the cat following repeated pelvic nerve sections. REFERENCES
1 Chung, J.M., Chung, K. and Wurster, R., Sympathetic preganglionic neurons of the cat spinal cord: horseradish peroxidase study~ Brain Res., 91 (1975) 126--131. 2 Coggeshall, R.E., Emery, D.G., Ito, H. and Maynard, C.W., Unmyelinated and small myelinated axons in rat ventral roots, J. comp. Neurol., 173 (1977) 175--184. 3 Cummings, J.F., Thoracolumbar preganglionic neurons and adrenal innervation in the dog, Acta Anat., 73 (1969) 27--37. 4 DeGroat, W.C. and Ryall, R.W., The identification and characteristics of sacral parasympathetic preganglionic neurones, J. Physiol. (Lond.), 196 (1968) 563--577. 5 Hancock, M.B. and Peveto, C.A., A preganglionic autonomic nucleus in the dorsal gray commissure of the lumbar spinal cord in the rat, J. comp. Neurol., in press. 6 Hardy, H. and Helmet, L., A safer and more sensitive substitute for diaminobenzidine in the light microscopic demonstration of retrograde and anterograde axonal transport of HRP, Neurosei. Le~t., 5 (1977) 235--240. 7 Huber, J.F., Nerve roots and nuclear groups in the spinal cord in the pigeon, J. comp. Neurol., 65 (1936) 43---91. 8 MacDonald, R.L. and Cohen, D.H., Cells of origin of sympathetic pre- and postganglionic cardioaceeleratory fibers in the pigeon, J. comp. Neurol., 140 (1970) 343--357. 9 Nadelhaft, I., Morgan, C., Schauble, T. and DeGroat, W.C., Localization of the sacral autonomic (parasympathetic) nucleus in the spinal cord of the cat and monkey by the horseradish peroxidase technique, Neurosci. A~str., 3 (1977) 24. 10 Oliver, J.E., Jr., Bradley, W.E. and Fletcher, T.F., Identification of preganglionic parasympathetic neurons in the sacral spinal cord of the cat, J. comp. Neurt~l., 137 (1969) 321--328. 11 Petras, J.M. and Fadert, A.I., The origin of sympathetic preganglionic neurons in the
dog, Brain. Res., 144 (1978) 353--357. 12 Sehnitzlein, H.M., Hoffman, H.H:, Hamlett, D.M. and Howell, E.M., A study of the sacral parasympathetic nucleus, J. comp. Neurol., 137 (1969) 329--346. 13 Schramm, L , Adair, J.R., Stribling, J. and Gray, L., Preganglionic innervation of the adrenal gland of the rat: a study using horseradish peroxidase, Exp. Ne,rol., 49 (1975) 540--553.
