- Email: [email protected]
Immunoreactivity for glutamic acid decarboxylase and several neuropeptides in the spinal cord of the raccoon
Brain
Research
Bullerin.
0361-9230190 $3.00 + .OO
Vol. 25. pp. 7X7-790. 0 Pergamon Press plc, 1990. Printed in the U.S.A
BRIEF COMMUNICATION
Immunoreactivity for Glutamic Acid Decarboxylase and Several Neuropeptides in the Spinal Cord of the Raccoon EDWARD K. MARK,;‘: DAVID H. HAUGE,” GREGG P. STANDAGE? AND GERNOT S. DOETSCH*$’ *Department of Surgery (Section of Neurosurgery), i-Department of Anatomy and $Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Received
GA 30912
27 July 1990
AND G. S. DOETSCH. hmunureactivifyfor glurumic acid decarboxylase and BRAIN RES BULL 25(5) 787-790, 1990.-The peroxidase-antiperoxidase method was used to examine major immunohistochemical features of the spinal cord of adult raccoons. The lateral portions of the ventral horn contained many large multipolar neurons that showed cholecystokinin-like immunoreactivity, suggesting the coexistence of cholecystokinin with acetylcholine in a subset of motoneurons. The dorsal horn revealed unique but overlapping patterns of immunoreactivity for glutamic acid decarboxylase. somatostatin, substance P, vasoactive intestinal polypeptide and cholecystokinin. The data imply that some of the peptides may coexist within the same dorsal root ganglion cells and their spinal cord processes. MARK, E. K., D. H. HAUGE.
several neuropeprides
Neurotransmitters
G. P. STANDAGE
in the spinal cord of rhe raccoon.
Neuropeptides
Spinal cord
Raccoon
stored in trays containing PBS. The free floating sections were prepared for immunohistochemistry using the peroxidase-antiperoxidase technique of Stemberger (13). Alternate sections were processed with primary antisera directed against choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), cholecystokinin (CCK), somatostatin (SOM), substance P (SP) or vasoactive intestinal polypeptide (VIP). (All antisera except that for GAD were obtained from INCSTAR Corp.; GAD antiserum was provided by D. E. Schmechel, Duke University.) Every 10th or 20th section was stained with cresyl violet for study of cytoarchitecture. The sections were mounted on glass slides and examined with light microscopy.
LITTLE is known about the localization of putative neurotransmitters and neuromodulators in the central nervous system of carnivores other than the domestic cat, and nothing is known about their distribution in the raccoon. This report summarizes some major immunohistochemical features of several neuroactive substances or their synthesizing enzymes in the spinal cord of the raccoon. METHOD
Data were obtained from cervical, thoracic and lumbosacral spinal cord segments of six adult raccoons (Procyon lotor). Each animal was deeply anesthetized with pentobarbital sodium and was perfused through the heart with 0.9% saline, followed by a fixative solution of either periodate-lysine-paraformaldehyde (12) or 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). About one hour later, the vasculature was perfused with cold 0.1 M PBS; the spinal cord was then removed and cut into several blocks which were placed in a cold solution of 30% sucrose in 0.1 M PBS and stored at 4°C. After the tissue had equilibrated, selected segments from each major spinal cord level were frozen and cut on a cryostat into 40 p,rn thick coronal sections that were
‘Requests for reprints should be addressed
to Dr. Gemot S. Doetsch.
RESULTS
AND DISCUSSION
As expected, the most prominent feature of immunoreactivity in the ventral and intermediate horns was the presence, at all spinal levels, of neurons positively stained for ChAT, the synthesizing enzyme for acetylcholine. Large and medium-sized ChATimmunoreactive cells, presumed to be motoneurons, were located in the lateral and medial portions of the ventral horn. These cells had multipolar somata and gave rise to ChAT-positive axons that
Section of Neurosurgery,
787
Medical College of Georgia,
Augusta,
GA 30912.
788
MARK, HAUGE, STANDAGE AND DOETSCH
FIG. 1. Photomicrographs showing cholecystokinin-like immunoreactivity in the ventral horn of transverse lumbosacral secttons of the raccoon spinal cord. (A) Low-power photograph of large and medium-sized CCK-positive neurons that are differentially located in the lateral part of the ventral horn. Scale = 0.5 mm. (B-D) High-power photographs of CCK-labeled multipolar neurons and their processes in the ventrolateral gray matter. Note the presence of beaded CCK-positive nerve fibers and terminals. Scale = 100 km.
could be traced out of the gray matter toward a ventral spinal nerve root. In tboracic and lumbosacral segments, small and medium-sized neurons immunoreactive for ChAT were located in the lateral portion of the intermediate gray matter; these cells were multipolar in shape and probably were preganglionic autonomic neurons. Surprisingly, me intermediate and ventral gray matter also contained neurons that demonstrated CCK-like immunoreactivity.
A few CCK-positive cells were scattered throughout the intermediate horn, both laterally and medially near the central canal. In contrast, many large and medium-sized multipolar cells in the ventral horn, with features characteristic of motoneurons, were labeled for CCK (Fig. 1). The vast majority of these neurons were located in the lateral part of the ventral gray matter; few were present in the ventromedial gray. This finding has not been reported previously in any species, and suggests that CCK may be colocalized with acetylcholine in a specific subpopulation of spinal cord motoneurons. Many CCK-positive fibers and terminals also were present in the ventral horn; some of the fibers appeared to terminate on CCK-containing neurons and others on unlabeled cells. The most salient feature of immunoreactivity in the dorsal horn was the existence of overlapping distributions of labeling for GAD and for each of the peptides in cervical, thoracic and lumbosacral segments (Fig. 2). In most cases, the immunoreactivity
was dominated by moderate to dense staining of nerve fibers and fiber terminals which tended to obscure the presence of scattered lightly stained neurons. More labeled neurons might have been seen if axoplasmic transport had been blocked by pretreatment with colchicine. Darkly stained GAD-immunoreactive nerve fibers and punctate terminals were located throughout Rexed’s lamina I (marginal zone), lamina II (substantia gelatinosa) and lamina III (Fig. 2A). The density of immunoreactivity typically was greater in laminae I and III than in lamina II. This produced a striped pattern of GAD immunoreactivity consisting of a thin dark band located dorsally and separated by a narrow light stripe from a wider dark band located more ventrally. Most of the staining consisted of densely packed GAD-positive terminals that tended to be arranged in clusters. However, transverse mediolaterally oriented GAD-immunoreactive fibers were present in lamina I; other fibers were found to cross the midline near the central canal. Immunoreactivity for the peptide SOM was also found in laminae I, II and III of the dorsal horn (Fig. 2B). Light to moderately stained nerve fibers and terminals were distributed throughout this region, with the most intense labeling usually present in lamina II. The resulting pattern of SOM-positive bands tended to be the converse of that for GAD, with lamina II being the darkest for SOM and the lightest for GAD. SP immunoreactivity was concentrated in lamina I and lamina
IMMUNOREACTIVITY
IN SPINAL
CORD
OF RACCOON
FIG. 2. Photomicrographs demonstrating the patterns of immunoreactivity of nerve fibers and terminals for five different substances in the dorsal horn of the raccoon spinal cord. B, C and E show transverse sections taken from lower lumbar levels; A and D from midsacral levels. (A) Glutamic acid decarboxylase; (B) somatostatin; (0 substance P; (D) vasoactive intestinal ~ly~ptide~ (E) cholecystokinin. Note the overlapping but differential dis~butions of i~unoreacfi~ity, Scale =0.5 mm.
790
MARK. HAUGE. STANDAGt:
II. forming a single band of labeling (Fig. 2C). Heavily stained and densely packed fiber terminals were located throughout this most superficial region of the dorsal horn. In addition, many transversely oriented SP-positive nerve fibers were found to curve ventrolaterally and to invade deeper parts of the dorsal gray matter, primarily laminae IV and V. Other fibers took a ventromedial course and crossed the midline near the central canal. Immunoreactivity for VIP (Fig. 2D) was found to be greater at lumbosacral levels than at more rostra1 levels of the spinal cord. Most VIP-labeled nerve fibers and terminals were lightly stained and were located in laminae I and II. Mediolaterally oriented fibers were found to travel ventrally into laminae IV and V and into even deeper regions near the central canal; a few fibers were found to cross the midline. CCK immunoreactivity (Fig. 2E) was distributed in a pattern similar to that for VIP. Light to moderately stained nerve fibers and terminals were located throughout laminae I and II, with the most intense labeling found in lamina I. Scattered CCK-positive terminals were also present in deeper regions of the dorsal horn. As with other peptides, CCK-immunoreactive fibers were found to travel ventrally into the gray matter of layers IV and V and some even deeper into the intermediate horn; a few fibers crossed the midline near the central canal. In summary, the present results indicate that the spinal cord of the raccoon contains many of the same neurotransmitters and neuropeptides found in other mammals (1-3, 5-IO), with similar spatial distributions. The data suggest that, in the raccoon, CCK may coexist with acetylcholine in a particular subset of lateral
AND DOETSCH
ventral horn motoneurons. A few peptides- but not CCK- have previously been found in spinal cord motoneurons of other species. For example, calcitonin gene-related present in motoneurons of a wide variety
peptide (CGRP, i> of mammals (4) ,mci
thyrotropin releasing hormone appears to be localized in some ventral horn cells of the human (2). Furthermore, motoneurons with immunoreactivity for VIP or SOM, ah well as CGRP, have been identified in the chick embryo (14). Further experiments are required to confirm the coexistence of peptides with acetylcholine in motoneurons of the raccoon. Finally, the results showed that nerve fibers and terminals in the dorsal horn display overlapping but differential patterns of immunoreactivity for neuropeptides SOM, SP. VIP and CCK and for the inhibitory transmitter gamma-aminobutyric acid which is synthesized by GAD. This finding suggests that some of these peptides may coexist in the same dorsal root ganglion cells and their central processes, as has been found in other species (11). All of the present data support the view that the dorsal horn is a region of complex information processing, where afferent signals can be modulated by local and intersegmental spinal cord circuits as well as descending influences from higher brain centers (8). ACKNOWLEDGEMENTS We are grateful to Dr. C.-S. Lin, S. M. Lu, P. Wade and L. Baker for their technical assistance and W. Taylor for typing the manuscript. This work was supported by NSF Grant BNS-8419035 to G.S.D. and NIH Grant NS21935 to G.P.S.
REFERENCES Barber, R. P.; Vaughn, J. E.; Saito, K.; McLaughlin, B. J.; Roberts, E. GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord. Brain Res. 141:35-55; 1978. Chung, K.; Briner, R. P.; Carlton, S. M.; Westlund, K. N. Immunohistochemical localization of seven different peptides in the human spinal cord. .I. Comp. Neurol. 280: 158-170; 1989. DiFiglia, M.; Aronin, N.; Leeman, S. E. Light microscopic and ultrastructural localization of immunoreactive substance P in the dorsal horn of monkey spinal cord. Neuroscience 7:1127-l 139; 1982. Gibson, S. J.; Polak, J. M.; Bloom, S. R.; Sabate, I. M.; Mulderry, P. M.; Ghatei, M. A.; McGregor, G. P.; Morrison, J. F. B.; Kelly, J. S.; Evans, R. M.; Rosenfeld, M. G. Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J. Neurosci. 4:3101-3111; 1984. Gibson, S. J.; Polak, J. M.; Bloom, S. R.; Wall, P. D. The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X). J. Comp. Neurol. 201:65-79; 1981. Ho, R. H.; Berelowitz, M. Somatostatin 28,_,, immunoreactivity in primary afferent neurons of the rat spinal cord. Neurosci. Lett. 46: 161-166; 1984. Kimura, H.; McGeer, P. L.; Peng, J. H.; McGeer, E. G. The central cholinergic system studied by choline acetyltransferase immuno-
histochemistry in the cat. J. Comp. Neurol. 200:151-201; 1981. 8. LaMotte, C. C. Organization of dorsal horn neurotransmitter systems. In: Yaksh, T. L., ed. Spinal afferent processing. New York: Plenum Press; 1986:97-l 16. 9. LaMotte, C. C.; de Lanerolle, N. C. VIP terminals, axons, and neurons: distribution throughout the length of monkey and cat spinal cord. J. Comp. Neurol. 249:133-145; 1986. 10. Larsson, L.-I.; Rehfeld, J. F. Localization and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system. Brain Res. 165:201-218; 1979. 11. Leah, J. D.; Cameron, A. A.; Kelly, W. L.; Snow. P. J. Coexistence of peptide immunoreactivity in sensory neurons of the cat. Neuroscience 16:683-690; 1985. 12. McLean, I. W.; Nakane, P. K. Periodate-lysine-paraformaldehyde fixative-a new fixative for immunoelectron-microscopy. J. Histochem. Cytochem. 22:1077-1083; 1974. 13. Stemberger, L. A. Immunocytochemistry. New York: J. Wiley and Sons; 1979. 14. Villar, M. J.; Huchet, M.; Hokfelt, T.; Changeux, J.-P.; Fahrenkrug, J.; Brown, J. C. Existence and coexistence of calcitonin gene-related peptide, vasoactive intestinal polypeptide- and somatostatin-like immunoreactivities in spinal cord motoneurons of developing embryos and post-hatch chicks. Neurosci. Lett. 86:114-l 18; 1988.
Research
Bullerin.
0361-9230190 $3.00 + .OO
Vol. 25. pp. 7X7-790. 0 Pergamon Press plc, 1990. Printed in the U.S.A
BRIEF COMMUNICATION
Immunoreactivity for Glutamic Acid Decarboxylase and Several Neuropeptides in the Spinal Cord of the Raccoon EDWARD K. MARK,;‘: DAVID H. HAUGE,” GREGG P. STANDAGE? AND GERNOT S. DOETSCH*$’ *Department of Surgery (Section of Neurosurgery), i-Department of Anatomy and $Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Received
GA 30912
27 July 1990
AND G. S. DOETSCH. hmunureactivifyfor glurumic acid decarboxylase and BRAIN RES BULL 25(5) 787-790, 1990.-The peroxidase-antiperoxidase method was used to examine major immunohistochemical features of the spinal cord of adult raccoons. The lateral portions of the ventral horn contained many large multipolar neurons that showed cholecystokinin-like immunoreactivity, suggesting the coexistence of cholecystokinin with acetylcholine in a subset of motoneurons. The dorsal horn revealed unique but overlapping patterns of immunoreactivity for glutamic acid decarboxylase. somatostatin, substance P, vasoactive intestinal polypeptide and cholecystokinin. The data imply that some of the peptides may coexist within the same dorsal root ganglion cells and their spinal cord processes. MARK, E. K., D. H. HAUGE.
several neuropeprides
Neurotransmitters
G. P. STANDAGE
in the spinal cord of rhe raccoon.
Neuropeptides
Spinal cord
Raccoon
stored in trays containing PBS. The free floating sections were prepared for immunohistochemistry using the peroxidase-antiperoxidase technique of Stemberger (13). Alternate sections were processed with primary antisera directed against choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), cholecystokinin (CCK), somatostatin (SOM), substance P (SP) or vasoactive intestinal polypeptide (VIP). (All antisera except that for GAD were obtained from INCSTAR Corp.; GAD antiserum was provided by D. E. Schmechel, Duke University.) Every 10th or 20th section was stained with cresyl violet for study of cytoarchitecture. The sections were mounted on glass slides and examined with light microscopy.
LITTLE is known about the localization of putative neurotransmitters and neuromodulators in the central nervous system of carnivores other than the domestic cat, and nothing is known about their distribution in the raccoon. This report summarizes some major immunohistochemical features of several neuroactive substances or their synthesizing enzymes in the spinal cord of the raccoon. METHOD
Data were obtained from cervical, thoracic and lumbosacral spinal cord segments of six adult raccoons (Procyon lotor). Each animal was deeply anesthetized with pentobarbital sodium and was perfused through the heart with 0.9% saline, followed by a fixative solution of either periodate-lysine-paraformaldehyde (12) or 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). About one hour later, the vasculature was perfused with cold 0.1 M PBS; the spinal cord was then removed and cut into several blocks which were placed in a cold solution of 30% sucrose in 0.1 M PBS and stored at 4°C. After the tissue had equilibrated, selected segments from each major spinal cord level were frozen and cut on a cryostat into 40 p,rn thick coronal sections that were
‘Requests for reprints should be addressed
to Dr. Gemot S. Doetsch.
RESULTS
AND DISCUSSION
As expected, the most prominent feature of immunoreactivity in the ventral and intermediate horns was the presence, at all spinal levels, of neurons positively stained for ChAT, the synthesizing enzyme for acetylcholine. Large and medium-sized ChATimmunoreactive cells, presumed to be motoneurons, were located in the lateral and medial portions of the ventral horn. These cells had multipolar somata and gave rise to ChAT-positive axons that
Section of Neurosurgery,
787
Medical College of Georgia,
Augusta,
GA 30912.
788
MARK, HAUGE, STANDAGE AND DOETSCH
FIG. 1. Photomicrographs showing cholecystokinin-like immunoreactivity in the ventral horn of transverse lumbosacral secttons of the raccoon spinal cord. (A) Low-power photograph of large and medium-sized CCK-positive neurons that are differentially located in the lateral part of the ventral horn. Scale = 0.5 mm. (B-D) High-power photographs of CCK-labeled multipolar neurons and their processes in the ventrolateral gray matter. Note the presence of beaded CCK-positive nerve fibers and terminals. Scale = 100 km.
could be traced out of the gray matter toward a ventral spinal nerve root. In tboracic and lumbosacral segments, small and medium-sized neurons immunoreactive for ChAT were located in the lateral portion of the intermediate gray matter; these cells were multipolar in shape and probably were preganglionic autonomic neurons. Surprisingly, me intermediate and ventral gray matter also contained neurons that demonstrated CCK-like immunoreactivity.
A few CCK-positive cells were scattered throughout the intermediate horn, both laterally and medially near the central canal. In contrast, many large and medium-sized multipolar cells in the ventral horn, with features characteristic of motoneurons, were labeled for CCK (Fig. 1). The vast majority of these neurons were located in the lateral part of the ventral gray matter; few were present in the ventromedial gray. This finding has not been reported previously in any species, and suggests that CCK may be colocalized with acetylcholine in a specific subpopulation of spinal cord motoneurons. Many CCK-positive fibers and terminals also were present in the ventral horn; some of the fibers appeared to terminate on CCK-containing neurons and others on unlabeled cells. The most salient feature of immunoreactivity in the dorsal horn was the existence of overlapping distributions of labeling for GAD and for each of the peptides in cervical, thoracic and lumbosacral segments (Fig. 2). In most cases, the immunoreactivity
was dominated by moderate to dense staining of nerve fibers and fiber terminals which tended to obscure the presence of scattered lightly stained neurons. More labeled neurons might have been seen if axoplasmic transport had been blocked by pretreatment with colchicine. Darkly stained GAD-immunoreactive nerve fibers and punctate terminals were located throughout Rexed’s lamina I (marginal zone), lamina II (substantia gelatinosa) and lamina III (Fig. 2A). The density of immunoreactivity typically was greater in laminae I and III than in lamina II. This produced a striped pattern of GAD immunoreactivity consisting of a thin dark band located dorsally and separated by a narrow light stripe from a wider dark band located more ventrally. Most of the staining consisted of densely packed GAD-positive terminals that tended to be arranged in clusters. However, transverse mediolaterally oriented GAD-immunoreactive fibers were present in lamina I; other fibers were found to cross the midline near the central canal. Immunoreactivity for the peptide SOM was also found in laminae I, II and III of the dorsal horn (Fig. 2B). Light to moderately stained nerve fibers and terminals were distributed throughout this region, with the most intense labeling usually present in lamina II. The resulting pattern of SOM-positive bands tended to be the converse of that for GAD, with lamina II being the darkest for SOM and the lightest for GAD. SP immunoreactivity was concentrated in lamina I and lamina
IMMUNOREACTIVITY
IN SPINAL
CORD
OF RACCOON
FIG. 2. Photomicrographs demonstrating the patterns of immunoreactivity of nerve fibers and terminals for five different substances in the dorsal horn of the raccoon spinal cord. B, C and E show transverse sections taken from lower lumbar levels; A and D from midsacral levels. (A) Glutamic acid decarboxylase; (B) somatostatin; (0 substance P; (D) vasoactive intestinal ~ly~ptide~ (E) cholecystokinin. Note the overlapping but differential dis~butions of i~unoreacfi~ity, Scale =0.5 mm.
790
MARK. HAUGE. STANDAGt:
II. forming a single band of labeling (Fig. 2C). Heavily stained and densely packed fiber terminals were located throughout this most superficial region of the dorsal horn. In addition, many transversely oriented SP-positive nerve fibers were found to curve ventrolaterally and to invade deeper parts of the dorsal gray matter, primarily laminae IV and V. Other fibers took a ventromedial course and crossed the midline near the central canal. Immunoreactivity for VIP (Fig. 2D) was found to be greater at lumbosacral levels than at more rostra1 levels of the spinal cord. Most VIP-labeled nerve fibers and terminals were lightly stained and were located in laminae I and II. Mediolaterally oriented fibers were found to travel ventrally into laminae IV and V and into even deeper regions near the central canal; a few fibers were found to cross the midline. CCK immunoreactivity (Fig. 2E) was distributed in a pattern similar to that for VIP. Light to moderately stained nerve fibers and terminals were located throughout laminae I and II, with the most intense labeling found in lamina I. Scattered CCK-positive terminals were also present in deeper regions of the dorsal horn. As with other peptides, CCK-immunoreactive fibers were found to travel ventrally into the gray matter of layers IV and V and some even deeper into the intermediate horn; a few fibers crossed the midline near the central canal. In summary, the present results indicate that the spinal cord of the raccoon contains many of the same neurotransmitters and neuropeptides found in other mammals (1-3, 5-IO), with similar spatial distributions. The data suggest that, in the raccoon, CCK may coexist with acetylcholine in a particular subset of lateral
AND DOETSCH
ventral horn motoneurons. A few peptides- but not CCK- have previously been found in spinal cord motoneurons of other species. For example, calcitonin gene-related present in motoneurons of a wide variety
peptide (CGRP, i> of mammals (4) ,mci
thyrotropin releasing hormone appears to be localized in some ventral horn cells of the human (2). Furthermore, motoneurons with immunoreactivity for VIP or SOM, ah well as CGRP, have been identified in the chick embryo (14). Further experiments are required to confirm the coexistence of peptides with acetylcholine in motoneurons of the raccoon. Finally, the results showed that nerve fibers and terminals in the dorsal horn display overlapping but differential patterns of immunoreactivity for neuropeptides SOM, SP. VIP and CCK and for the inhibitory transmitter gamma-aminobutyric acid which is synthesized by GAD. This finding suggests that some of these peptides may coexist in the same dorsal root ganglion cells and their central processes, as has been found in other species (11). All of the present data support the view that the dorsal horn is a region of complex information processing, where afferent signals can be modulated by local and intersegmental spinal cord circuits as well as descending influences from higher brain centers (8). ACKNOWLEDGEMENTS We are grateful to Dr. C.-S. Lin, S. M. Lu, P. Wade and L. Baker for their technical assistance and W. Taylor for typing the manuscript. This work was supported by NSF Grant BNS-8419035 to G.S.D. and NIH Grant NS21935 to G.P.S.
REFERENCES Barber, R. P.; Vaughn, J. E.; Saito, K.; McLaughlin, B. J.; Roberts, E. GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord. Brain Res. 141:35-55; 1978. Chung, K.; Briner, R. P.; Carlton, S. M.; Westlund, K. N. Immunohistochemical localization of seven different peptides in the human spinal cord. .I. Comp. Neurol. 280: 158-170; 1989. DiFiglia, M.; Aronin, N.; Leeman, S. E. Light microscopic and ultrastructural localization of immunoreactive substance P in the dorsal horn of monkey spinal cord. Neuroscience 7:1127-l 139; 1982. Gibson, S. J.; Polak, J. M.; Bloom, S. R.; Sabate, I. M.; Mulderry, P. M.; Ghatei, M. A.; McGregor, G. P.; Morrison, J. F. B.; Kelly, J. S.; Evans, R. M.; Rosenfeld, M. G. Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J. Neurosci. 4:3101-3111; 1984. Gibson, S. J.; Polak, J. M.; Bloom, S. R.; Wall, P. D. The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X). J. Comp. Neurol. 201:65-79; 1981. Ho, R. H.; Berelowitz, M. Somatostatin 28,_,, immunoreactivity in primary afferent neurons of the rat spinal cord. Neurosci. Lett. 46: 161-166; 1984. Kimura, H.; McGeer, P. L.; Peng, J. H.; McGeer, E. G. The central cholinergic system studied by choline acetyltransferase immuno-
histochemistry in the cat. J. Comp. Neurol. 200:151-201; 1981. 8. LaMotte, C. C. Organization of dorsal horn neurotransmitter systems. In: Yaksh, T. L., ed. Spinal afferent processing. New York: Plenum Press; 1986:97-l 16. 9. LaMotte, C. C.; de Lanerolle, N. C. VIP terminals, axons, and neurons: distribution throughout the length of monkey and cat spinal cord. J. Comp. Neurol. 249:133-145; 1986. 10. Larsson, L.-I.; Rehfeld, J. F. Localization and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system. Brain Res. 165:201-218; 1979. 11. Leah, J. D.; Cameron, A. A.; Kelly, W. L.; Snow. P. J. Coexistence of peptide immunoreactivity in sensory neurons of the cat. Neuroscience 16:683-690; 1985. 12. McLean, I. W.; Nakane, P. K. Periodate-lysine-paraformaldehyde fixative-a new fixative for immunoelectron-microscopy. J. Histochem. Cytochem. 22:1077-1083; 1974. 13. Stemberger, L. A. Immunocytochemistry. New York: J. Wiley and Sons; 1979. 14. Villar, M. J.; Huchet, M.; Hokfelt, T.; Changeux, J.-P.; Fahrenkrug, J.; Brown, J. C. Existence and coexistence of calcitonin gene-related peptide, vasoactive intestinal polypeptide- and somatostatin-like immunoreactivities in spinal cord motoneurons of developing embryos and post-hatch chicks. Neurosci. Lett. 86:114-l 18; 1988.