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Spinothalamic cells in the rat lumbar cord with collaterals to the medullary reticular formation
Brain Research, 238 (1982) 181-185
181
Elsevier Biomedical Press
Short Communications Spinothalamic cells in the rat lumbar cord with collaterals to the medullary reticular formation
GOLDA ANNE KEVETTER and WILLIAM D. WILLIS Marine Biomedical Institute, Departments of Anatomy and of Physiology and Biophysics, University of Texas Medical Branch, 200 University Boulevard, Galveston, TX 77550 (U.S.A.)
(Accepted December 24th, 1981) Key words: spinoreticular tract -- spinothalamic tract - - pain pathways - - spinal systems
Cells with dual projections to both the thalamus and the medullary reUcular formation were identified in the rat lumbar cord after separate injections of different retrogradely transported markers into each area. Most double-labeled cells were located in the intermediate zone or in lamina V of the contralateral spinal cord. The distribution in the spinal cord of the cells which project to either the thalamus 7,17 or the reticular formationl,Z,5,9-11,14 has been examined for several mammalian species. Although the distributions differ in some respects, cells in each pathway are located in lamina V of the dorsal horn and dorsomedially in the ventral horn1,2,5,9-11, ~7. This overlap in distribution supports Mehler's proposal that cells projecting in the phylogenetically oldest part of the spinothalamic tract also send collaterals into the medial medullary reticular formation lz,1~. Spinothalamic cells with collaterals in the reticular formation have been demonstrated with antidromic activation techniques6,S, 9. This report examines the distribution of cells in the lumbar cord of the rat which project to both the thalamus and to the medullary reticular formation. Albino rats weighing 250-500 g were anesthetized with sodium pentobarbital (36 mg/kg) and positioned in a stereotaxic apparatus. The fourth ventricle was exposed and 0.5/~1 of either 1 ~o Nuclear Yellow (Hoechst Co.) or 30 ~o horseradish peroxidase (HRP, Miles Laboratory) was injected into the medullary reticular formation through a micropipette. Two 0.5 /A injections of 1 ~ 4',6-diamidino-2-phenylindole 2HC1 (DAPI, Sigma) were stereotaxically injected into both the medial and lateral thalamus. After a 6-day survival period, the animals were reanesthetized, perfused intracardially with warm physiological saline, followed by cold 3.5~o paraformaldehyde in 0.1 M phosphate buffer, and then by 25 ~o sucrose in 0.1 M phosphate buffer is. The brain and spinal cord were removed and stored in the sucrose-phosphate solution in the refrigerator overnight. Thirty/zm sections through the lumbar spinal cord and the injection sites were cut on a freezing microtome. In the animals (n = 3) in which H R P was injected into the reticular formation, the spinal sections were reacted with
182
DAP
Z
B
~SRT C
Fig. 1. A and B: schematic representation of injection of fluorescent dye, DAPI, into both the medial and lateral thalamus. C: representation of injection of HRP into the medial medullary reticular formation. The most concentrated area of injection is shown in black, the intermediate areas are stippled, while areas adjacent to the injection site are outlined by dashed lines. D-F: distribution of labeled ceils in a representative section of lumbar cord. D: distribution of spinothalamic cells, labeled after injection of DAPI into the thalamus. E: distribution of cells in the lumbar cord which contained label from both injections shown in A-C. These cells project to both the thalamus and medullary reticular formation. F: distribution of ceils in the lumbar cord which contained HRP reaction product after HRP injection into the medial reticular formation shown in C. C, nucleus cuneatus, Cn, central nucleus of the medulla; DM, dorsomedial nucleus of the thalamus; G, nucleus gracilis; IP, interpeduncular nucleus; L, lateral reticular nucleus; LG, lateral geniculate; LHA, lateral hypothalamic area; MG, medial geniculate; ML, medial lemniscus; P, pyramids; PC, cerebral peduncles; VE, ventral N of thalamus; VD, ventral nucleus pars dorsalis of thalamus; Vsp, spinal nucleus of the trigeminal; XII, hypoglossal nucleus. tetramethylbenzidine ( T M B ) is and the injection sites with diaminobenzidine 4. H R P labeled neurons were identified with light microscopy. Fluorescence was observed with a Zeiss P h o t o m i c r o s c o p e II equipped with an epifluorescence illuminator with appropriate filters. B o t h Nuclear Yellow and D A P I were observed with a 405 nm excitation wavelength. H R P and the fluorescent dye(s) in the same cell could be accurately identified by alternating between fluorescent illumination and light microscopy. The injections o f D A P I into the thalamus included the intralaminar and ventrobasal complexes (Fig. 1A, B). Cells retrogradely labeled with D A P I from the thalamus contained blue granular fluorescence t h r o u g h the cytoplasm (Fig. 2A, right). Injections o f H R P or Nuclear Yellow into the reticular formation were centered in the central nucleus o f the medulla and the nucleus gigantocellularis (Fig. 1C). Cells labeled f r o m the reticular formation contained either a bright fluorescence uniformly distri-
183
Fig. 2. A: fluorescent photomicrograph of cell labeled with Nuclear Yellow (on left) and adjacent cell labeled with DAPI. Nuclear Yellow was injected into the reticular formation and DAPI was injected into the thalamus. Both of these cells are located in lamina X. B: double-labeled cell located in lamina VII on the side contralateral to the injections. C: labeled cell containing both Nuclear Yellow and DAPI. This cell was in lamina VII, ipsilateral to the injection. D: Double-labeled cell in lamina VII contralateral to the site of the injection.
buted in the cell nucleus (Nuclear Yellow, Fig. 2A, left) or blue TMB reaction product from H R P histochemistry. Labeled cells that projected to either the thalamus or the reticular formation or both were observed in the lumbar cord of each animal. The majority of labeled cells was found on the side contralateral to the injections (Fig. 1D-F), although a few labeled cells were found on the ipsilateral side. Spinothalamic cells were located throughout the dorsal horn and also ventromedially at the border of laminae VII and VIII of Rexed 16 (Fig. 1D). Spinoreticular cells, on the other hand, were concentrated in the ventromedial intermediate area with few labeled cells in the dorsal horn (Fig. IF). The labeled dorsal horn cells were located in the lateral aspect of lamina V. These distributions are in agreement with previous studies in rat 2,7. Those neurons that contained H R P or Nuclear Yellow transported from the reticular formation and D A P I from the thalamus were located in areas of overlap between the distributions of spinoreticular and spinothalamic tract cells. These cells were generally multipolar. Fig. 2 B - D shows examples of cells which contained both retrogradely transported D A P I and Nuclear Yellow. The double-labeled neurons were concentrated ventrally in the intermediate and ventral horn area (Fig. 1E), corres-
184 ponding to laminae VII (Fig. 2B, D) and VIII. A few double-labeled cells were also found in lamina V, especially the more lateral, reticulated part. Most double-labeled cells were located contralateral to the side o f injections (Fig. l E), although a few cells were found on the ipsilateral side (Fig. 2C). in one animal double-labeled cells were f o u n d bilaterally in the lateral spinal nucleus. There were always cells labeled from either the thalamus or reticular formation that did not project to both of these terminal fields. In this report we d o c u m e n t the existence o f a population of spinal neurons that project to both the thalamus and the medullary reticular formation. The majority of our double-labeled cells project to the contralateral brainstem and thalamus and are found in cord laminae typical for spinothalamic and spinoreticular tract neurons. The neurons that have dual projections appear to be a significant subset o f the population o f each pathway. The spinoreticular system may play a critical role in the central processing o f noxious stimuli 6,9,14. However, neurons o f this system are unlikely to be involved in discriminative functions due to their complex receptive field characteristics and polymodal response properties 3,s,9,11. Spinal cells which project to both the reticular formation and the thalamus may be involved in alerting or affecting responses to peripheral stimuli, including those associated with painS, 11-13. Cells which project exclusively to the thalamus, however, may be responsible for the more discriminative aspects o f painful, as well as non-painful, stimuli3, sAS. The authors would like to thank G. Gonzales, B. M o o r e and H. Willcockson for their fine technical help and P. W a l d r o p for typing the manuscript. This work was supported by Research Grants NS 09743 and NS 11255, by Training Grant NS 07185 f r o m the National Institutes o f Health and a grant from The M o o d y Foundation.
i Abols, I. A. and Basbaum, A. I., Afferent connections of the rostral medulla of the cat : a neural substrate for midbrain-medullary interactions in the modulation of pain, J. camp. Neural., 201 (1981) 285-297. 2 Andrezi, K. J. A., Chan-Palay, V. and Falay, S. L., q-he nucleus paragigantocellularis in the rat : demonstration of afferents by the retrograde transport of horseradish peroxidase, Anat. Embryol., 161 (1981) 373-390. 3 Casey, K. L. and Jones, E. G., An overview of ascending pathways: brainstem and thalamus, Neurosci. Res. Progr. Bull., 16 (1978) 103-118. 4 Colman, D., Scalia, F. and Cabrales, E., Light and electron microscopic observations of the anterograde transport of horseradish peroxidase m the optic pathway in the mouse and rat, Brain Research, 102 (1976) 589-596. 5 Corvaja, No, Grofova, I., Pompeiano, O. and Walberg, F., The lateral reticular nucleus in the cat - - I. An experimental anatomical study of its spinal and supraspinal afferent connections, Neuroscience, 2 (1977) 541-551. 6 Fields, H. L., Clanton, C. H. and Anderson, S. D., Somatosensory properties of spinoreticular neurons in the cat, Brain Research, 120 (1975) 49--66. 7 Giesler, G. J., Men6trey, D. and Basbaum, A. I., Differential origins of spinothalamic tract projections to medial and lateral thalamus in the rat, J. camp. Neural., 184 (1979) 107-125. 8 Giesler, G. J., Jr., Yezierski, R. P., Gerhart, K. D. and Willis, W. D., Spinothalamic tract neurons that project to medial and/or lateral thalamic nuclei: evidence for a physiologically novel population of spinal cord neurons, J. Neurophysiol., 46 (1981) 1285-1308.
185 9 Haber, L. H., Moore, B. D. and Willis, W. D., Electrophysiological response properties of spinoreticular neurons in the monkey, J. comp. Neurol., in press. l0 Kevetter, G. A., Haber, L. H., Yezierski, R. P., Chung, J. M., Martin, R. F. and Willis, W. D., Cells of origin of the spinoreticular tract in the monkey, J. comp. Neurol., in press. I l Maunz, R. A., Pitts, N. G. and Peterson, B. W., Cat spinoreticular neurons: Locations, responses and changes in responses during repetitive stimulation, Brain Research, 148 (1978) 365-379. 12 Mehler, W. R., Some neurological species differences - - a posteriori, Ann. N.Y. Acad. Sci., 167 (1969) 424-468. 13 Mehler, W. R., Feferman, M. E. and Nauta, W. J. H., Ascending axon degeneration following anterolateral cordotomy. An experimental study in the monkey, Brain, 83 (1960) 718-751. 14 Men6trey, D., Chaouch, A. and Besson, J. M., Location and properties of dorsal horn neurons at origin of spinoreticular tract in lumbar enlargement of the rat, J. Neurophysiol., 44 (1980) 862877. 15 Price, D. D. and Dubner, R., Neurons that subserve the sensoridiscriminative aspects of pain, Pain, 3 (1977) 307-338. 16 Rexed, B., The cytoarchitectonic organization of the spinal cord in the cat, J. comp. Neurol., 96 (1952) 415-496. 17 Willis, W. D., Kenshalo, D. R., Jr. and Leonard, R. B., The cells of origin of the primate spinothalamic tract, J. comp. Neurol., 188 (1979) 543-573. 18 Yezierski, R. P. and Bowker, R. M., A retrograde double label tracing technique using horseradish peroxidase and the fluorescent dye 4',6-diamidino-2-phenylindole 2HC1 (DAPI), J. Neurosci. Meth., 4 (1981) 53-62.
181
Elsevier Biomedical Press
Short Communications Spinothalamic cells in the rat lumbar cord with collaterals to the medullary reticular formation
GOLDA ANNE KEVETTER and WILLIAM D. WILLIS Marine Biomedical Institute, Departments of Anatomy and of Physiology and Biophysics, University of Texas Medical Branch, 200 University Boulevard, Galveston, TX 77550 (U.S.A.)
(Accepted December 24th, 1981) Key words: spinoreticular tract -- spinothalamic tract - - pain pathways - - spinal systems
Cells with dual projections to both the thalamus and the medullary reUcular formation were identified in the rat lumbar cord after separate injections of different retrogradely transported markers into each area. Most double-labeled cells were located in the intermediate zone or in lamina V of the contralateral spinal cord. The distribution in the spinal cord of the cells which project to either the thalamus 7,17 or the reticular formationl,Z,5,9-11,14 has been examined for several mammalian species. Although the distributions differ in some respects, cells in each pathway are located in lamina V of the dorsal horn and dorsomedially in the ventral horn1,2,5,9-11, ~7. This overlap in distribution supports Mehler's proposal that cells projecting in the phylogenetically oldest part of the spinothalamic tract also send collaterals into the medial medullary reticular formation lz,1~. Spinothalamic cells with collaterals in the reticular formation have been demonstrated with antidromic activation techniques6,S, 9. This report examines the distribution of cells in the lumbar cord of the rat which project to both the thalamus and to the medullary reticular formation. Albino rats weighing 250-500 g were anesthetized with sodium pentobarbital (36 mg/kg) and positioned in a stereotaxic apparatus. The fourth ventricle was exposed and 0.5/~1 of either 1 ~o Nuclear Yellow (Hoechst Co.) or 30 ~o horseradish peroxidase (HRP, Miles Laboratory) was injected into the medullary reticular formation through a micropipette. Two 0.5 /A injections of 1 ~ 4',6-diamidino-2-phenylindole 2HC1 (DAPI, Sigma) were stereotaxically injected into both the medial and lateral thalamus. After a 6-day survival period, the animals were reanesthetized, perfused intracardially with warm physiological saline, followed by cold 3.5~o paraformaldehyde in 0.1 M phosphate buffer, and then by 25 ~o sucrose in 0.1 M phosphate buffer is. The brain and spinal cord were removed and stored in the sucrose-phosphate solution in the refrigerator overnight. Thirty/zm sections through the lumbar spinal cord and the injection sites were cut on a freezing microtome. In the animals (n = 3) in which H R P was injected into the reticular formation, the spinal sections were reacted with
182
DAP
Z
B
~SRT C
Fig. 1. A and B: schematic representation of injection of fluorescent dye, DAPI, into both the medial and lateral thalamus. C: representation of injection of HRP into the medial medullary reticular formation. The most concentrated area of injection is shown in black, the intermediate areas are stippled, while areas adjacent to the injection site are outlined by dashed lines. D-F: distribution of labeled ceils in a representative section of lumbar cord. D: distribution of spinothalamic cells, labeled after injection of DAPI into the thalamus. E: distribution of cells in the lumbar cord which contained label from both injections shown in A-C. These cells project to both the thalamus and medullary reticular formation. F: distribution of ceils in the lumbar cord which contained HRP reaction product after HRP injection into the medial reticular formation shown in C. C, nucleus cuneatus, Cn, central nucleus of the medulla; DM, dorsomedial nucleus of the thalamus; G, nucleus gracilis; IP, interpeduncular nucleus; L, lateral reticular nucleus; LG, lateral geniculate; LHA, lateral hypothalamic area; MG, medial geniculate; ML, medial lemniscus; P, pyramids; PC, cerebral peduncles; VE, ventral N of thalamus; VD, ventral nucleus pars dorsalis of thalamus; Vsp, spinal nucleus of the trigeminal; XII, hypoglossal nucleus. tetramethylbenzidine ( T M B ) is and the injection sites with diaminobenzidine 4. H R P labeled neurons were identified with light microscopy. Fluorescence was observed with a Zeiss P h o t o m i c r o s c o p e II equipped with an epifluorescence illuminator with appropriate filters. B o t h Nuclear Yellow and D A P I were observed with a 405 nm excitation wavelength. H R P and the fluorescent dye(s) in the same cell could be accurately identified by alternating between fluorescent illumination and light microscopy. The injections o f D A P I into the thalamus included the intralaminar and ventrobasal complexes (Fig. 1A, B). Cells retrogradely labeled with D A P I from the thalamus contained blue granular fluorescence t h r o u g h the cytoplasm (Fig. 2A, right). Injections o f H R P or Nuclear Yellow into the reticular formation were centered in the central nucleus o f the medulla and the nucleus gigantocellularis (Fig. 1C). Cells labeled f r o m the reticular formation contained either a bright fluorescence uniformly distri-
183
Fig. 2. A: fluorescent photomicrograph of cell labeled with Nuclear Yellow (on left) and adjacent cell labeled with DAPI. Nuclear Yellow was injected into the reticular formation and DAPI was injected into the thalamus. Both of these cells are located in lamina X. B: double-labeled cell located in lamina VII on the side contralateral to the injections. C: labeled cell containing both Nuclear Yellow and DAPI. This cell was in lamina VII, ipsilateral to the injection. D: Double-labeled cell in lamina VII contralateral to the site of the injection.
buted in the cell nucleus (Nuclear Yellow, Fig. 2A, left) or blue TMB reaction product from H R P histochemistry. Labeled cells that projected to either the thalamus or the reticular formation or both were observed in the lumbar cord of each animal. The majority of labeled cells was found on the side contralateral to the injections (Fig. 1D-F), although a few labeled cells were found on the ipsilateral side. Spinothalamic cells were located throughout the dorsal horn and also ventromedially at the border of laminae VII and VIII of Rexed 16 (Fig. 1D). Spinoreticular cells, on the other hand, were concentrated in the ventromedial intermediate area with few labeled cells in the dorsal horn (Fig. IF). The labeled dorsal horn cells were located in the lateral aspect of lamina V. These distributions are in agreement with previous studies in rat 2,7. Those neurons that contained H R P or Nuclear Yellow transported from the reticular formation and D A P I from the thalamus were located in areas of overlap between the distributions of spinoreticular and spinothalamic tract cells. These cells were generally multipolar. Fig. 2 B - D shows examples of cells which contained both retrogradely transported D A P I and Nuclear Yellow. The double-labeled neurons were concentrated ventrally in the intermediate and ventral horn area (Fig. 1E), corres-
184 ponding to laminae VII (Fig. 2B, D) and VIII. A few double-labeled cells were also found in lamina V, especially the more lateral, reticulated part. Most double-labeled cells were located contralateral to the side o f injections (Fig. l E), although a few cells were found on the ipsilateral side (Fig. 2C). in one animal double-labeled cells were f o u n d bilaterally in the lateral spinal nucleus. There were always cells labeled from either the thalamus or reticular formation that did not project to both of these terminal fields. In this report we d o c u m e n t the existence o f a population of spinal neurons that project to both the thalamus and the medullary reticular formation. The majority of our double-labeled cells project to the contralateral brainstem and thalamus and are found in cord laminae typical for spinothalamic and spinoreticular tract neurons. The neurons that have dual projections appear to be a significant subset o f the population o f each pathway. The spinoreticular system may play a critical role in the central processing o f noxious stimuli 6,9,14. However, neurons o f this system are unlikely to be involved in discriminative functions due to their complex receptive field characteristics and polymodal response properties 3,s,9,11. Spinal cells which project to both the reticular formation and the thalamus may be involved in alerting or affecting responses to peripheral stimuli, including those associated with painS, 11-13. Cells which project exclusively to the thalamus, however, may be responsible for the more discriminative aspects o f painful, as well as non-painful, stimuli3, sAS. The authors would like to thank G. Gonzales, B. M o o r e and H. Willcockson for their fine technical help and P. W a l d r o p for typing the manuscript. This work was supported by Research Grants NS 09743 and NS 11255, by Training Grant NS 07185 f r o m the National Institutes o f Health and a grant from The M o o d y Foundation.
i Abols, I. A. and Basbaum, A. I., Afferent connections of the rostral medulla of the cat : a neural substrate for midbrain-medullary interactions in the modulation of pain, J. camp. Neural., 201 (1981) 285-297. 2 Andrezi, K. J. A., Chan-Palay, V. and Falay, S. L., q-he nucleus paragigantocellularis in the rat : demonstration of afferents by the retrograde transport of horseradish peroxidase, Anat. Embryol., 161 (1981) 373-390. 3 Casey, K. L. and Jones, E. G., An overview of ascending pathways: brainstem and thalamus, Neurosci. Res. Progr. Bull., 16 (1978) 103-118. 4 Colman, D., Scalia, F. and Cabrales, E., Light and electron microscopic observations of the anterograde transport of horseradish peroxidase m the optic pathway in the mouse and rat, Brain Research, 102 (1976) 589-596. 5 Corvaja, No, Grofova, I., Pompeiano, O. and Walberg, F., The lateral reticular nucleus in the cat - - I. An experimental anatomical study of its spinal and supraspinal afferent connections, Neuroscience, 2 (1977) 541-551. 6 Fields, H. L., Clanton, C. H. and Anderson, S. D., Somatosensory properties of spinoreticular neurons in the cat, Brain Research, 120 (1975) 49--66. 7 Giesler, G. J., Men6trey, D. and Basbaum, A. I., Differential origins of spinothalamic tract projections to medial and lateral thalamus in the rat, J. camp. Neural., 184 (1979) 107-125. 8 Giesler, G. J., Jr., Yezierski, R. P., Gerhart, K. D. and Willis, W. D., Spinothalamic tract neurons that project to medial and/or lateral thalamic nuclei: evidence for a physiologically novel population of spinal cord neurons, J. Neurophysiol., 46 (1981) 1285-1308.
185 9 Haber, L. H., Moore, B. D. and Willis, W. D., Electrophysiological response properties of spinoreticular neurons in the monkey, J. comp. Neurol., in press. l0 Kevetter, G. A., Haber, L. H., Yezierski, R. P., Chung, J. M., Martin, R. F. and Willis, W. D., Cells of origin of the spinoreticular tract in the monkey, J. comp. Neurol., in press. I l Maunz, R. A., Pitts, N. G. and Peterson, B. W., Cat spinoreticular neurons: Locations, responses and changes in responses during repetitive stimulation, Brain Research, 148 (1978) 365-379. 12 Mehler, W. R., Some neurological species differences - - a posteriori, Ann. N.Y. Acad. Sci., 167 (1969) 424-468. 13 Mehler, W. R., Feferman, M. E. and Nauta, W. J. H., Ascending axon degeneration following anterolateral cordotomy. An experimental study in the monkey, Brain, 83 (1960) 718-751. 14 Men6trey, D., Chaouch, A. and Besson, J. M., Location and properties of dorsal horn neurons at origin of spinoreticular tract in lumbar enlargement of the rat, J. Neurophysiol., 44 (1980) 862877. 15 Price, D. D. and Dubner, R., Neurons that subserve the sensoridiscriminative aspects of pain, Pain, 3 (1977) 307-338. 16 Rexed, B., The cytoarchitectonic organization of the spinal cord in the cat, J. comp. Neurol., 96 (1952) 415-496. 17 Willis, W. D., Kenshalo, D. R., Jr. and Leonard, R. B., The cells of origin of the primate spinothalamic tract, J. comp. Neurol., 188 (1979) 543-573. 18 Yezierski, R. P. and Bowker, R. M., A retrograde double label tracing technique using horseradish peroxidase and the fluorescent dye 4',6-diamidino-2-phenylindole 2HC1 (DAPI), J. Neurosci. Meth., 4 (1981) 53-62.