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Dendritic bundles exist
BRAIN RESEARCH
273
D E N D R I T I C B U N D L E S EXIST
RICHARD CHARLES MARSH, LLOYD MATLOVSKY AND MELVIN W. STROMBERG Department of Psychology, Utah State University, Logan, Utah 84321, Los Angeles County Hospital, Los Angeles, Calif. 90033 and Department of Veterinary Anatomy, Purdue University, Lafayette, Ind. 47907 (U.S.A.)
(Accepted April 30th, 1971)
INTRODUCTION In the area of anatomical interrelationships of neurons, there have been recent investigations of dendrites, e.g., dendritic arborizations 1, spines on dendrites a, and dendritic fields 1°. The electron microscope has been used to search for the occurrence of junctions between dendrites and somasL It is also known that the apical dendrites of lower motoneurons tend to travel as a group for substantial distances before final branching s . However, none of these previous studies illuminate the spatial interrelations between individual dendrites from different cells. Such three-dimensional reconstructions with the electron microscope are so tedious that it is not realistic to investigate more than several cubic micra in this manner. In light microscopy the difficulty has been the absence of a suitable stain for dendrites, With the aid of a staining technique (Marsh, in preparation), we have been able to explore the course of dendrites from different cells for approximately 100/~m. The present study examines the spatial interrelations of dendrites in the spinal cord using the special staining technique outlined below. Individual cross sections reveal numerous dendrites near one another apparently arranged in bundles. To further explore the possibility that dendrites tend to course in closely packed bundles, we have prepared serial sections of the spinal cord. METHODS Five to 7-week-old pigs provided the spinal cords used for this study. Staining of dendrites was accomplished through the use of a Nissl method utilizing toluidine blue 0 and a complex ethanol differentiation procedure. The complex nature of differentiation (after Rexed 6) is the only difference between this technique and a standard Nissl. After fixation and a water bath, the tissue pieces were placed in 70~o ethanol at 37°C for a number of hours. This step was followed by embedding, sectioning, and deparatfination. The slides were stained in 0.5 ~o toluidine blue 0 in distilled water for 15 min. Immediately after staining, differentiation was completed by (a) a quick wash in a large volume of water (approximately one liter), (b) a 1-min 7 0 ~ Brain Research, 33 (1971) 273-277
274
R. (7. MARSHet ai.
Fig. 1. Serial analysis of somas. Two neurons sectioned into layers are mounted upright and photographed at an angle set to suggest fusion back to the original three-dimensional cells. In the foreground, the two cells provide one dendrite each to the formation of a dendritic bundle. The total depth of each cell is 63 #m. Note also the bisection of dendrites in each cell in the nuclear region of SO1TIR.
ethanol bath and, (c) a 5-min 70 ~ ethanol bath. Then the slides were treated routinely. The main advantage of this technique is the intense staining of Nissl bodies. As seen in Fig. 1, the Nissl substance of both the soma and dendrite is stained. This method stains Nissl bodies in dendrites so clearly that dendrites can be followed in serial sections. In longitudinal section, Nissl bodies appear as dots or, less often, irregular polygons. They stain light to very intense blue. Ten serial sections at 7 #m were cut from the last lumbar and first 3 sacral cord segments. Photographs were taken from the serial sections in an area thought to contain dendritic bundles. Each photograph was mounted on graph paper and a common rectangular coordinate system (field)was established for all photographs in the series. This was accomplished by finding reference points between consecutive pairs of sections. Five c o m m o n reference items were necessary to determine a field linkup. Dendrites were never used as reference points. Tracings were then made of all the dendrites cut in or near cross section. The angle of the section was judged from the obliqueness of the dendrite circumference z and from staining properties. The sections were then color coded. Subsequently, the complement of dendrites in each section was transferred to a common rectangular grid. The different colors identified the sections so that when this was concluded, the Brain Research, 33 (1971) 273-277
Fig. 2. Serial analysis of dendrites. A, Serial sections showing a common field of 155 p m × 98 p m in nine 7 p m histological sections. Nissl stain was used for staining of dendrites (see text). Original microscopic magnification: × 310. B, Tracings of 4 dendrites common to all sections in 'A' (one exception). Markings within outlines are Nissl bodies. Spatial relations are maintained. The fibers range from 7 to 14 p m in diameter.
276
~. c . MARSH Cl a/,
course of a given dendrite could be followed in 3 dimensions from its location and the changes of color. Dendrites that were not identifiable on at least 50~;; of the sections were excluded from further processing. Somas were mapFed three-dimensionally as were dendrites. Dendrites that were structurally confused with or fused into somas were also screened from further analysis. In this manner, areas containing dendrites situated closely together were identified. Photographic enlargements of these areas were printed and processed as before. For each of the dendrites in this final set, the photographic analysis described above was verified microscopically under direct observation. All dendrites were followed through the thickness of each section before the data were considered acceptable. RESULTS
From the study of somas, it can be shown that different neurons do send dendrites into a common path. Fig. 1 shows two nerve cells. Each cellis broken into several layers which represent its consecutive two-dimensional projections created by the process of sectioning. The sequential serial sections were pinned up and photographed at an angle set to suggest fusion back to the three-dimensional original. At the lower left of the field, two dendrites are near one another. The two fibers begin to travel along a c o m m o n path. The serial following of dendrites is shown in Fig. 2. The dendrites are identified from the photographs of the sections (Fig. 2A) and traced (Fig. 2B). The 4 fibers followed here form a bundle for at least 63/~m. Considering the bundle as a cylinder, the dimensions are 55-85 # m (diameter) x 63 ,urn (length). The diameters of fibers range from 7 to 14/zm. The distance between fibers in most cases is less than 10/~m and at their closest approach, the distance is < 1 #m (section Nos. 2 and 9). DISCUSSION
The case illustrated in Fig. 2 is not unique. Bundles with as many as 12 dendrites have been seen in cross sections. They are found at all the levels investigated in the lateral extreme of the ventrolateral nucleus of the spinal cord, i.e., lower motor nucleus or Rexed's lamina IX 7. It seems advisable to define dendritic bundles so that they must contain a minimum number of fibers and to set a minimum length. We suggest that dendritic bundles be formally considered as groups of 3 or more dendrites that course the same path for a distance greater than 3 times the diameter of the largest fiber in the group. In this way, crossings of dendrites cannot be construed as bundles. TP,e frequency of occurrence and localization of these units is only preliminarily known. Their function must await further investigation. However, even at this stage it seems clear that dendritic bundles form a most favorable anatomical substrate for ephaptic spread between the individual elements in each bundle. I f these dendrites originate in different cells, and if the extracellular fluid and/or glia between the dendrites do not completely shunt current spread between these graded membranes (an unlikely possibility4), an effect amounting to non-synaptic information transfer between neurons is predictable. Such ephapsis within overlapping dendrites may add another dimension to informaBrain Research, 33 (1971) 273-277
DENDRITIC BUNDLES EXIST
277
tion processing in the nervous system. Thus, for instance, the present observations help in understanding the physiological observations of neuronal field effects5. SUMMARY
Within dendritic fields of lower motoneurons, dendrites from different cells bundle together. The techniques used here show bundles as long as 63 #m which contain as many as four 7-14/~m diameter dendrites. The nearness of fibers within a bundle can be < 1/~m. These dendritic interrelationships provide an enabling condition for information transfer between neurons and may help to further elucidate neural processes. NOTE ADDED IN PROOF
Two recent articles supplement and corroborate the findings reported here: SCHEmEL, M., AND SCHEmEL, A., Organization of spinal motoneuron dendrites in bundles, Exp. Neurol., 28 (1970) 106-112. HINSMAN, E. J., AND STROMBERG,M. W., Ultrastructure of the ventral horn neuron of the calf spinal cord, Anat. Rec., 157 (1967) 260. ACKNOWLEDGEMENTS
The senior author wishes to thank Dr. Rafael Elul, Department of Anatomy, UCLA, for proofreading the manuscript.
REFERENCES 1 COLEMAN,P., AND RIESEN,A., Environmental effects on cortical dendritic fields, J. Anat. (Lond.), 102 (1968) 363-374. 2 ELIAS, H., AND PAULY,J., Human Microanatomy, Davis, Philadelphia, 1966, pp. 335-348. 3 GLOBUS,A., AND SCHEIBEL,A., Loss of dendritic spines as an index of presynaptic terminal patterns, Nature (Lond.), 212 (1966) 463--465. 4 KUFFLER, S., AND POTTER, D., Glia in the leech czntral nervous system: Physiological properties and neuron-glia relationship, J. Neurophysiol., 27 (1964) 290-320. 5 NELSON,P. G., Interaction between motoneurons of the cat, J. Neurophysiol., 29 (1966) 275-287. 6 REXED, B., The cytoarchitectonic organization of the spinal cord in the cat, J. comp. Neurol., 96 (1952) 415-495. 7 REXED, B., A cytoarchitectonic atlas of the spinal cord in the cat, J. comp. Neurol., 100 (1954) 297-329. 8 SCHEIBEL,M., AND SCHEIBEL,A., Terminal patterns in the cat spinal cord. III. Primary afferent collaterals, Brain Research, 13 (1969) 417-443. 9 SETALA,G., AND SZEKELY,G., The presence of membrane specializations indicative of somatodendritic synaptic junctions in the optic tectum of the frog, Exp. Brain Res., 4 (1967) 237-242. 10 SHOLL, D. A., Dendritic organization in the neurons of the visual and motor cortices of the cat, J. Anat. (Lond.), 87 (1953) 387-407.
Brain Research, 33 (1971) 273-277
273
D E N D R I T I C B U N D L E S EXIST
RICHARD CHARLES MARSH, LLOYD MATLOVSKY AND MELVIN W. STROMBERG Department of Psychology, Utah State University, Logan, Utah 84321, Los Angeles County Hospital, Los Angeles, Calif. 90033 and Department of Veterinary Anatomy, Purdue University, Lafayette, Ind. 47907 (U.S.A.)
(Accepted April 30th, 1971)
INTRODUCTION In the area of anatomical interrelationships of neurons, there have been recent investigations of dendrites, e.g., dendritic arborizations 1, spines on dendrites a, and dendritic fields 1°. The electron microscope has been used to search for the occurrence of junctions between dendrites and somasL It is also known that the apical dendrites of lower motoneurons tend to travel as a group for substantial distances before final branching s . However, none of these previous studies illuminate the spatial interrelations between individual dendrites from different cells. Such three-dimensional reconstructions with the electron microscope are so tedious that it is not realistic to investigate more than several cubic micra in this manner. In light microscopy the difficulty has been the absence of a suitable stain for dendrites, With the aid of a staining technique (Marsh, in preparation), we have been able to explore the course of dendrites from different cells for approximately 100/~m. The present study examines the spatial interrelations of dendrites in the spinal cord using the special staining technique outlined below. Individual cross sections reveal numerous dendrites near one another apparently arranged in bundles. To further explore the possibility that dendrites tend to course in closely packed bundles, we have prepared serial sections of the spinal cord. METHODS Five to 7-week-old pigs provided the spinal cords used for this study. Staining of dendrites was accomplished through the use of a Nissl method utilizing toluidine blue 0 and a complex ethanol differentiation procedure. The complex nature of differentiation (after Rexed 6) is the only difference between this technique and a standard Nissl. After fixation and a water bath, the tissue pieces were placed in 70~o ethanol at 37°C for a number of hours. This step was followed by embedding, sectioning, and deparatfination. The slides were stained in 0.5 ~o toluidine blue 0 in distilled water for 15 min. Immediately after staining, differentiation was completed by (a) a quick wash in a large volume of water (approximately one liter), (b) a 1-min 7 0 ~ Brain Research, 33 (1971) 273-277
274
R. (7. MARSHet ai.
Fig. 1. Serial analysis of somas. Two neurons sectioned into layers are mounted upright and photographed at an angle set to suggest fusion back to the original three-dimensional cells. In the foreground, the two cells provide one dendrite each to the formation of a dendritic bundle. The total depth of each cell is 63 #m. Note also the bisection of dendrites in each cell in the nuclear region of SO1TIR.
ethanol bath and, (c) a 5-min 70 ~ ethanol bath. Then the slides were treated routinely. The main advantage of this technique is the intense staining of Nissl bodies. As seen in Fig. 1, the Nissl substance of both the soma and dendrite is stained. This method stains Nissl bodies in dendrites so clearly that dendrites can be followed in serial sections. In longitudinal section, Nissl bodies appear as dots or, less often, irregular polygons. They stain light to very intense blue. Ten serial sections at 7 #m were cut from the last lumbar and first 3 sacral cord segments. Photographs were taken from the serial sections in an area thought to contain dendritic bundles. Each photograph was mounted on graph paper and a common rectangular coordinate system (field)was established for all photographs in the series. This was accomplished by finding reference points between consecutive pairs of sections. Five c o m m o n reference items were necessary to determine a field linkup. Dendrites were never used as reference points. Tracings were then made of all the dendrites cut in or near cross section. The angle of the section was judged from the obliqueness of the dendrite circumference z and from staining properties. The sections were then color coded. Subsequently, the complement of dendrites in each section was transferred to a common rectangular grid. The different colors identified the sections so that when this was concluded, the Brain Research, 33 (1971) 273-277
Fig. 2. Serial analysis of dendrites. A, Serial sections showing a common field of 155 p m × 98 p m in nine 7 p m histological sections. Nissl stain was used for staining of dendrites (see text). Original microscopic magnification: × 310. B, Tracings of 4 dendrites common to all sections in 'A' (one exception). Markings within outlines are Nissl bodies. Spatial relations are maintained. The fibers range from 7 to 14 p m in diameter.
276
~. c . MARSH Cl a/,
course of a given dendrite could be followed in 3 dimensions from its location and the changes of color. Dendrites that were not identifiable on at least 50~;; of the sections were excluded from further processing. Somas were mapFed three-dimensionally as were dendrites. Dendrites that were structurally confused with or fused into somas were also screened from further analysis. In this manner, areas containing dendrites situated closely together were identified. Photographic enlargements of these areas were printed and processed as before. For each of the dendrites in this final set, the photographic analysis described above was verified microscopically under direct observation. All dendrites were followed through the thickness of each section before the data were considered acceptable. RESULTS
From the study of somas, it can be shown that different neurons do send dendrites into a common path. Fig. 1 shows two nerve cells. Each cellis broken into several layers which represent its consecutive two-dimensional projections created by the process of sectioning. The sequential serial sections were pinned up and photographed at an angle set to suggest fusion back to the three-dimensional original. At the lower left of the field, two dendrites are near one another. The two fibers begin to travel along a c o m m o n path. The serial following of dendrites is shown in Fig. 2. The dendrites are identified from the photographs of the sections (Fig. 2A) and traced (Fig. 2B). The 4 fibers followed here form a bundle for at least 63/~m. Considering the bundle as a cylinder, the dimensions are 55-85 # m (diameter) x 63 ,urn (length). The diameters of fibers range from 7 to 14/zm. The distance between fibers in most cases is less than 10/~m and at their closest approach, the distance is < 1 #m (section Nos. 2 and 9). DISCUSSION
The case illustrated in Fig. 2 is not unique. Bundles with as many as 12 dendrites have been seen in cross sections. They are found at all the levels investigated in the lateral extreme of the ventrolateral nucleus of the spinal cord, i.e., lower motor nucleus or Rexed's lamina IX 7. It seems advisable to define dendritic bundles so that they must contain a minimum number of fibers and to set a minimum length. We suggest that dendritic bundles be formally considered as groups of 3 or more dendrites that course the same path for a distance greater than 3 times the diameter of the largest fiber in the group. In this way, crossings of dendrites cannot be construed as bundles. TP,e frequency of occurrence and localization of these units is only preliminarily known. Their function must await further investigation. However, even at this stage it seems clear that dendritic bundles form a most favorable anatomical substrate for ephaptic spread between the individual elements in each bundle. I f these dendrites originate in different cells, and if the extracellular fluid and/or glia between the dendrites do not completely shunt current spread between these graded membranes (an unlikely possibility4), an effect amounting to non-synaptic information transfer between neurons is predictable. Such ephapsis within overlapping dendrites may add another dimension to informaBrain Research, 33 (1971) 273-277
DENDRITIC BUNDLES EXIST
277
tion processing in the nervous system. Thus, for instance, the present observations help in understanding the physiological observations of neuronal field effects5. SUMMARY
Within dendritic fields of lower motoneurons, dendrites from different cells bundle together. The techniques used here show bundles as long as 63 #m which contain as many as four 7-14/~m diameter dendrites. The nearness of fibers within a bundle can be < 1/~m. These dendritic interrelationships provide an enabling condition for information transfer between neurons and may help to further elucidate neural processes. NOTE ADDED IN PROOF
Two recent articles supplement and corroborate the findings reported here: SCHEmEL, M., AND SCHEmEL, A., Organization of spinal motoneuron dendrites in bundles, Exp. Neurol., 28 (1970) 106-112. HINSMAN, E. J., AND STROMBERG,M. W., Ultrastructure of the ventral horn neuron of the calf spinal cord, Anat. Rec., 157 (1967) 260. ACKNOWLEDGEMENTS
The senior author wishes to thank Dr. Rafael Elul, Department of Anatomy, UCLA, for proofreading the manuscript.
REFERENCES 1 COLEMAN,P., AND RIESEN,A., Environmental effects on cortical dendritic fields, J. Anat. (Lond.), 102 (1968) 363-374. 2 ELIAS, H., AND PAULY,J., Human Microanatomy, Davis, Philadelphia, 1966, pp. 335-348. 3 GLOBUS,A., AND SCHEIBEL,A., Loss of dendritic spines as an index of presynaptic terminal patterns, Nature (Lond.), 212 (1966) 463--465. 4 KUFFLER, S., AND POTTER, D., Glia in the leech czntral nervous system: Physiological properties and neuron-glia relationship, J. Neurophysiol., 27 (1964) 290-320. 5 NELSON,P. G., Interaction between motoneurons of the cat, J. Neurophysiol., 29 (1966) 275-287. 6 REXED, B., The cytoarchitectonic organization of the spinal cord in the cat, J. comp. Neurol., 96 (1952) 415-495. 7 REXED, B., A cytoarchitectonic atlas of the spinal cord in the cat, J. comp. Neurol., 100 (1954) 297-329. 8 SCHEIBEL,M., AND SCHEIBEL,A., Terminal patterns in the cat spinal cord. III. Primary afferent collaterals, Brain Research, 13 (1969) 417-443. 9 SETALA,G., AND SZEKELY,G., The presence of membrane specializations indicative of somatodendritic synaptic junctions in the optic tectum of the frog, Exp. Brain Res., 4 (1967) 237-242. 10 SHOLL, D. A., Dendritic organization in the neurons of the visual and motor cortices of the cat, J. Anat. (Lond.), 87 (1953) 387-407.
Brain Research, 33 (1971) 273-277