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The entopeduncular nucleus: Golgi morphometrics of serially reconstructed neurons in adult cats
Brain Research, 375 (1986) 395-400 Elsevier
395
BRE 21595
The entopeduncular nucleus: Golgi morphometrics of serially reconstructed neurons in adult cats C.L. DVERGSTEN, C.D. HULL, M.S. LEVINE, A.M. ADINOLFI, R.S. FISHER and N.A. BUCHWALD Mental Retardation Research Center, University of California, School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted February 18th, 1986) Key words: basal ganglion - - pallidal segment - - dendrite - - morphometry - - Golgi method
Computer-assisted morphometrics were used to characterize mature somatodendritic architecture in Golgi-stained neurons of the entopeduncular nucleus (EN) of the adult cat. Only one form of adult EN neuron was apparent and characterized by common features including: (1) relatively large conical somata, (2) long aspiny and moderately branched dendrites and (3) discoid to spherical dendritic fields oriented randomly within the EN. These results indicate that feline EN neurons have some properties in common with large neurons of the primate medial pallidal segment. The entopeduncular nucleus (EN) in rodent and carnivore brains is considered generally to be homologous to the medial pailidal segment of primate brains because of similarities of cytoarchitecture, connectivity and ultrastructure ],2'5,8-1°. However, the somatodendritic morphology of the E N revealed by Golgi methods has been obtained mainly from immature animals due to favorable silver impregnation prior to myelination of the fiber fascicles of the cerebral peduncle coursing through the region 2']°'11'15'17. The present investigation was designed to assess the full extent of mature somatodendritic architecture in the E N by using computer-assisted m o r p h o m e t r y of serial reconstructions of Golgi-stained E N neurons in the adult cat. The rapid Golgi method was employed to impregnate EN neurons with silver 6'14. Adequately stained neurons were produced in 6 adult cats. These animals were all of known age ( 1 - 3 years). They were born and reared under standard conditions in the kitten breeding colony of the Mental Retardation Research Center at U C L A . The cats were sacrificed by barbiturate overdose and perfused transcardially with aldehyde fixative. Qualitative observations were obtained from well
over 100 E N neurons scattered throughout the nucleus in each cat. Quantitative measurements were derived from 9 neurons in 4 cats. These latter neurons were also dispersed widely in different regions of the EN. They were selected because of the apparent fullness of their silver impregnation and the completeness of their dendritic field reconstruction from serial coronal sections (100 /zm, E p o n embedment) 6'14. Neurons were reconstructed from a minimum of two to a maximum of 10 serial sections. Neurons located at the border of the lateral hypothalamus or embedded in the fiber fascicles of the cerebral peduncle were excluded from this sample. Computer-assisted morphometric methods were used to assess somatic dimensions, dendritic lengths and dendritic branching patterns in 3 dimensions among the serially reconstructed neurons 6. Dendritic branching was defined by a centrifugal ordering method 2°. Golgi-stained neurons were also characterized by photomicrographs and camera lucida drawings using brightfield transmission combined occasionally with angled incident illumination for 'optical sectioning' of neuronal somata viewed from both tissue section faces 19. Our major new finding was that the E N of the adult
Correspondence: M.S. Levine, Mental Retardation Research Center, RM 58-258 NPI, University of California, Los Angeles, CA 90024, U.S.A.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
396 cat contains only one basic form of neuron. In essence, mature EN neurons have rather uniform somatodendritic morphology characterized by moderately large conical somata and long, modestly branched aspinous dendrites. Overall, EN neurons had large dendritic fields oriented randomly within the confines of peduncular fascicles coursing through the nucleus. The somatodendritic architecture of adult EN neurons corresponded well with (1) larger intensely chromophilic EN cells stained by Nissl methods and (2) EN neurons projecting to the thalamus, substantia nigra, globus pallidus and/or mesencephalic pedunculopontine regions demonstrated by retrograde axonal transport of horseradish peroxidaset2,20, 24.
mata were situated mainly in neuropil regions forming a matrix around heavily myelinated cerebral peduncular fiber fascicles (Fig. 1A). While transitions of somata into proximal dendrites were gradual and ill-defined, cell body profiles were clearly of moderately large size (average diameter = 43 + 3/xm, range = 36-59/xm). These somatic profiles were only modestly polymorphic and were typically ovoid or fusiform in shape as reflected by an approximate 2:1 ratio between major and minor somatic axis values (ranges = 49-71/xm vs 25-46ktm, Fig. 1A-C), These neuronal cell bodies generally gave rise to 5 stout primary dendrites (overall range = 3-7 primary dendrites, 4-10/xm diameter, Table I). These processes tapered gradually through their long radiations distally (Fig. 1A-C). Dendritic spines and appendages were found rarely. While short lengths of
Golgi-stained EN neuronal cell bodies had few or no spines along the lengths of their profiles. These so-
2 0 0 pm
B ~
V •..........
. ( . . . .
. . . . . . . . . . . .
.
•
..
. . . . . . . . . . . . . . . . . . . . . . . . . . .
- .....
b
Fig. 1. A: inset shows 3 neurons in a region of the entopeduncular nucleus where the density of myelinated fiber bundles (mb) is high. Dendrites appear to bundle together (arrows in inset) between the fiber bundles. B, C: two serially reconstructed neurons from adult cats. Bold-faced arrows point to terminal arborizations of the dendrites, vb, ventral border of the entopeduncular nucleus.
397 TABLE I Quantitative analysis of E N neurons
Ranges and means (+__S.E.M.) of dendritic parameters of reconstructed EN neurons. Length measurements are in micrometers. Parameter
Morphometric data
No. of dendrites Total dendritic length Dendritic length Dendritic field radius Branches per neuron Branches per dendrite
Mean + S.E.M.
Range
5.0 _+0.3 9434 + 913 1907 + 193 582 + 30 63.3 _+9.8 13.1 _+2.6
4-6 5898-14,204 188-6222 149-1300 36-133 3-78
Branch order
Branch order analysis Branch length (~m)
Number of branches
1
105 + 23* 162 + 20 195 + 17 163 + 11 124 + 10 128 + 29 87 + 21
5.0 + 0.3 9.8 + 0.5 13.8 + 1.1 13.4 + 1.6 8.4 + 1.3 5.5 + 2.3 7.3 + 5.9
2 3 4 5 6 7
* Values are means + S.E.M.
varicose dendrites were observed infrequently, varicosites were not apparent in many silver impregnated neurons. Morphometric analyses demonstrated that individual EN neurons possessed considerable total dendritic length (approximately 9.5 ram, Table I). These extensive processes were segmented by modestly prevalent branching (over 60 branches/cell, Table I). Individual dendrites were about 2 mm long and had roughly 13 branches (Table I). Overall, the dendritic fields of EN neurons were large as reflected by average dendritic field radii of nearly 600/zm (Table I). Branching patterns varied widely in individual dendrites as well as between different cells. For example, some dendrites branched at regular intervals while others branched mainly in proximity to the parent cell bodies. Additionally, dendrites with extensive distal branching (i.e. tufts or thickets) were fairly common (Fig. 1B). By morphometric analyses, a maximum of 7 branch orders accounted for nearly all of the segmentation of individual dendrites of mature EN neurons (Table I). Third and fourth order branches were most prevalent. These branch orders also tended to have the longest branch lengths. Re-
gardless of branch order, dendritic branch lengths usually ranged between 100 and 200/~m. The large extensive dendritic arbors of EN neurons radiated in all directions away from parent cell bodies (Fig. 1A, B). The dendritic fields of different neurons occupied overlapping territory confined, for the most part, within the EN neuropil (Fig. 1A). The stained dendrites wove their course through but did not penetrate the outer borders of the EN. The course of individual dendrites was closely associated with the pattern and density of peduncular fiber fascicles (Fig. 1A). Within the neuropil, dendrites assumed a straight course. When a fiber bundle was encountered, dendrites followed the outer contour of the bundle. When fiber bundles were closely adjacent, dendrites aggregated into bundles running through small neuropil channels interspersed between axonal fascicles. While dendritic branching could occur anywhere in the neuropil, branching seemed to be more prevalent when dendrites encountered fiber fascicles. As an exception to the apparent random radiation of EN dendrites within their available neuropil zones, these processes were unable to penetrate the ansa lenticularis (Fig. 1B). When somata lay in the ventral border of the EN, their dendrites would follow the border and/or radiate centrally within the nucleus. Due to their large size, the dendritic fields of some EN neurons spread across the entire dorsal-ventral extent of the nucleus (maximum dimension -1.0-1.5 mm). However, these dendritic fields failed to penetrate the entire rostral caudal or medial-lateral extents of the EN (maximum dimensions = 2.53.0 mm). On the whole, dendritic fields of EN neurons were oriented randomly within the nucleus in reference to the 3 cardinal planes of section (Fig. 2A-C). The dendritic fields of particular EN neurons displayed a variety of shapes and orientations. Spherical dendritic fields were commonly encountered (Fig. 2A). Cells with flattened spherical or discoid fields were also evident (Fig. 2B). Finally, cells with intermediate field forms were readily apparent (Fig. 2C). In general, the dendritic morphology of adult EN neurons was very similar to earlier descriptions of neurons in the reticulate zone of the substantia nigra of the adult cat 13'18'22. These results are in good agreement with earlier developmental morphometric studies of Golgi mate-
398 n e u r o n s attain m a t u r e s o m a t o d e n d r i t i c m o r p h o l o g y
rial f r o m the feline E N p e r f o r m e d b o t h in o u r o w n and o t h e r l a b o r a t o r i e s 4-6'9-11A5-17'26. In t h e cat, E N
by 9 0 - 1 2 0 days postnatal 6. B o t h the a f f e r e n t and el-
FRONTAL
SAGITTAL
D
D
R
V
V
C
HORIZONTAL
Cell A
Cell B
Cell C
500 pm Fig. 2. Three serially reconstructed neurons from adult cats as they appear in the 3 cardinal planes of section. Cells were entered into the computer in the frontal plane. Sagittal and horizontal views of these cells were obtained by computer rotation. Cell A was reconstructed from 9 serial sections. Cell B was reconstructed from 4 serial sections. Cell C was reconstructed from 8 serial sections.
399 ferent connections of these neurons are established and functional by the day of birth even though considerable neurotransmitter differentiation, terminal morphogenesis and subsequent increases in synaptic security occur during the early postnatal period2,7,8,12. However, our observation of somatodendritic uniformity among mature E N neurons has considerable developmental implications. A m o n g E N neurons stained by Golgi, Nissl and peroxidase connectivity methods, there is extensive polymorphism during early development in the cat both between particular neurons and among the different dendrites of any single neuron 6. Assuming that most, if not all, of these cells survive into adulthood, these widely variable neuronal forms assume a c o m m o n structure as morphogenesis terminated. Therefore, morphogenesis may be heterogeneous among the E N neurons 3,5,6. Additionally, in the mature cat EN, neurons may have only some properties in c o m m o n to those of the medial pallidal segment of primates. We observed only larger neuronal somata with extensive dendritic fields in the adult E N while various investigators have found large, medium and small neurons in Golgi studies of the medial pallidal segment 5,9-11. While it is possible that we failed to stain smaller E N neurons because of the capricious nature of Golgi methods, our results are consistent with the uniform appearance of E N neurons of the adult cat obtained by more
1 Adinolfi, A.M., The fine structure of neurons and synapses in the entopeduncular nucleus of the cat, J. Comp. Neurol., 135 (1969) 225-248. 2 Adinolfi, A.M., Levine, M.S. and Hull, C.D., Early postnatal development of entopeduncular neurons and their neostriatal connections, Exp. Neurol., 70 (1980) 463-474. 3 Berry, M., Bradley, P. and Borges, S., Environmental and genetic determinants of connectivity in the central nervous system - - an approach through dendritic field analysis. In M.A. Corner, R.E. Baker, N.E. Van de Poi, D.F. Swaab and H.B.M. Uylings (Eds.), Maturation of the Nervous System. Progress in Brain Research, Vol. 48, Elsevier, Amsterdam, 1978, pp. 133-146. 4 Chang, H.T., Wilson, C.J. and Kitai, S.T., Single neostriatal efferent axons in the globus pallidus: a light and electron microscopic study, Science, 213 (1981) 915-918. 5 DiFiglia, M., Pasik, P. and Pasik, T., Golgi and ultrastructural study of the monkey globus pallidus, J. Comp. Neurol., 212 (1982) 53-75. 6 Dvergsten, C.L., Hull, C.D., Levine, M.S., Adinolfi, A.M. and Buchwald, N.A., Postnatal differentiation and
reliable Nissl and connectivity staining methods 8A2'24. Thus, the larger pallidal neurons may be the homologs of the adult feline E N neurons. In both cats and primates, the large and rather randomly oriented dendritic trees are situated to receive numerous convergent inputs particularly from wide areas of the neostriatum 4,8,21. The somatodendritic surfaces of these neurons are heavily ensheathed mainly by striatopallidal terminals containing 7-amino-butyric acid 1'2'7. There are other differences in the dendritic architecture of E N and larger medial pallidal segment neurons that should be noted. E N neurons have almost twice as many branches as medial pallidal neurons 5'9-11'26. A possible cause of this difference may be that previous studies using primates did not analyze serially reconstructed neurons. Furthermore, E N neurons have discoid to spherical dendritic fields while medial pallidal neurons have fields that tend to be more flattened. The similarities of somatodendritic architecture between E N and larger medial pallidal segment neurons suggest a high degree of phylogenetic preservation of these characteristics 3,23. In contrast, differences in dendritic patterning may be more phylogenetically plastic due to rearrangements of cerebral peduncular fibers in conjunction with neocortical elaboration. Supported by U S P H S A G 01558, H D 05958.
growth of cat entopeduncular neurons. A transient spiny period associated with branch elongation, Dev. Brain Res., 24 (1986) 47-62. 7 Fisher, R.S., Hull, C.D., Adinolfi, A.M. and Buchwald, N.A., Development of GABAergic terminals in the cat brain, Anat. Rec., 205 (1983) 57A. 8 Fisher, R.S., Levine, M.S., Hull, C.D. and Buchwald, N.A., Postnatal ontogeny of connectivity in the basal ganglia of the cat: output neurons of the caudate nucleus, Soc. Neurosci. Abstr., 8 (1982) 964. 9 Fox, C.A., Andrade, A.N., LuQui, I.J. and Rafols, J.A., The primate globus pallidus: a Golgi and electron microscopic study, J. Hirnforsch., 15 (1974) 75-93. 10 Fox, C.A., Hillman, D.E., Siegesmund, K.A. and Sether, L.A., The primate globus pallidus and its feline and avian homologues: a Golgi and electron microscopic study. In R. Hassler and H. Stephan (Eds.), Evolution of the Forebrain -- Phylogenesis and Ontogenesis of the Forebrain, Georg Thieme Verlag, Stuttgart, 1966, pp. 237-248. 11 Francois, C., Percheron, G., Yelnik, J. and Heyner, S., A Golgi analysis of the primate globus pallidus. I. Inconstant
400 processes of large neurons, other neuronal types, and afferent axons, J. Comp. Neurol., 227 (1984) 182-199. 12 Gazzara, R.A., Levine, M.S. and Hull, C.D., Postnatal ontogeny of afferent inputs to the ventral anterior and ventral lateral thalamis nuclei in cats, Soc. Neurosci. Abstr., 9 (1982) 1229. 13 Grofova, I., Denian, J.M. and Kitai, S.T., Morphology of the substantia nigra pars reticulata projection neurons intracellularly labeled with HRP, J. Comp. Neurol., 208 (1982) 352-368. 14 Hull, C.D., McAllister, J.P., Levine, M.S. and Adinolfi, A.M., Quantitative developmental studies of feline neostriatal spiny neurons, Dev. Brain Res., 1 (1981) 309-332. 15 Iwahori, N. and Mizuno, N., Entopeduncular nucleus of the cat: a Golgi study, Exp. Neurol., 72 (1981) 654-661. 16 Iwahori, N. and Mizuno, N., A Golgi study on the globus pallidus of the mouse, J. Comp. Neurol., 197 (1981) 29-43. 17 Larsen, K.D. and McBride, R.L., The organization of feline entopeduncular nucleus projections: anatomical studies, J. Comp. Neurol., 184 (1979) 293-308. 18 Leontovich, T.A. and Zhukova, G.P., The specificity of the general structure and topography of the reticular formation in the brain and spinal cord of carnivora, J. Comp. Neurol., 121 (1963) 347-379. 19 Mannen, H., Three-dimensional reconstruction of individual neurons in higher mammals, Int. Rev. Cytol Suppl., 7 (1978) 329-372. 20 McGuiness, C.M. and Krauthamer, G.M., The afferent projections to the centrum medianum of the cat as demon-
strated by retrograde transport of horseradish peroxidase, Brain Research, 184 (1980) 255-269. 21 Park, M.R., Falls, W.M. and Kitai, S.T., An intraeellular HRP study of the rat globus pallidus. I. Response and light microscopic analysis, J. Comp. Neurol., 211 (1982) 284-294. 22 Phelps, P., Adinolfi, A.M. and Levine, M.S., Development of the kitten substantia nigra: a rapid Golgi study of the early postnatal period, Dev. Brain Res., 10 (1983) 1-19. 23 Rakic, P., Intrinsic and extrinsic factors influencing the shape of neurons and their assembly into neuronal circuits. In P. Seeman and G.M. Brown (Eds.), Frontiers in Neurology and Neuroscience Research, University of Toronto, Toronto, 1974, pp. 112-132. 24 Schneider, J.S., Hull, C.D., Buchwald, N.A., Fisher, R.S. and Levine, M.S., Interactions between basal ganglia, parabrachial and trigeminal nuclei: a functional and anatomical study, Anat. Rec., 208 (1984) 158A. 25 Uylings, H.B.M., Smith, G.J. and Veltman, W.A.B., Ordering methods in quantitative analysis of branching structure of dendritic trees. In G.W. Kreutzberg (Ed.), Physiology and Pathology of Dendrites, Advances in Neurology, Vol. 12, Raven Press, New York, 1975, pp. 247-254. 26 Yelnik, J., Percheron, G. and Francois, C., A Golgi analysis of the primate globus pallidus. II. Quantitative morphology and spatial orientation of dendritic arborizations, J. Comp. Neurol., 227 0984) 200-213.
395
BRE 21595
The entopeduncular nucleus: Golgi morphometrics of serially reconstructed neurons in adult cats C.L. DVERGSTEN, C.D. HULL, M.S. LEVINE, A.M. ADINOLFI, R.S. FISHER and N.A. BUCHWALD Mental Retardation Research Center, University of California, School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted February 18th, 1986) Key words: basal ganglion - - pallidal segment - - dendrite - - morphometry - - Golgi method
Computer-assisted morphometrics were used to characterize mature somatodendritic architecture in Golgi-stained neurons of the entopeduncular nucleus (EN) of the adult cat. Only one form of adult EN neuron was apparent and characterized by common features including: (1) relatively large conical somata, (2) long aspiny and moderately branched dendrites and (3) discoid to spherical dendritic fields oriented randomly within the EN. These results indicate that feline EN neurons have some properties in common with large neurons of the primate medial pallidal segment. The entopeduncular nucleus (EN) in rodent and carnivore brains is considered generally to be homologous to the medial pailidal segment of primate brains because of similarities of cytoarchitecture, connectivity and ultrastructure ],2'5,8-1°. However, the somatodendritic morphology of the E N revealed by Golgi methods has been obtained mainly from immature animals due to favorable silver impregnation prior to myelination of the fiber fascicles of the cerebral peduncle coursing through the region 2']°'11'15'17. The present investigation was designed to assess the full extent of mature somatodendritic architecture in the E N by using computer-assisted m o r p h o m e t r y of serial reconstructions of Golgi-stained E N neurons in the adult cat. The rapid Golgi method was employed to impregnate EN neurons with silver 6'14. Adequately stained neurons were produced in 6 adult cats. These animals were all of known age ( 1 - 3 years). They were born and reared under standard conditions in the kitten breeding colony of the Mental Retardation Research Center at U C L A . The cats were sacrificed by barbiturate overdose and perfused transcardially with aldehyde fixative. Qualitative observations were obtained from well
over 100 E N neurons scattered throughout the nucleus in each cat. Quantitative measurements were derived from 9 neurons in 4 cats. These latter neurons were also dispersed widely in different regions of the EN. They were selected because of the apparent fullness of their silver impregnation and the completeness of their dendritic field reconstruction from serial coronal sections (100 /zm, E p o n embedment) 6'14. Neurons were reconstructed from a minimum of two to a maximum of 10 serial sections. Neurons located at the border of the lateral hypothalamus or embedded in the fiber fascicles of the cerebral peduncle were excluded from this sample. Computer-assisted morphometric methods were used to assess somatic dimensions, dendritic lengths and dendritic branching patterns in 3 dimensions among the serially reconstructed neurons 6. Dendritic branching was defined by a centrifugal ordering method 2°. Golgi-stained neurons were also characterized by photomicrographs and camera lucida drawings using brightfield transmission combined occasionally with angled incident illumination for 'optical sectioning' of neuronal somata viewed from both tissue section faces 19. Our major new finding was that the E N of the adult
Correspondence: M.S. Levine, Mental Retardation Research Center, RM 58-258 NPI, University of California, Los Angeles, CA 90024, U.S.A.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
396 cat contains only one basic form of neuron. In essence, mature EN neurons have rather uniform somatodendritic morphology characterized by moderately large conical somata and long, modestly branched aspinous dendrites. Overall, EN neurons had large dendritic fields oriented randomly within the confines of peduncular fascicles coursing through the nucleus. The somatodendritic architecture of adult EN neurons corresponded well with (1) larger intensely chromophilic EN cells stained by Nissl methods and (2) EN neurons projecting to the thalamus, substantia nigra, globus pallidus and/or mesencephalic pedunculopontine regions demonstrated by retrograde axonal transport of horseradish peroxidaset2,20, 24.
mata were situated mainly in neuropil regions forming a matrix around heavily myelinated cerebral peduncular fiber fascicles (Fig. 1A). While transitions of somata into proximal dendrites were gradual and ill-defined, cell body profiles were clearly of moderately large size (average diameter = 43 + 3/xm, range = 36-59/xm). These somatic profiles were only modestly polymorphic and were typically ovoid or fusiform in shape as reflected by an approximate 2:1 ratio between major and minor somatic axis values (ranges = 49-71/xm vs 25-46ktm, Fig. 1A-C), These neuronal cell bodies generally gave rise to 5 stout primary dendrites (overall range = 3-7 primary dendrites, 4-10/xm diameter, Table I). These processes tapered gradually through their long radiations distally (Fig. 1A-C). Dendritic spines and appendages were found rarely. While short lengths of
Golgi-stained EN neuronal cell bodies had few or no spines along the lengths of their profiles. These so-
2 0 0 pm
B ~
V •..........
. ( . . . .
. . . . . . . . . . . .
.
•
..
. . . . . . . . . . . . . . . . . . . . . . . . . . .
- .....
b
Fig. 1. A: inset shows 3 neurons in a region of the entopeduncular nucleus where the density of myelinated fiber bundles (mb) is high. Dendrites appear to bundle together (arrows in inset) between the fiber bundles. B, C: two serially reconstructed neurons from adult cats. Bold-faced arrows point to terminal arborizations of the dendrites, vb, ventral border of the entopeduncular nucleus.
397 TABLE I Quantitative analysis of E N neurons
Ranges and means (+__S.E.M.) of dendritic parameters of reconstructed EN neurons. Length measurements are in micrometers. Parameter
Morphometric data
No. of dendrites Total dendritic length Dendritic length Dendritic field radius Branches per neuron Branches per dendrite
Mean + S.E.M.
Range
5.0 _+0.3 9434 + 913 1907 + 193 582 + 30 63.3 _+9.8 13.1 _+2.6
4-6 5898-14,204 188-6222 149-1300 36-133 3-78
Branch order
Branch order analysis Branch length (~m)
Number of branches
1
105 + 23* 162 + 20 195 + 17 163 + 11 124 + 10 128 + 29 87 + 21
5.0 + 0.3 9.8 + 0.5 13.8 + 1.1 13.4 + 1.6 8.4 + 1.3 5.5 + 2.3 7.3 + 5.9
2 3 4 5 6 7
* Values are means + S.E.M.
varicose dendrites were observed infrequently, varicosites were not apparent in many silver impregnated neurons. Morphometric analyses demonstrated that individual EN neurons possessed considerable total dendritic length (approximately 9.5 ram, Table I). These extensive processes were segmented by modestly prevalent branching (over 60 branches/cell, Table I). Individual dendrites were about 2 mm long and had roughly 13 branches (Table I). Overall, the dendritic fields of EN neurons were large as reflected by average dendritic field radii of nearly 600/zm (Table I). Branching patterns varied widely in individual dendrites as well as between different cells. For example, some dendrites branched at regular intervals while others branched mainly in proximity to the parent cell bodies. Additionally, dendrites with extensive distal branching (i.e. tufts or thickets) were fairly common (Fig. 1B). By morphometric analyses, a maximum of 7 branch orders accounted for nearly all of the segmentation of individual dendrites of mature EN neurons (Table I). Third and fourth order branches were most prevalent. These branch orders also tended to have the longest branch lengths. Re-
gardless of branch order, dendritic branch lengths usually ranged between 100 and 200/~m. The large extensive dendritic arbors of EN neurons radiated in all directions away from parent cell bodies (Fig. 1A, B). The dendritic fields of different neurons occupied overlapping territory confined, for the most part, within the EN neuropil (Fig. 1A). The stained dendrites wove their course through but did not penetrate the outer borders of the EN. The course of individual dendrites was closely associated with the pattern and density of peduncular fiber fascicles (Fig. 1A). Within the neuropil, dendrites assumed a straight course. When a fiber bundle was encountered, dendrites followed the outer contour of the bundle. When fiber bundles were closely adjacent, dendrites aggregated into bundles running through small neuropil channels interspersed between axonal fascicles. While dendritic branching could occur anywhere in the neuropil, branching seemed to be more prevalent when dendrites encountered fiber fascicles. As an exception to the apparent random radiation of EN dendrites within their available neuropil zones, these processes were unable to penetrate the ansa lenticularis (Fig. 1B). When somata lay in the ventral border of the EN, their dendrites would follow the border and/or radiate centrally within the nucleus. Due to their large size, the dendritic fields of some EN neurons spread across the entire dorsal-ventral extent of the nucleus (maximum dimension -1.0-1.5 mm). However, these dendritic fields failed to penetrate the entire rostral caudal or medial-lateral extents of the EN (maximum dimensions = 2.53.0 mm). On the whole, dendritic fields of EN neurons were oriented randomly within the nucleus in reference to the 3 cardinal planes of section (Fig. 2A-C). The dendritic fields of particular EN neurons displayed a variety of shapes and orientations. Spherical dendritic fields were commonly encountered (Fig. 2A). Cells with flattened spherical or discoid fields were also evident (Fig. 2B). Finally, cells with intermediate field forms were readily apparent (Fig. 2C). In general, the dendritic morphology of adult EN neurons was very similar to earlier descriptions of neurons in the reticulate zone of the substantia nigra of the adult cat 13'18'22. These results are in good agreement with earlier developmental morphometric studies of Golgi mate-
398 n e u r o n s attain m a t u r e s o m a t o d e n d r i t i c m o r p h o l o g y
rial f r o m the feline E N p e r f o r m e d b o t h in o u r o w n and o t h e r l a b o r a t o r i e s 4-6'9-11A5-17'26. In t h e cat, E N
by 9 0 - 1 2 0 days postnatal 6. B o t h the a f f e r e n t and el-
FRONTAL
SAGITTAL
D
D
R
V
V
C
HORIZONTAL
Cell A
Cell B
Cell C
500 pm Fig. 2. Three serially reconstructed neurons from adult cats as they appear in the 3 cardinal planes of section. Cells were entered into the computer in the frontal plane. Sagittal and horizontal views of these cells were obtained by computer rotation. Cell A was reconstructed from 9 serial sections. Cell B was reconstructed from 4 serial sections. Cell C was reconstructed from 8 serial sections.
399 ferent connections of these neurons are established and functional by the day of birth even though considerable neurotransmitter differentiation, terminal morphogenesis and subsequent increases in synaptic security occur during the early postnatal period2,7,8,12. However, our observation of somatodendritic uniformity among mature E N neurons has considerable developmental implications. A m o n g E N neurons stained by Golgi, Nissl and peroxidase connectivity methods, there is extensive polymorphism during early development in the cat both between particular neurons and among the different dendrites of any single neuron 6. Assuming that most, if not all, of these cells survive into adulthood, these widely variable neuronal forms assume a c o m m o n structure as morphogenesis terminated. Therefore, morphogenesis may be heterogeneous among the E N neurons 3,5,6. Additionally, in the mature cat EN, neurons may have only some properties in c o m m o n to those of the medial pallidal segment of primates. We observed only larger neuronal somata with extensive dendritic fields in the adult E N while various investigators have found large, medium and small neurons in Golgi studies of the medial pallidal segment 5,9-11. While it is possible that we failed to stain smaller E N neurons because of the capricious nature of Golgi methods, our results are consistent with the uniform appearance of E N neurons of the adult cat obtained by more
1 Adinolfi, A.M., The fine structure of neurons and synapses in the entopeduncular nucleus of the cat, J. Comp. Neurol., 135 (1969) 225-248. 2 Adinolfi, A.M., Levine, M.S. and Hull, C.D., Early postnatal development of entopeduncular neurons and their neostriatal connections, Exp. Neurol., 70 (1980) 463-474. 3 Berry, M., Bradley, P. and Borges, S., Environmental and genetic determinants of connectivity in the central nervous system - - an approach through dendritic field analysis. In M.A. Corner, R.E. Baker, N.E. Van de Poi, D.F. Swaab and H.B.M. Uylings (Eds.), Maturation of the Nervous System. Progress in Brain Research, Vol. 48, Elsevier, Amsterdam, 1978, pp. 133-146. 4 Chang, H.T., Wilson, C.J. and Kitai, S.T., Single neostriatal efferent axons in the globus pallidus: a light and electron microscopic study, Science, 213 (1981) 915-918. 5 DiFiglia, M., Pasik, P. and Pasik, T., Golgi and ultrastructural study of the monkey globus pallidus, J. Comp. Neurol., 212 (1982) 53-75. 6 Dvergsten, C.L., Hull, C.D., Levine, M.S., Adinolfi, A.M. and Buchwald, N.A., Postnatal differentiation and
reliable Nissl and connectivity staining methods 8A2'24. Thus, the larger pallidal neurons may be the homologs of the adult feline E N neurons. In both cats and primates, the large and rather randomly oriented dendritic trees are situated to receive numerous convergent inputs particularly from wide areas of the neostriatum 4,8,21. The somatodendritic surfaces of these neurons are heavily ensheathed mainly by striatopallidal terminals containing 7-amino-butyric acid 1'2'7. There are other differences in the dendritic architecture of E N and larger medial pallidal segment neurons that should be noted. E N neurons have almost twice as many branches as medial pallidal neurons 5'9-11'26. A possible cause of this difference may be that previous studies using primates did not analyze serially reconstructed neurons. Furthermore, E N neurons have discoid to spherical dendritic fields while medial pallidal neurons have fields that tend to be more flattened. The similarities of somatodendritic architecture between E N and larger medial pallidal segment neurons suggest a high degree of phylogenetic preservation of these characteristics 3,23. In contrast, differences in dendritic patterning may be more phylogenetically plastic due to rearrangements of cerebral peduncular fibers in conjunction with neocortical elaboration. Supported by U S P H S A G 01558, H D 05958.
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