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The internal organization of the mouse caudate nucleus: Evidence for cell clustering and regional variation
Brain Research, 137 (1977) 53-66
53
© Elsevier/North-Holland Biomedical Press
T H E I N T E R N A L O R G A N I Z A T I O N OF T H E M O U S E C A U D A T E N U C L E U S : E V I D E N C E F O R CELL C L U S T E R I N G A N D R E G I O N A L V A R I A T I O N *
PATRICIA L. MENSAH Department of Anatomy, University of Southern California, School of Medicine, Los Angeles, Calif. 90033 (U.S.A.)
(Accepted March 10th, 1977)
SUMMARY From paraffin-embedded brains of adult mice, ten-micron sections were taken through the entire extent of the caudate nucleus. Coronal, horizontal or sagittal sections stained with cresyl violet were used to examine two aspects of the internal organization of the nucleus: regional variation and cell clustering. A core of large cells located centrally in the head of the nucleus and ventrally in the body was described. This core is bounded by zones containing medium cells. Slightly larger medium cells occur laterally in the head of the nucleus than elsewhere. Based on these results, three zones have been described in the head of the nucleus: a lateral periphery, a central core area and a medial periphery. The dorsal area of the body of the nucleus probably corresponds most nearly to the lateral periphery, and the ventral area to the central core. Cell clustering is the dominant pattern of organization within each zone. Only one large cell was seen per clumped cell grouping, and this cell could occupy either a peripheral or central position in the cluster. Medium cells participated in the clumped arrangement of cells, and also formed rings around fibers of the internal capsule.
INTRODUCTION Nissl staining of the caudate nucleus is often reported to present a homogeneous picture. The predominant cell type, a medium cell with moderate Nissl substance, is said to occur randomly throughout the neuropil. Yet N a m b a 12 was able to divide the caudate nucleus into several subregions based upon statistically significant differences in medium cell density. In addition, small 'clusters' or 'aggregations' of neo-
* The results reported here were presented in part at the sixth annual meeting of the Society for Neuroscience, Toronto, Canada, 1976.
54 striatal cell bodies have been noted in Nissl material2,8,13. Clusters of medium cells characterize both rodent and carnivore neostriatum and show some relationship to the axon fascicles of internal capsular fibers 4. Whether or not other cell types, particularly the large caudatal cell, also participate in cluster formation has not been reported. Both of these parameters, that is, regional variation and cellular aggregation, could represent the corollary to two commonly reported features of the caudate neuropil: topographically organized afferent input and synaptic island formation, respectively. Although the corticostriate projection is massive3,7,18,19, anterior areas project to anterior caudate-putamen, and postcentral cortices to more caudal regions. Rigid topography exists as well in the nigrostriatal system, suggesting that connections occur between discrete regions of both structures 17. Furthermore, the clustering of the synaptic endings of individual afferent or intrinsic systems, more commonly referred to as 'synaptic islands', has been reportedS,9,16. The weight of the evidence, then, supports the concept of microorganization within the caudate nucleus of mammals. The cellular basis for this suborganization is a question that will be considered in this report. MATERIALS AND METHODS Seven normal adult mice of the species C57/BL supplied by Simonson Laboratories (Gilroy, California) were anesthetized with Nembutal and perfused intracardially with a 10% formol-saline solution. Brains were removed and blocked for the entire extent of the caudate nucleus. The material was processed for paraffin embedding using dioxane as both dehydrant and clearing agent 10. Ten-micron sections were taken at regular intervals through the caudate nucleus, three brains being sectioned coronally, two horizontally and one sagittally. One brain was used to prepare 30 #m frozen sections soon after fixation for comparison with the paraffin sectioned material. Groups of sections were stained with cresyl violet; and in one sagittal and two coronal brains, a duplicate series of sections was prepared and stained using a silver stain for unmyelinated fibers 14. Sections in each of the three planes were examined carefully for evidence of cell clustering. In addition, a quantitative approach was taken to the question of homo- or heterogeneity along the rostrocaudal and dorsoventral axes of the caudate nucleus. Ten-micron coronal sections from two animals were used for this aspect of the data analysis. A standard section through the caudate was taken to be the nucleus at the level of the crossing of the anterior commissure, a level at which the head of the nucleus presents sizeable extent. The total area of the caudate in this section was divided by two lines, one sagittal and one transverse. Therefore, using this procedure (for more detail, see Burandt et al.1), this standard section was divided into four quadrants: dorsolateral (dl), ventrolateral (vl), dorsomedial (dm) and ventromedial (vm). The areas comprising each of these quadrants were kept constant and applied directly to other levels of the nucleus. Five other levels were selected for detailed analysis: (l) rostral caudate containing only dl, dm and vm quadrants (level l ; note that cells of the claustrum invade the vl quadrant, making scoring of striatal cells
55 here impossible); (2) a more caudal section through the anterior aspect of the nucleus in which the lateral and medial septum are well developed and all 4 quadrants are present (level 2); (3) anterior region of the body of the nucleus, a level at which the pallidum has begun to invade the vm quadrant (level 4); (4) a section representing an intermediate region of the body of the nucleus (level 5); and (5) a section through the posterior body of the nucleus (level 6). For levels 5 and 6, note that only dl and vl quadrants are unequivocally present. Using this procedure, three standard sections occur through the head of the caudate nucleus and three from what has been called the caudate-putamen nucleus of rodents by Gurdjian 6 and the body of the caudate by many workers. A vertical line through the center of each quadrant divided each quadrant into medial and lateral portions. Therefore, a total of 8 equal areas could be delimited on the standard section denoted level 3 (see Fig. 3): VL1 and VLm, DL1 and DLm, DM1 and DMm, and VM1 and VMm. Every attempt was made to be sure, under the microscope, that these areas were equal. Sections from one of two coronally sectioned brains which most closely approximated the standard level were used to accumulate the raw data. Two types of sampling procedure were used. In the first procedure, only large cells, that is, cells with long axes ranging from 20 to 30 # m in length, were noted. The total number of large cells occurring in each of the 8 areas, or in as many areas as occurred in the section, were counted (see Fig. 3). The second procedure included an examination of the medium cell population of the nucleus. Medium cells were selected randomly from each division of each quadrant. Using an eyepiece micrometer, the long axes of these cells were measured and the lengths recorded. To be sure the cells were selected randomly, the scale of the eyepiece micrometer was moved into the field. Cells at positions 1 or 2, 5 or 6 and 9 or l0 were scored if they satisfied three criteria: (1) had a clear nucleolus; (2) the cell membrane was clear; and (3) the long axis of the cell fell into the 10-19/~m range. Using this procedure, the medium cell population could be analyzed without regard to the small populations of small and large cells. Small cells represent less than 1 ~ of the total cell population and have been ignored in this report. Ten measurements were taken from each region, providing a total of 20 measurements of medium cell size per quadrant. Means of the l0 measurements per sector were calculated from the raw scores; means listed in Tables 5-8 represent means of the sample to be tested in the statistical analysis. Calculations for large cell variation appear in Tables 1-4 and the medium cell results appear in Tables 5-8. A t-test for uncorrelated means z0 provided dorsal vs. ventral, medial vs. lateral, core vs. periphery and anterior vs. posterior comparisons of (1) the frequency of large cells and (2) sizes of medium cells. In addition, the distribution of large cells was plotted on projection drawings of each standard section (Fig. 3). Horizontal sections through the nucleus were also used to provide a pictorial analysis of the distribution of large cells (see Fig. 4). RESULTS
Description of cell types and cell clustering Except for the subependymal region of the lateral ventricle, groups of nerve
56
D
I_A
Fig. 1. Cell clusters in adult mouse caudate, sagittal section. Photomicrograph to left indicates a large area of caudate. Notice that many cells form aggregates, but scattered cells also occur. Bar --: 100 /~m. 1-3 are high power photographs of cell clusters in brackets I 3 respectively. 1 : clumped mass of cells containing one large and several medium cells. 2: clumped mass of medium cells. 3: linear arrangement of medium cells that might represent part of ring formation of cells seen in coronal section. Bar in 1 applies to pictures 1-4 and represents 25/bin. Arrowheads point out the same cells at each of the two magnifications as an aid to orientation of the cell cluster. 4: cell cluster containing a cell 'pair', located ventral to the area shown in picture at left. Marking at lower left denotes dorsal and anterior directions and applies to all photomicrographs.
cells occur t h r o u g h o u t the nucleus. The overwhelming majority of cells of the nucleus fall into one of two size categories: 10-14 or 15-20 # m in diameter. Both categories of m e d i u m cells have large, pale nuclei, s u r r o u n d e d by a thin rim of cytoplasm a n d very little Nissl substance (see Fig. 1). Cells larger t h a n 20 # m c o n t a i n a large a m o u n t of Nissl substance and, often, indented nuclei. These cells are multipolar or fusiform in appearance, and, although a small percentage of the total population, are quite p r o m i n e n t due to their size a n d Nissl content. In all regions of the nucleus, cell clustering occurs, groups of cells being bordered by the fibers of the bundles of the internal capsule. Scattered cells lie between cell clusters, but the impression of cell clustering is evident in all three planes of section. Ten to 15 cells are c o m m o n l y present in a cell cluster. Often, a n d in all planes of section, cell clusters may take the form of a clumped mass of cells. Clumps composed of one large and several m e d i u m cells or of only medium cells occur. The m e d i u m cells in these clumpings are from both size categories of m e d i u m cells a n d no pattern or
57
Fig. 2. A. Notice several unmyelinated and lightly myelinated fibers coursing through the caudate. Area at open arrows contains many of these fibers in a circular arrangement. Note similarity of distribution of these fibers and patterning of cell clusters shown in Fig. 1. B, C, D are high power photomicrographs of fibers denoted by arrows to A. Bar in A represents 50 #m for A and 12.5/~m for B, C, D. Unmyelinated fiber stain, Scalia et al. 14, coronal section. Darkly stained areas are fascicles of the internal capsule in cross-section. Dorsal at top, medial to the right.
difference could be n o t e d in the occurrence o f m e d i u m - s m a l l or m e d i u m - l a r g e cells. Large cells, on the other hand, c a n n o t occur in every cell cluster due to their low frequency o f occurrence. I n the clusters in which they are present, these cells m a y o c c u p y either a central or a p e r i p h e r a l position. However, no m o r e t h a n one large cell seems to occur in any cell cluster. In the c l u m p e d a r r a n g e m e n t o f m e d i u m cells, one o r a few cells m a y be centrally l o c a t e d a n d they m a y be either m e d i u m - s m a l l or m e d i u m - l a r g e or both. E x a m i n a t i o n s o f sections p r e p a r e d using the u n m y e l i n a t e d fiber stain show a c o r r e s p o n d e n c e in the intrastriatal t r a j e c t o r y o f these fibers to areas c o n t a i n i n g the c l u m p e d a r r a n g e m e n t o f cells (Fig. 2). Often, within individual clusters, p a i r i n g occurs. In the thick sections used in this study (10 #m), the p l a s m a m e m b r a n e s o f the two cells are extremely close a n d often a p p e a r fused (see Fig. 1). Occasionally, a t r i a d a r r a n g e m e n t also occurs in which one cell is centrally located a n d b o u n d e d laterally by two cells. A l t h o u g h frequency estimates for cell p a i r i n g o r the triad a r r a n g e m e n t were n o t a t t e m p t e d , cell p a i r i n g occurs often b u t n o t in every cell cluster, a n d is m o r e likely to occur t h a n the triad arrangement. A n alternate a r r a n g e m e n t o f the cell cluster also occurs. M e d i u m cells m a y occur single file a n d in ring-like f o r m a t i o n a r o u n d a fascicle o f the internal capsule. The ring-like f o r m a t i o n o f m e d i u m cells was m o s t easily seen in c o r o n a l sections b u t
58
O
A Fig. 3. Pictorial analysis of large cell distribution-~oronal section. Each of the 6 standard levels is drawn. Note solid horizontal and solid vertical lines which divide each section into 4 quadrants. Dashed lines subdivide each quadrant into a lateral and a medial zone. Only a few structures are labelled (see Sidman et al. 15, plates 21, 28, 32, 34, 38 and 42 for more detail). Abbreviations: A, amygdala, lateral nucleus; AC, anterior commissure; CC, corpus callosum; CL, claustrum; GL, globus paUidus; R, reticular nucleus of the thalamus; S, septum. The lateral ventricle is present in each drawing.
also occurred in horizontally sectioned material. In sagittal sections, this a r r a n g e m e n t of cell somata c a n n o t be d e m o n s t r a t e d clearly. These three types of clusters, that is, clumped m e d i u m a n d large cells, clumped m e d i u m cells, and m e d i u m cells in ring-like a r r a n g e m e n t occur t h r o u g h o u t the anteroposterior a n d dorsoventral extent of the nucleus.
59
Fig. 4. Pictorial analysis of large cell distribution--horizontal section. The three sections drawn represent dorsal, intermediate and ventral sections through the caudate nucleus. (For more fully labeled sections, see Sidman et al. 15, plates 119, 125 and 131) Abbreviations: CC, corpus callosum; F, fornix of the hippocampus; GP, globus pallidus; R, reticular nucleus of the thalamus; S, septum. The internal capsule is oulined but not labeled in the bottom drawing.
Description of the quadrant analysis 1. Large cell distribution. The statistical re suits concur completely with the pictorial analysis o f the data (Figs. 3 and 4). There is no difference in the dorsoventral distribution o f large 20-30-/~m cells in sections t h r o u g h the caudate anterior to the crossing o f the anterior commissure. However, the difference in large cell content between dorsal and ventral areas posterior to the anterior commissure is significant (see Table I). The mediolateral large cell distribution presents an interesting picture (see Fig. 3 and Tables lI and III). There is no difference in probability o f large cell occurrence if lateral is defined as D L and V L and medial as D M and V M quadrants. However, if, as the pictorial analysis implies, medial is defined as a 'core' area consisting o f the
6O TABLE ! Raw score for each sector can be obtained from Fig. 3. Data analysis
Dorsoventral variation - - Large cell (DLL+M + DML+M/ VLL+M + VML+M)
Number of cells (N) Standard deviation (S)
Entire caudate
Levels 1, 2, 3
Levels" 4, 5, 6
Dorsal
Ventral
Dorsal
Ventral
Dorsal
Ventral
20 1.93
17 1.50
12 2.22
10 1.15
8 1.06
7 1.70
Estimated sample variance (Sp~)* To
* Given by equation
3.05 0.14
3.31 1.28
1.94 2.31 **
(N1 - - 1)S21 + ( N 2 - l)SCz N1 ~- N2 - - 2
** Significant at 0.05 confidence level. TABLE II Raw score for each sector can be obtained from Fig. 3. These results are not significant. Data analysis
Mediolateral variation - - Large cell (DML+M + VML+M / DLL+M + VLL+M)
Number of cells (N) Standard deviation (S) Estimated sample variance (Sp2)* To
* Given by equation
Whole caudate
Levels 1, 2, 3
Medial
Lateral
Medial
Lateral
15 1.77
22 1.70
12 1.88
10 1.89
2.99 1.04
3.55 0.35
(N1 - - 1)SZl + (N2 - - 1)$22 N1 + N 2 - - 2
centrally located portions of all 4 quadrants, DLm + VLm + DMI + VM1, a n d lateral includes the two peripheral zones bordering the 'core' area, namely, DL~ + VL1 a n d D M m + VMm, a highly significant result is obtained for both the whole caudate (P < 0.01) and for the three anterior sections (P -< 0.001). O n the other h a n d , there is n o core-peripheral difference whatsoever in the large cell d i s t r i b u t i o n posterior to the anterior commissure. Also note that n o difference exists in lateral (DLm + VLm) vs. medial (DMI + VM1) core or in lateral (DLI + VL1) vs. medial (DMm + VMm) periphery anterior to the commissure. This difference could n o t be tested posterior to the commissure due to the reduction in the mediolateral extent of the nucleus in this area. A n t e r o p o s t e r i o r differences appear n o t to play a m a j o r role in the mouse caudate
Core
2.03 3.92***
11 0.50
N1 + N a -
2
(N1 - - 1)$21 -}- ( N 2 - 1)$22
2.39 3.13"*
11 1.95
** Significant at 0.01 confidence level. *** Significant at the 0.001 confidence level.
*Given by equation
Estimated sample variation (Sp 2)* To
18 1.14
8
7 1.68
2.68 0.58
1.60
11 1.75 3.57 0.51
8 2.07
Medial
Entire caudate
Periphery Lateral
Levels 4, 5, 6
Periphery Core
Levels 1, 2, 3
Periphery Core
Entire caudate
5 2.17 4.25 0.10
Lateral 6 1.97
Medial
Levels 1, 2, 3
Lateral vs. medical core
1.18 1.64
11 1.34
Lateral
7 0.38
Medial
Entire caudate
5 0.55 0.23 1.49
Lateral
6 0.41
Medial
Levels 1, 2, 3
Lateral vs. medial periphery
Mediolateral variation - - Large cell (Core vs. periphery) (DLM ÷ DML ÷ VLM q- V A I L / D L L ~- D M ~ q- VLL q- VMM)
19 Number of cells (N) Standard deviation (S) 1.85
Data analysis
Raw score for each sector can be obtained from Fig. 3.
TABLE Ili
62 TABLE IV Raw score for each sector can be obtained from Fig. 3. Data analysis
Anteroposterior variation - - Large cell (Levels 1, 2, 3 vs. levels 4, 5, 6)
Number of cells (N) Standard deviation (S)
All sectors
Dorsal sectors Ventral sectors Core
Periphery
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
22 1.84
12 2.22
10 1.15
11 1.95
11 0.50
Estimated sample variation (Sp2) * To
15 1.59
8 1.06
3.04 0.26
* Given by equation
3.45 1.62
7 1.70 1.95 1.87"*
8 1.60
7 1.68
3.29 1.30
1.21 1.46
(N1 - - 1)$21 + ( N 2 - 1)S22 N1 + Ne - - 2
** Significant at 0.10 confidence level only. n u c l e u s ( T a b l e IV). N o differences w e r e seen w i t h all a r e a s c o u n t e d , b e t w e e n d o r s a l a r e a s only, b e t w e e n the t w o cores, o r b e t w e e n the t w o p e r i p h e r a l zones. H o w e v e r , sections p o s t e r i o r to the a n t e r i o r c o m m i s s u r e are m o r e likely to c o n t a i n large cells in v e n t r a l a r e a s t h a n are sections a n t e r i o r to the c r o s s i n g o f the c o m m i s s u r e ( P < 0.1). T h e s e findings are a p p a r e n t in the p r o j e c t i o n
drawings
of
cell s i z e variation. T h e statistical analysis o f the c o m p a r i s o n
of
h o r i z o n t a l sections in Fig. 4. 2. M e d i u m
a v e r a g e cell size a m o n g the different q u a d r a n t s was m o s t interesting. N o d o r s o v e n t r a l differences o c c u r in m e d i u m cell size c h a r a c t e r i s t i c s in e i t h e r the entire c a u d a t e o r TABLE V The mean listed above is the mean of the sample in question. The number of measurements (N) corresponds with the number of sectors involved in each sample. Data analysis
Dorsoventral variation - - Medium cell size (DLL+M + DML+M / VLL+M -~ VML+M)
Number of measurements (N) Mean, in ttm (~) Standard deviation (S) Estimated sample variation (Sp 2)* To
* Given by equation
Entire caudate
Levels 1, 2, 3
Levels 4, 5, 6
Dorsal
Ventral
Dorsal
Ventral
DorsM
VeJltral
20 15.05 0.98
17 15.14 1.32
12 15.23 0.91
10 14.58 1.18
8 14.76 1.07
7 15.93 1.15
1.32 0.24
(N1 - - 1)521 + ( N 2 - 1)522 NI + N2 - - 2
** Significant at the 0.10 confidence level only.
1.08
1.23
1.46
2.04**
63 T A B L E VI T h e m e a n listed above is the m e a n o f the sample in question. T h e n u m b e r o f m e a s u r e m e n t s (N) c o r r e s p o n d s to the n u m b e r o f sectors involved in each sample. These results were n o t significant.
Mediolateral variation - - Medium cell size (DML+M -}- VML+M / DLL+M q- VLL+M)
Data analysis
Number of measurements (N) Mean, in/~m (~) Standard deviation (S) Estimated sample variation (Sp2)* To
* Given by e q u a t i o n
Whole caudate
Levels 1, 2, 3
Medial
Lateral
Medial
Lateral
15 14.75 1.09
22 15.31 1.13
12 14.61 1.07
10 15.33 0.98
1.24 1.51
1.06 1.63
(N1 - - 1)S21 + ( N 2 - - 1)S22 N1 + N2 - - 2
in the rostral three sections (Table V). However, more caudally, a slight difference between the two areas exists (P < 0.1) with more medium-large cells tending to occur in the ventral quadrants. When medial and lateral quadrants were compared statistically, no significant difference in medium cell size could be noted (Table VI). Further, no core vs. periphery differences existed (Table VII); and there was no statistically significant difference between the medium-sized cell population of the lateral core vs. that of the medial core. Yet, microscopic inspection of the slides seemed to indicate a gradual decrease in cell size from lateral to medial areas. This qualitative impression was upheld only by the statistical analysis of the difference between lateral vs. medial periphery, a difference that is highly significant. The only anteroposterior difference occurred ventrally (Table VIII). Mediumlarge cells are more likely to occur ventrally caudal to level 3 than at or rostral to it. No other statistically significant difference exists along the rostrocaudal axis of the nucleus. DISCUSSION
The foregoing analysis has attempted to describe the caudate nucleus according to several easily defined features in Nissl stained material. Two aspects of the internal organization of the nucleus are clear. First, the nucleus is divided into cellular territories, and secondly, within each territory, cellular aggregation or clustering is the prevailing pattern of organization. From the large cell analysis, it is clear that a core located centrally in the head of the nucleus is surrounded laterally by smaller zones rarely (but occasionally) containing large cells. The lateral peripheral zone is much more likely to contain medium-large cells than the peripheral zone bordering the ependyma of the lateral
Core
11 15.05 1.01
N1 ~- N 2 - - 2
(N1 - - I)S '~ % ( N 2 - -
1.32 0.56
** Significant at 0.05 confidence level. *** Significant at 0.01 confidence level.
* G i v e n by equation
Estimated sample variance (Sp2) * To
18 15.19 1.27
1)$22
1.19 0.50
11 14.82 1.17
8 14.89 1.09 1.38 1.48
7 15.79 1.27
11 14.77 0.97 1.02 1.09
8 15.28 1.07
Medial
Entire caudate
Periphery Lateral
Levels 4, 5, 6
Periphery Core
Levels 1, 2, 3
Periphery Core
Entire caudate
5 15.04 0.98
6 15.07 1.12
Medial
1.12 0.05
Lateral
Levels 1, 2, 3
Lateral vs. medial core
11 15.85 1.06
7 14.16 0.80
Medial
0.94 3.60***
Lateral
Entire caudate
5 15.62 0.99
0.87 2.61 **
6 14.15 0.88
Lateral Medial
Levels 1, 2, 3
Lateral vs. medial periphery
Mediolateral variation- Medium cell size (Core vs. periphery) (DLM + D ML -r VLM + VML / DLL + D MM + VLL + VMM)
N u m b e r of measurem e n t s (N) 19 M e a n , in/~m (,'~) 14.98 Standard deviation (S) 1.02
Data analysis
T h e m e a n listed above is the m e a n of the s a m p l e in question. T h e n u m b e r o f m e a s u r e m e n t s (N) c o r r e s p o n d s to the n u m b e r o f sectors involved in each sample.
T A B L E VII
65 TABLE VIII The mean listed above is the mean of the sample in question. The number of measurements (N) corresponds to the number of sectors involved in each sample. Data analysis
Anteroposterior variation - - Medium cell size (Levels 1, 2, 3 vs. levels 4, 5, 6) All sectors
Dorsal sectors
Ventral sectors Core
1,2,3
1,2,3
1,2,3
4, 5,6
4, 5,6
4, 5,6
1,2,3
Periphery 4, 5,6
1, 2,3
4, 5,6
Number of measurements (N) 22 15 12 8 10 7 11 8 11 7 Mean, in #m (~) 14.94 15.31 15.23 14.76 14.58 15.93 15.05 14.89 14.82 15.79 Standard deviation (S) 1.07 1.23 0.91 1.07 1.18 1.15 1.01 1.09 1.17 1.27 Estimated sample variation (Sp~)* To
1.29 0.97
0.95 1.06
1.36
1.09
1.46
2.35**
0.33
1.66
(N1 - - 1)S~1 + ( N s - 1)S~2 NI + N z - 2 ** Significant at 0.05 confidence level. * Given by equation
ventricle. Medium-small cells, on the other hand, occupy areas that are large-cell free and tend to occur more medially than the other two cell types. Therefore, three zones occur in the head of the nucleus. A lateral zone bordering the corpus callosum contains mainly medium-large, some medium-small and very few large cells. A larger zone medial to this includes the entire central area of the head of the nucleus, and contains almost all of the large cells occurring at this level and many medium-large cells. The third zone, the small medial margin, is clearly different from the other zones and contains cells smaller than the other two areas. The only consistent difference in the body of the nucleus (that is, levels caudal to the crossing of the anterior commissure) is a dorsoventral one. The ventral quadrant (usually denoted VL in the drawings at this level) more often contains large and medium-large cells than the dorsal area. Large cells in this ventral region belong to a ventral column of cells extending throughout the nucleus (see Fig. 4). Medium cells in this area are significantly larger than ventrally located medium cells of the head of the nucleus. The dorsal area, on the other hand, has a low frequency of large cell occurrence, and approximates the cell population of a peripheral zone, particularly the lateral periphery. Although generalization from one species to another is always difficult, these results may well be characteristic of the mammalian neostriatum. Chronister et al. 4 demonstrated both the clumped arrangement of medium cells and medium cells in ringed formation in Golgi preparations of mouse and rat neostriatum. In the same study, cell clustering was shown to be a dominant aspect of the organization of the feline caudate as well, and in spite of several significant differences between carnivore and rodent material. These authors did not comment upon large cell participation
66 in cell clusters. Large cells represent a small percentage of the total cell p o p u l a t i o n of both rat 11 a n d cat 8 caudate. Since cell clustering of m e d i u m cells occurs in these two animals, large cells p r o b a b l y also participate in this a r r a n g e m e n t in m u c h the same was as they do in mouse neostriatum. Whether the caudate nucleus of primates presents a h o m o l o g o u s condition c a n n o t be stated from the available literature. The work of G o l d m a n a n d N a u t a 5 d e m o n s t r a t i n g synaptic island f o r m a t i o n by prefrontal cortical afferents to the head of the nucleus is the closest a p p r o x i m a t i o n to date of this concept. The bulk of the evidence, then, suggests morphological diversity in the internal organization of the caudate. F u r t h e r study is warranted. ACKNOWLEDGEMENTS This research was supported by a seed grant from the School of Medicine of the University of Southern California. I am very grateful to Dr. Richard Binggeli for his m a n y helpful suggestions during preparation of this paper. Mrs. Caroline Brown's thoughtful typing of the m a n u s c r i p t is also gratefully appreciated.
REFERENCES 1 Burandt, D. C., French, G. M. and Akert, K., Relationships between the caudate nucleus and the frontal cortex in Macaca mMatta, Confin. neuroL (Basel), 21 (1961) 289-306. 2 Butcher, L. L. and Hodge, G. K., Post natal development of acetylcholines~erasein the caudateputamen nucleus and substantia nigra of rats, Brain Research, 106 (1976) 223-240. 3 Carmen, J. B., Cowan, W. M. and Powell, T. P. S., The organization of tbe corticostriate connections in the rabbit, Brain, 86 0963) 525-562. 4 Chronister, R. B., Farnell, K. E., Marco, L. A. and White, L. E., Jr., The rodent neostriatum: a Golgi analysis, Brain Research, 108 (1976) 3746. 5 Goldman, P. S. and Nauta, W. J. H., An intricately patterned prefronto-caudate projection in the rhesus monkey, J. comp. NeuroL, 171 (1977) 369-386. 6 Gurdjian, E. S., The corpus striatum of the cat, J. comp. Neurol., 45 (1928) 249-281. 7 Kemp, J. M. and Powell, T. P. S, The corticostriate projections in the monkey, Brain, 93 (1970) 526-546. 8 Kemp, J. M. and Powell, T. P. S., The structure of the caudate nucleus of the cat: light and electron microscopy, Phil. Trans. B, 262 (1971) 383-401. 9 Kemp, J. M. and Powell, T. P. S., The site of termination of afferent fibres in the caudate nucleus, Phil. Trans. B, 262 (1971) 413427. 10 Luna, L. G. (Ed.), Manual o f Histologic Staining Methods Of the Armed Forces Institute o f PathoIogy, 3rd ed., New York, 1968. 11 Mensah, P. and Deadwyler, S., The caudate nucleus of the rat: cell types and the demonstrationofa commissural system, J. Anat. (Lond.), 117 (1974) 281-293. 12 Namba, M., Cytoarchitektonische Untersuchungen am Striatum, J. Hirnforsch., 3 (1957) 2448. 13 Papez, J. W., A summary of the fiber connections of the basal ganglia with each other and with portions of the brain, Res. Publ. Ass. herr. merit. Dts., 21 (1942) 21-68. 14 Scalia, F., Halpern, M., Knapp, H. and Riss, W., The efferent connexions of the olfactory bulb in the frog: A study of degenerating unmyelinated fibres, J. Anat. (Lond.), 103 (1968) 245-262. 15 Sidman, R. L., Angevine, J. B., Jr. and Pierce, E. T., Atlas o f the Mouse Brain and Spinal Cord, Cambridge, Massachusetts, 1971. 16 Tennyson, V. M. and Marco, L. A., Intrinsic connections of caudate neurons. 11. Fluorescence and electron microscopy following chronic isolation, Brain Research, 53 (1973) 307-317. 17 Usunoff, K.G., Hassler, R., Romansky, K., Usunova, P. and Wagner, A., The nigrostriatal projection in the cat: Part 1. Silver impregnation study, J. neurol. Sci., 28 (1976) 265-288. 18 Webster, K. E., Cortico-striate interrelations in the albino rat, J. Anat. (Lond.), 95 (1961) 532-544. 19 Webster, K. E., The cortico-striatal projection in the cat, J. Anat. (Lond.), 99 (1965) 329-337. 20 Zuwaylif, F. H., General .4ppJied Statistics, Menlo Park, California, 1972.
53
© Elsevier/North-Holland Biomedical Press
T H E I N T E R N A L O R G A N I Z A T I O N OF T H E M O U S E C A U D A T E N U C L E U S : E V I D E N C E F O R CELL C L U S T E R I N G A N D R E G I O N A L V A R I A T I O N *
PATRICIA L. MENSAH Department of Anatomy, University of Southern California, School of Medicine, Los Angeles, Calif. 90033 (U.S.A.)
(Accepted March 10th, 1977)
SUMMARY From paraffin-embedded brains of adult mice, ten-micron sections were taken through the entire extent of the caudate nucleus. Coronal, horizontal or sagittal sections stained with cresyl violet were used to examine two aspects of the internal organization of the nucleus: regional variation and cell clustering. A core of large cells located centrally in the head of the nucleus and ventrally in the body was described. This core is bounded by zones containing medium cells. Slightly larger medium cells occur laterally in the head of the nucleus than elsewhere. Based on these results, three zones have been described in the head of the nucleus: a lateral periphery, a central core area and a medial periphery. The dorsal area of the body of the nucleus probably corresponds most nearly to the lateral periphery, and the ventral area to the central core. Cell clustering is the dominant pattern of organization within each zone. Only one large cell was seen per clumped cell grouping, and this cell could occupy either a peripheral or central position in the cluster. Medium cells participated in the clumped arrangement of cells, and also formed rings around fibers of the internal capsule.
INTRODUCTION Nissl staining of the caudate nucleus is often reported to present a homogeneous picture. The predominant cell type, a medium cell with moderate Nissl substance, is said to occur randomly throughout the neuropil. Yet N a m b a 12 was able to divide the caudate nucleus into several subregions based upon statistically significant differences in medium cell density. In addition, small 'clusters' or 'aggregations' of neo-
* The results reported here were presented in part at the sixth annual meeting of the Society for Neuroscience, Toronto, Canada, 1976.
54 striatal cell bodies have been noted in Nissl material2,8,13. Clusters of medium cells characterize both rodent and carnivore neostriatum and show some relationship to the axon fascicles of internal capsular fibers 4. Whether or not other cell types, particularly the large caudatal cell, also participate in cluster formation has not been reported. Both of these parameters, that is, regional variation and cellular aggregation, could represent the corollary to two commonly reported features of the caudate neuropil: topographically organized afferent input and synaptic island formation, respectively. Although the corticostriate projection is massive3,7,18,19, anterior areas project to anterior caudate-putamen, and postcentral cortices to more caudal regions. Rigid topography exists as well in the nigrostriatal system, suggesting that connections occur between discrete regions of both structures 17. Furthermore, the clustering of the synaptic endings of individual afferent or intrinsic systems, more commonly referred to as 'synaptic islands', has been reportedS,9,16. The weight of the evidence, then, supports the concept of microorganization within the caudate nucleus of mammals. The cellular basis for this suborganization is a question that will be considered in this report. MATERIALS AND METHODS Seven normal adult mice of the species C57/BL supplied by Simonson Laboratories (Gilroy, California) were anesthetized with Nembutal and perfused intracardially with a 10% formol-saline solution. Brains were removed and blocked for the entire extent of the caudate nucleus. The material was processed for paraffin embedding using dioxane as both dehydrant and clearing agent 10. Ten-micron sections were taken at regular intervals through the caudate nucleus, three brains being sectioned coronally, two horizontally and one sagittally. One brain was used to prepare 30 #m frozen sections soon after fixation for comparison with the paraffin sectioned material. Groups of sections were stained with cresyl violet; and in one sagittal and two coronal brains, a duplicate series of sections was prepared and stained using a silver stain for unmyelinated fibers 14. Sections in each of the three planes were examined carefully for evidence of cell clustering. In addition, a quantitative approach was taken to the question of homo- or heterogeneity along the rostrocaudal and dorsoventral axes of the caudate nucleus. Ten-micron coronal sections from two animals were used for this aspect of the data analysis. A standard section through the caudate was taken to be the nucleus at the level of the crossing of the anterior commissure, a level at which the head of the nucleus presents sizeable extent. The total area of the caudate in this section was divided by two lines, one sagittal and one transverse. Therefore, using this procedure (for more detail, see Burandt et al.1), this standard section was divided into four quadrants: dorsolateral (dl), ventrolateral (vl), dorsomedial (dm) and ventromedial (vm). The areas comprising each of these quadrants were kept constant and applied directly to other levels of the nucleus. Five other levels were selected for detailed analysis: (l) rostral caudate containing only dl, dm and vm quadrants (level l ; note that cells of the claustrum invade the vl quadrant, making scoring of striatal cells
55 here impossible); (2) a more caudal section through the anterior aspect of the nucleus in which the lateral and medial septum are well developed and all 4 quadrants are present (level 2); (3) anterior region of the body of the nucleus, a level at which the pallidum has begun to invade the vm quadrant (level 4); (4) a section representing an intermediate region of the body of the nucleus (level 5); and (5) a section through the posterior body of the nucleus (level 6). For levels 5 and 6, note that only dl and vl quadrants are unequivocally present. Using this procedure, three standard sections occur through the head of the caudate nucleus and three from what has been called the caudate-putamen nucleus of rodents by Gurdjian 6 and the body of the caudate by many workers. A vertical line through the center of each quadrant divided each quadrant into medial and lateral portions. Therefore, a total of 8 equal areas could be delimited on the standard section denoted level 3 (see Fig. 3): VL1 and VLm, DL1 and DLm, DM1 and DMm, and VM1 and VMm. Every attempt was made to be sure, under the microscope, that these areas were equal. Sections from one of two coronally sectioned brains which most closely approximated the standard level were used to accumulate the raw data. Two types of sampling procedure were used. In the first procedure, only large cells, that is, cells with long axes ranging from 20 to 30 # m in length, were noted. The total number of large cells occurring in each of the 8 areas, or in as many areas as occurred in the section, were counted (see Fig. 3). The second procedure included an examination of the medium cell population of the nucleus. Medium cells were selected randomly from each division of each quadrant. Using an eyepiece micrometer, the long axes of these cells were measured and the lengths recorded. To be sure the cells were selected randomly, the scale of the eyepiece micrometer was moved into the field. Cells at positions 1 or 2, 5 or 6 and 9 or l0 were scored if they satisfied three criteria: (1) had a clear nucleolus; (2) the cell membrane was clear; and (3) the long axis of the cell fell into the 10-19/~m range. Using this procedure, the medium cell population could be analyzed without regard to the small populations of small and large cells. Small cells represent less than 1 ~ of the total cell population and have been ignored in this report. Ten measurements were taken from each region, providing a total of 20 measurements of medium cell size per quadrant. Means of the l0 measurements per sector were calculated from the raw scores; means listed in Tables 5-8 represent means of the sample to be tested in the statistical analysis. Calculations for large cell variation appear in Tables 1-4 and the medium cell results appear in Tables 5-8. A t-test for uncorrelated means z0 provided dorsal vs. ventral, medial vs. lateral, core vs. periphery and anterior vs. posterior comparisons of (1) the frequency of large cells and (2) sizes of medium cells. In addition, the distribution of large cells was plotted on projection drawings of each standard section (Fig. 3). Horizontal sections through the nucleus were also used to provide a pictorial analysis of the distribution of large cells (see Fig. 4). RESULTS
Description of cell types and cell clustering Except for the subependymal region of the lateral ventricle, groups of nerve
56
D
I_A
Fig. 1. Cell clusters in adult mouse caudate, sagittal section. Photomicrograph to left indicates a large area of caudate. Notice that many cells form aggregates, but scattered cells also occur. Bar --: 100 /~m. 1-3 are high power photographs of cell clusters in brackets I 3 respectively. 1 : clumped mass of cells containing one large and several medium cells. 2: clumped mass of medium cells. 3: linear arrangement of medium cells that might represent part of ring formation of cells seen in coronal section. Bar in 1 applies to pictures 1-4 and represents 25/bin. Arrowheads point out the same cells at each of the two magnifications as an aid to orientation of the cell cluster. 4: cell cluster containing a cell 'pair', located ventral to the area shown in picture at left. Marking at lower left denotes dorsal and anterior directions and applies to all photomicrographs.
cells occur t h r o u g h o u t the nucleus. The overwhelming majority of cells of the nucleus fall into one of two size categories: 10-14 or 15-20 # m in diameter. Both categories of m e d i u m cells have large, pale nuclei, s u r r o u n d e d by a thin rim of cytoplasm a n d very little Nissl substance (see Fig. 1). Cells larger t h a n 20 # m c o n t a i n a large a m o u n t of Nissl substance and, often, indented nuclei. These cells are multipolar or fusiform in appearance, and, although a small percentage of the total population, are quite p r o m i n e n t due to their size a n d Nissl content. In all regions of the nucleus, cell clustering occurs, groups of cells being bordered by the fibers of the bundles of the internal capsule. Scattered cells lie between cell clusters, but the impression of cell clustering is evident in all three planes of section. Ten to 15 cells are c o m m o n l y present in a cell cluster. Often, a n d in all planes of section, cell clusters may take the form of a clumped mass of cells. Clumps composed of one large and several m e d i u m cells or of only medium cells occur. The m e d i u m cells in these clumpings are from both size categories of m e d i u m cells a n d no pattern or
57
Fig. 2. A. Notice several unmyelinated and lightly myelinated fibers coursing through the caudate. Area at open arrows contains many of these fibers in a circular arrangement. Note similarity of distribution of these fibers and patterning of cell clusters shown in Fig. 1. B, C, D are high power photomicrographs of fibers denoted by arrows to A. Bar in A represents 50 #m for A and 12.5/~m for B, C, D. Unmyelinated fiber stain, Scalia et al. 14, coronal section. Darkly stained areas are fascicles of the internal capsule in cross-section. Dorsal at top, medial to the right.
difference could be n o t e d in the occurrence o f m e d i u m - s m a l l or m e d i u m - l a r g e cells. Large cells, on the other hand, c a n n o t occur in every cell cluster due to their low frequency o f occurrence. I n the clusters in which they are present, these cells m a y o c c u p y either a central or a p e r i p h e r a l position. However, no m o r e t h a n one large cell seems to occur in any cell cluster. In the c l u m p e d a r r a n g e m e n t o f m e d i u m cells, one o r a few cells m a y be centrally l o c a t e d a n d they m a y be either m e d i u m - s m a l l or m e d i u m - l a r g e or both. E x a m i n a t i o n s o f sections p r e p a r e d using the u n m y e l i n a t e d fiber stain show a c o r r e s p o n d e n c e in the intrastriatal t r a j e c t o r y o f these fibers to areas c o n t a i n i n g the c l u m p e d a r r a n g e m e n t o f cells (Fig. 2). Often, within individual clusters, p a i r i n g occurs. In the thick sections used in this study (10 #m), the p l a s m a m e m b r a n e s o f the two cells are extremely close a n d often a p p e a r fused (see Fig. 1). Occasionally, a t r i a d a r r a n g e m e n t also occurs in which one cell is centrally located a n d b o u n d e d laterally by two cells. A l t h o u g h frequency estimates for cell p a i r i n g o r the triad a r r a n g e m e n t were n o t a t t e m p t e d , cell p a i r i n g occurs often b u t n o t in every cell cluster, a n d is m o r e likely to occur t h a n the triad arrangement. A n alternate a r r a n g e m e n t o f the cell cluster also occurs. M e d i u m cells m a y occur single file a n d in ring-like f o r m a t i o n a r o u n d a fascicle o f the internal capsule. The ring-like f o r m a t i o n o f m e d i u m cells was m o s t easily seen in c o r o n a l sections b u t
58
O
A Fig. 3. Pictorial analysis of large cell distribution-~oronal section. Each of the 6 standard levels is drawn. Note solid horizontal and solid vertical lines which divide each section into 4 quadrants. Dashed lines subdivide each quadrant into a lateral and a medial zone. Only a few structures are labelled (see Sidman et al. 15, plates 21, 28, 32, 34, 38 and 42 for more detail). Abbreviations: A, amygdala, lateral nucleus; AC, anterior commissure; CC, corpus callosum; CL, claustrum; GL, globus paUidus; R, reticular nucleus of the thalamus; S, septum. The lateral ventricle is present in each drawing.
also occurred in horizontally sectioned material. In sagittal sections, this a r r a n g e m e n t of cell somata c a n n o t be d e m o n s t r a t e d clearly. These three types of clusters, that is, clumped m e d i u m a n d large cells, clumped m e d i u m cells, and m e d i u m cells in ring-like a r r a n g e m e n t occur t h r o u g h o u t the anteroposterior a n d dorsoventral extent of the nucleus.
59
Fig. 4. Pictorial analysis of large cell distribution--horizontal section. The three sections drawn represent dorsal, intermediate and ventral sections through the caudate nucleus. (For more fully labeled sections, see Sidman et al. 15, plates 119, 125 and 131) Abbreviations: CC, corpus callosum; F, fornix of the hippocampus; GP, globus pallidus; R, reticular nucleus of the thalamus; S, septum. The internal capsule is oulined but not labeled in the bottom drawing.
Description of the quadrant analysis 1. Large cell distribution. The statistical re suits concur completely with the pictorial analysis o f the data (Figs. 3 and 4). There is no difference in the dorsoventral distribution o f large 20-30-/~m cells in sections t h r o u g h the caudate anterior to the crossing o f the anterior commissure. However, the difference in large cell content between dorsal and ventral areas posterior to the anterior commissure is significant (see Table I). The mediolateral large cell distribution presents an interesting picture (see Fig. 3 and Tables lI and III). There is no difference in probability o f large cell occurrence if lateral is defined as D L and V L and medial as D M and V M quadrants. However, if, as the pictorial analysis implies, medial is defined as a 'core' area consisting o f the
6O TABLE ! Raw score for each sector can be obtained from Fig. 3. Data analysis
Dorsoventral variation - - Large cell (DLL+M + DML+M/ VLL+M + VML+M)
Number of cells (N) Standard deviation (S)
Entire caudate
Levels 1, 2, 3
Levels" 4, 5, 6
Dorsal
Ventral
Dorsal
Ventral
Dorsal
Ventral
20 1.93
17 1.50
12 2.22
10 1.15
8 1.06
7 1.70
Estimated sample variance (Sp~)* To
* Given by equation
3.05 0.14
3.31 1.28
1.94 2.31 **
(N1 - - 1)S21 + ( N 2 - l)SCz N1 ~- N2 - - 2
** Significant at 0.05 confidence level. TABLE II Raw score for each sector can be obtained from Fig. 3. These results are not significant. Data analysis
Mediolateral variation - - Large cell (DML+M + VML+M / DLL+M + VLL+M)
Number of cells (N) Standard deviation (S) Estimated sample variance (Sp2)* To
* Given by equation
Whole caudate
Levels 1, 2, 3
Medial
Lateral
Medial
Lateral
15 1.77
22 1.70
12 1.88
10 1.89
2.99 1.04
3.55 0.35
(N1 - - 1)SZl + (N2 - - 1)$22 N1 + N 2 - - 2
centrally located portions of all 4 quadrants, DLm + VLm + DMI + VM1, a n d lateral includes the two peripheral zones bordering the 'core' area, namely, DL~ + VL1 a n d D M m + VMm, a highly significant result is obtained for both the whole caudate (P < 0.01) and for the three anterior sections (P -< 0.001). O n the other h a n d , there is n o core-peripheral difference whatsoever in the large cell d i s t r i b u t i o n posterior to the anterior commissure. Also note that n o difference exists in lateral (DLm + VLm) vs. medial (DMI + VM1) core or in lateral (DLI + VL1) vs. medial (DMm + VMm) periphery anterior to the commissure. This difference could n o t be tested posterior to the commissure due to the reduction in the mediolateral extent of the nucleus in this area. A n t e r o p o s t e r i o r differences appear n o t to play a m a j o r role in the mouse caudate
Core
2.03 3.92***
11 0.50
N1 + N a -
2
(N1 - - 1)$21 -}- ( N 2 - 1)$22
2.39 3.13"*
11 1.95
** Significant at 0.01 confidence level. *** Significant at the 0.001 confidence level.
*Given by equation
Estimated sample variation (Sp 2)* To
18 1.14
8
7 1.68
2.68 0.58
1.60
11 1.75 3.57 0.51
8 2.07
Medial
Entire caudate
Periphery Lateral
Levels 4, 5, 6
Periphery Core
Levels 1, 2, 3
Periphery Core
Entire caudate
5 2.17 4.25 0.10
Lateral 6 1.97
Medial
Levels 1, 2, 3
Lateral vs. medical core
1.18 1.64
11 1.34
Lateral
7 0.38
Medial
Entire caudate
5 0.55 0.23 1.49
Lateral
6 0.41
Medial
Levels 1, 2, 3
Lateral vs. medial periphery
Mediolateral variation - - Large cell (Core vs. periphery) (DLM ÷ DML ÷ VLM q- V A I L / D L L ~- D M ~ q- VLL q- VMM)
19 Number of cells (N) Standard deviation (S) 1.85
Data analysis
Raw score for each sector can be obtained from Fig. 3.
TABLE Ili
62 TABLE IV Raw score for each sector can be obtained from Fig. 3. Data analysis
Anteroposterior variation - - Large cell (Levels 1, 2, 3 vs. levels 4, 5, 6)
Number of cells (N) Standard deviation (S)
All sectors
Dorsal sectors Ventral sectors Core
Periphery
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
1,2,3 4 , 5 , 6
22 1.84
12 2.22
10 1.15
11 1.95
11 0.50
Estimated sample variation (Sp2) * To
15 1.59
8 1.06
3.04 0.26
* Given by equation
3.45 1.62
7 1.70 1.95 1.87"*
8 1.60
7 1.68
3.29 1.30
1.21 1.46
(N1 - - 1)$21 + ( N 2 - 1)S22 N1 + Ne - - 2
** Significant at 0.10 confidence level only. n u c l e u s ( T a b l e IV). N o differences w e r e seen w i t h all a r e a s c o u n t e d , b e t w e e n d o r s a l a r e a s only, b e t w e e n the t w o cores, o r b e t w e e n the t w o p e r i p h e r a l zones. H o w e v e r , sections p o s t e r i o r to the a n t e r i o r c o m m i s s u r e are m o r e likely to c o n t a i n large cells in v e n t r a l a r e a s t h a n are sections a n t e r i o r to the c r o s s i n g o f the c o m m i s s u r e ( P < 0.1). T h e s e findings are a p p a r e n t in the p r o j e c t i o n
drawings
of
cell s i z e variation. T h e statistical analysis o f the c o m p a r i s o n
of
h o r i z o n t a l sections in Fig. 4. 2. M e d i u m
a v e r a g e cell size a m o n g the different q u a d r a n t s was m o s t interesting. N o d o r s o v e n t r a l differences o c c u r in m e d i u m cell size c h a r a c t e r i s t i c s in e i t h e r the entire c a u d a t e o r TABLE V The mean listed above is the mean of the sample in question. The number of measurements (N) corresponds with the number of sectors involved in each sample. Data analysis
Dorsoventral variation - - Medium cell size (DLL+M + DML+M / VLL+M -~ VML+M)
Number of measurements (N) Mean, in ttm (~) Standard deviation (S) Estimated sample variation (Sp 2)* To
* Given by equation
Entire caudate
Levels 1, 2, 3
Levels 4, 5, 6
Dorsal
Ventral
Dorsal
Ventral
DorsM
VeJltral
20 15.05 0.98
17 15.14 1.32
12 15.23 0.91
10 14.58 1.18
8 14.76 1.07
7 15.93 1.15
1.32 0.24
(N1 - - 1)521 + ( N 2 - 1)522 NI + N2 - - 2
** Significant at the 0.10 confidence level only.
1.08
1.23
1.46
2.04**
63 T A B L E VI T h e m e a n listed above is the m e a n o f the sample in question. T h e n u m b e r o f m e a s u r e m e n t s (N) c o r r e s p o n d s to the n u m b e r o f sectors involved in each sample. These results were n o t significant.
Mediolateral variation - - Medium cell size (DML+M -}- VML+M / DLL+M q- VLL+M)
Data analysis
Number of measurements (N) Mean, in/~m (~) Standard deviation (S) Estimated sample variation (Sp2)* To
* Given by e q u a t i o n
Whole caudate
Levels 1, 2, 3
Medial
Lateral
Medial
Lateral
15 14.75 1.09
22 15.31 1.13
12 14.61 1.07
10 15.33 0.98
1.24 1.51
1.06 1.63
(N1 - - 1)S21 + ( N 2 - - 1)S22 N1 + N2 - - 2
in the rostral three sections (Table V). However, more caudally, a slight difference between the two areas exists (P < 0.1) with more medium-large cells tending to occur in the ventral quadrants. When medial and lateral quadrants were compared statistically, no significant difference in medium cell size could be noted (Table VI). Further, no core vs. periphery differences existed (Table VII); and there was no statistically significant difference between the medium-sized cell population of the lateral core vs. that of the medial core. Yet, microscopic inspection of the slides seemed to indicate a gradual decrease in cell size from lateral to medial areas. This qualitative impression was upheld only by the statistical analysis of the difference between lateral vs. medial periphery, a difference that is highly significant. The only anteroposterior difference occurred ventrally (Table VIII). Mediumlarge cells are more likely to occur ventrally caudal to level 3 than at or rostral to it. No other statistically significant difference exists along the rostrocaudal axis of the nucleus. DISCUSSION
The foregoing analysis has attempted to describe the caudate nucleus according to several easily defined features in Nissl stained material. Two aspects of the internal organization of the nucleus are clear. First, the nucleus is divided into cellular territories, and secondly, within each territory, cellular aggregation or clustering is the prevailing pattern of organization. From the large cell analysis, it is clear that a core located centrally in the head of the nucleus is surrounded laterally by smaller zones rarely (but occasionally) containing large cells. The lateral peripheral zone is much more likely to contain medium-large cells than the peripheral zone bordering the ependyma of the lateral
Core
11 15.05 1.01
N1 ~- N 2 - - 2
(N1 - - I)S '~ % ( N 2 - -
1.32 0.56
** Significant at 0.05 confidence level. *** Significant at 0.01 confidence level.
* G i v e n by equation
Estimated sample variance (Sp2) * To
18 15.19 1.27
1)$22
1.19 0.50
11 14.82 1.17
8 14.89 1.09 1.38 1.48
7 15.79 1.27
11 14.77 0.97 1.02 1.09
8 15.28 1.07
Medial
Entire caudate
Periphery Lateral
Levels 4, 5, 6
Periphery Core
Levels 1, 2, 3
Periphery Core
Entire caudate
5 15.04 0.98
6 15.07 1.12
Medial
1.12 0.05
Lateral
Levels 1, 2, 3
Lateral vs. medial core
11 15.85 1.06
7 14.16 0.80
Medial
0.94 3.60***
Lateral
Entire caudate
5 15.62 0.99
0.87 2.61 **
6 14.15 0.88
Lateral Medial
Levels 1, 2, 3
Lateral vs. medial periphery
Mediolateral variation- Medium cell size (Core vs. periphery) (DLM + D ML -r VLM + VML / DLL + D MM + VLL + VMM)
N u m b e r of measurem e n t s (N) 19 M e a n , in/~m (,'~) 14.98 Standard deviation (S) 1.02
Data analysis
T h e m e a n listed above is the m e a n of the s a m p l e in question. T h e n u m b e r o f m e a s u r e m e n t s (N) c o r r e s p o n d s to the n u m b e r o f sectors involved in each sample.
T A B L E VII
65 TABLE VIII The mean listed above is the mean of the sample in question. The number of measurements (N) corresponds to the number of sectors involved in each sample. Data analysis
Anteroposterior variation - - Medium cell size (Levels 1, 2, 3 vs. levels 4, 5, 6) All sectors
Dorsal sectors
Ventral sectors Core
1,2,3
1,2,3
1,2,3
4, 5,6
4, 5,6
4, 5,6
1,2,3
Periphery 4, 5,6
1, 2,3
4, 5,6
Number of measurements (N) 22 15 12 8 10 7 11 8 11 7 Mean, in #m (~) 14.94 15.31 15.23 14.76 14.58 15.93 15.05 14.89 14.82 15.79 Standard deviation (S) 1.07 1.23 0.91 1.07 1.18 1.15 1.01 1.09 1.17 1.27 Estimated sample variation (Sp~)* To
1.29 0.97
0.95 1.06
1.36
1.09
1.46
2.35**
0.33
1.66
(N1 - - 1)S~1 + ( N s - 1)S~2 NI + N z - 2 ** Significant at 0.05 confidence level. * Given by equation
ventricle. Medium-small cells, on the other hand, occupy areas that are large-cell free and tend to occur more medially than the other two cell types. Therefore, three zones occur in the head of the nucleus. A lateral zone bordering the corpus callosum contains mainly medium-large, some medium-small and very few large cells. A larger zone medial to this includes the entire central area of the head of the nucleus, and contains almost all of the large cells occurring at this level and many medium-large cells. The third zone, the small medial margin, is clearly different from the other zones and contains cells smaller than the other two areas. The only consistent difference in the body of the nucleus (that is, levels caudal to the crossing of the anterior commissure) is a dorsoventral one. The ventral quadrant (usually denoted VL in the drawings at this level) more often contains large and medium-large cells than the dorsal area. Large cells in this ventral region belong to a ventral column of cells extending throughout the nucleus (see Fig. 4). Medium cells in this area are significantly larger than ventrally located medium cells of the head of the nucleus. The dorsal area, on the other hand, has a low frequency of large cell occurrence, and approximates the cell population of a peripheral zone, particularly the lateral periphery. Although generalization from one species to another is always difficult, these results may well be characteristic of the mammalian neostriatum. Chronister et al. 4 demonstrated both the clumped arrangement of medium cells and medium cells in ringed formation in Golgi preparations of mouse and rat neostriatum. In the same study, cell clustering was shown to be a dominant aspect of the organization of the feline caudate as well, and in spite of several significant differences between carnivore and rodent material. These authors did not comment upon large cell participation
66 in cell clusters. Large cells represent a small percentage of the total cell p o p u l a t i o n of both rat 11 a n d cat 8 caudate. Since cell clustering of m e d i u m cells occurs in these two animals, large cells p r o b a b l y also participate in this a r r a n g e m e n t in m u c h the same was as they do in mouse neostriatum. Whether the caudate nucleus of primates presents a h o m o l o g o u s condition c a n n o t be stated from the available literature. The work of G o l d m a n a n d N a u t a 5 d e m o n s t r a t i n g synaptic island f o r m a t i o n by prefrontal cortical afferents to the head of the nucleus is the closest a p p r o x i m a t i o n to date of this concept. The bulk of the evidence, then, suggests morphological diversity in the internal organization of the caudate. F u r t h e r study is warranted. ACKNOWLEDGEMENTS This research was supported by a seed grant from the School of Medicine of the University of Southern California. I am very grateful to Dr. Richard Binggeli for his m a n y helpful suggestions during preparation of this paper. Mrs. Caroline Brown's thoughtful typing of the m a n u s c r i p t is also gratefully appreciated.
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