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Nucleus raphe dorsalis: A morphometric golgi study in rats of three age groups
Brain Research, 207 (1981) 1-16 © Elsevier/North-Holland Biomedical Press
1
Research Reports
NUCLEUS RAPHE DORSALIS: A MORPHOMETRIC GOLGI STUDY IN RATS OF T H R E E A G E G R O U P S
S. DIAZ-CINTRA,* L. CINTRA,* T. KEMPER, O. RESNICK and P. J. MORGANE Worcester Foundation for Experimental Biology, Shrewsbury, Mass. and Neurological Unit, Boston City Hospital, Boston, Mass. (U.S.A.)
(Accepted July 31st, 1980) Key words: nucleus raphe dorsalis - - reticular neurons - - Golgi morphometric study - - spine density -
-
dendritic architecture - - raphe cell-vascular relations - - raphe dorsalis cells
SUMMARY Using Rapid Golgi and Nissl techniques, three major cell types: fusiform, multipolar and ovoid-shaped were identified in the nucleus raphe dorsalis of male rats at 30, 90, and 220 days of age. We have described the orientation and dendritic architecture of raphe cells as to type and the relationships of these cells to blood vessels and surrounding structures. For each cell type, the origin of the axon is characteristic. One hundred neurons per age group were measured at their maximal linear extent and the number of spines on the somal surface was counted. Dendritic number, linear extent, diameter and the number of spines along a 50/~m segment near the mid- point of dendritic length in an equal number of primary and secondary dendrites were quantified in each age group. The most striking age-related changes in the multipolar and ovoid-shaped cells were dendritic number, diameter and spine number as well as the number of perisomatic spines. The fusiform cells showed the least age-related changes. In general, the nucleus raphe dorsalis is organized as a reticular nucleus with neurons having few, straight and poorly ramified dendrites.
INTRODUCTION In recent years great interest has developed in the anatomical, physiological and chemical organization of the nucleus raphe dorsalis (NRD). Ram6n y CajaP 4, using several Golgi methods, examined and described cells of the 'central magnocellular nucleus of the raphe' (corresponding to the nucleus raphe dorsalis) in the cat and rabbit * On leave from: Departamento de Fisiologia, Instituto de Investigaciones Biom6dicas, Universidad Nacional Aut6noma de M6xico, M6xico 20, D.F., M6xico.
as being large, fusiform, triangular, or starlike in shape with multiple diverging dendrites that were covered with spines and tended to be grouped into vertical fasciculi. Axons of these cells were described as following an ascending course which, after giving off collaterals, were followed into several brain areas, including the medial longitudinal fasciculus (MLF). Recently, Leontovich and Zhukova 11 described the cells in the raphe nuclei as typical reticular neurons based on their appearance in Golgi stained sections. In a cytoarchitectonic study of the raphe nuclei in the cat, Taber is described two cell types in the N R D : medium-sized multipolar neurons and fusiform cells. Azmitia 1, likewise, recognized in the rat two main classes of neurons in the N R D : a medium-sized fusiform type cell that comprised about three-quarters of the total number of cells and a large ovoid type cell that accounted for the remaining one-quarter. Serotoninergic neurons in the N R D were identified by Dahlstr6m and Fuxe3just above and medial to the M L F and were classified as cell group B7 in their fluorescence mapping studies. In a recent study in the rabbit Felten and Cummings 4, and Felten and Harrigan6, 7, using Golgi and cresyl violet stains and histofluorescence techniques, described serotonin-containing neurons in the N R D as medium-sized oval and fusiform cells oriented vertically in the midline, within and tangential to the MLF. In the nucleus linearis rostralis and the nucleus raphe pontis a neurovascular proximity between raphe dendrites and blood vessels was described by Scheibel et al. 16. Felten and Crutcher 5, in a combined fluorescence, histochemical, and electron microscopic study in the N R D and nucleus raphe medialis in the squirrel monkey and rhesus monkey, demonstrated direct apposition of blood vessels to both the perikarya and dendrites of the serotoninergic neurons. These particular neurovascular relationships were present in relation to 20-30 ~ of the small vessels in the NRD. Felten and Crutcher postulated that the serotonin-containing neurons of the raphe may thus be influenced directly by hormones or other substances in the blood. The objective of the present study was to define and describe the cell types in the NRD, using quantitative techniques on rapid Golgi impregnated neurons, in male rats of three different ages. MATERIALS AND METHODS The material in this study was obtained from male Charles River C.D. Sprague-Dawley rats fed a 25 ~o~ casein diet according to the paradigm used in the Worcester Foundation Protein Program Project 13. Since in that project we are carrying out ontogenetic studies at certain critical ages, we have chosen these ages for the present anatomical analyses. Accordingly, at 30, 90 and 220 days of age the animals were weighed, anesthetized with pentobarbital, perfused through the heart with 10 ~ neutral buffered formalin and, on the following day, the brains removed and weighed. Slices of midbrain tissue were cut into 3 mm thick blocks in the frontal and sagittal planes and post-fixed in a potassium dichromate 4 ~o~ solution in buffered formalin. Each block was then transferred to classical rapid Golgi fixative for 6-11 days and silvered in a 0 . 7 5 ~ silver nitrate solution for 18-48 h. For some blocks in
220-day-old animals double or triple impregnation was used according to the method of Valverde 19. Finally the blocks were embedded in low viscosity nitrocellulose and cut in serial section at a thickness of 120-180 #m. One hundred neurons from 4 well impregnated brains per age group were selected for quantitative studies at random from all parts of the nucleus raphe dorsalis, including the anterior and posterior poles and the middle third of the nucleus. This area corresponds to the region of the N R D in the stereotaxic atlas of Krnig and Klippel lo as well as the region outlined in histofluorescence studies by Dahlstrrm and FuxeL All measurements were made at 400 x using a calibrated ocular reticle. Each cell chosen was outlined in pencil with the aid of a camera lucida and the following measurements were made: (1)the major and minor axes of the cell body were measured at their maximal extent; and (2) the number of spines on the somal surface was counted by focusing through several planes. In addition, dendrites were divided into primary and secondary branches according to the method illustrated in Fig. 2. Primary dendrites were defined as those arising from the perikarya, whereas secondary dendrites were defined by their origin from primary dendrites, their thinner diameters, and a lesser density of spines. Then the linear extent per cell of two primary dendrites and their secondary dendrites was measured. The linear extent of dendrites within the focal plane was measured directly by establishing the distance from the dendritic point of origin on the perikaryon to the tip of the dendrite. Those not in the plane of section were estimated by triangulation according to the method of Bok z and Kemper et al. 9. Measurements of the diameter of primary and secondary dendrites was made at the midpoint between their origin and tip. From the same middle segment the number of spines per 5 0 / t m of dendritic length was counted. Two additional rats from each age group were perfused with 1 0 ~ neutral buffered formalin, their brains embedded in albumin-gelatin and serially sectioned in the frontal and sagittal plane, respectively, at a thickness of 30 #m. These sections were stained with cresyl violet and examined for the general morphology of cell bodies, extent of the nucleus and various cytoarchitectural details. From this material the major and minor axes of the perikarya of 100 cells, including the three types selected at random from anterior, middle and posterior parts of the N R D per age group, were also measured and compared to similar naeasures in the Golgi material. Statistical significance of all measurements was determined by using the Student's t-test. RESULTS In the rat the N R D extends rostrally to the caudal pole of the trochlear nucleus and caudally to the posterior pole of the dorsal tegmental nucleus of Gudden, with its long axis directed rostro-caudally. In this long axis, it measures 1.6, 1.9 and 2.2 mm, respectively, at 30, 90 and 220 days. Throughout the entire extent of the nucleus 3 cell types were found which, based primarily on the shape of their perikaryon, are referred to as fusiform, multipolar and ovoid-shaped cells (Fig. 1). The appearance of all 3 types of cells at 3 different rostro-caudal levels of the nucleus raphe dorsalis are shown in Figs. 3-5.
~ la a( ÷
A
I
B
I
30 p.
Fig. 1. Camera lucida drawings of the perikaryon of fnsiform (la), multipolar (1 b), and ovoid (lc) cells in 90-day-old rats. The cells in column A are the largest examples encountered and those in B the smallest. On the right the major and minor axes of each cell type are shown schematically, a, axons.
/
S
p~-
[ I00
jim
i
p
Fig. 2. Camera lucida drawing of the cellular types in the nucleus raphe dorsalis. Primary dendrites (P) were defined as the largest extention of the processes on the perikarya, secondary dendrites (S) were defined as originating from the primary dendrites. Brackets show the dendritic extent on which spines were counted. The arrows indicate the loci for thickness measurement in the primary and secondary dendrites. Abbreviations: la, fusiform cell; lb,'_multipolar cell; lc, ovoid cell; a, axons.
The fusiform cells were the most frequently encountered type in the Golgi impregnated material at all levels in the N R D but are more heavily concentrated in the middle one-third o f the nucleus. Their perikarya showed the most marked differences between the major and minor axis (Fig. 1 and Table I). The primary dendrites arise from polar regions o f the cell, thereby further emphasizing their fusiform shape. A t lhe middle one-third o f the nucleus fusiform-shaped neurons situated on the superior aspect o f the M L F and the superior cerebellar peduncle show a tangential orientation o f their perikarya to these bundles. Dendrites o f some o f these fusiform cells penetrate deeply into both o f those fiber systems and radiate widely into other parts o f the N R D (Figs. 3 and 4). In the most caudal part o f the nucleus raphe dorsalis the fusiform cells are more vertically oriented. The dendritic process o f the fusiform cells extend
Fig. 3. Camera lucida drawing of the NRD at the level of the trochlear nucleus (Nu IV). Asterisks identify ceils with special characteristics discussed in the text. The single headed arrow shows the dendrites of an ovoid cell wrapped around a blood vessel. The double headed arrows show dendritic tips of a multipolar cell in close relation to a blood vessel. The triple headed arrow shows the close relationship of a fusiform cell dendrite to the fibers of the MLF. Abbreviations: l a, fusiform cell; l b, multipolar cell; I c, ovoid cell, aq, aqueduct; CG, central gray; dr, nucleus raphe dorsalis; DSCP, superior cerebellar peduncle decussation; MLF, medial longitudinal fasciculus; mr, nucleus raphe medianus.
f' /
\
\ VG
.
RF
100 jim
~
\
z
Fig. 4. Camera lucida drawing of the NRD midway between its caudal and rostral poles. The dendrites of the ovoid cell indicated by the arrow are following the vertical course of a blood vessel. Note the overlap of dendrites of multipolar cells (1b) with those of the neurons of the ventral tegmental nucleus of Gudden (VG) and nucleus raphe medianus (mr). Abbreviations: la, fusiform cell; lb, multipolar cell; lc, ovoid cell; RF, reticular formation; SCP, superior cerebellar peduncle.
vertically between the two M L F bundles and into the dorsal and ventral parts of the N R D coming into close relation with dendrites of the nucleus raphe medianus. Other dendrites extend into the M F L at this level (Fig. 5). In the rostral third of the nucleus raphe dorsalis the fusiform cells are largely oriented similar to those at the middle third of the nucleus. Here the dendrites disperse widely into other parts of the nucleus raphe dorsalis and tend to be more horizontally oriented than in the middle third of the nucleus. The axons arise primarily from a proximal dendrite in 75 ~o of the fusiform cells while on most of the remainder the axon arises from the soma. The multipolar cell was the next most frequently encountered cell in the N R D . This cell is found frequently at all levels in the N R D but are most heavily concentrated
7
aq
....
LOC
dr
.....~:-: SCP
,,
iV
~'lltF ' 100 pro'
Fig. 5. Camera lucida drawing of the NRD near its caudal pole. Note the close relationship of the dendrites of the fusiform ceils (la) to the medial longitudinal fasciculus (MLF). Also note the vertical orientation of the dendrites of these fusiform cells in the midline. Abbreviations: la, fusiform cell; 1b, multipolar cell; lc, ovoid cell; aq, aqueduct; C, cerebellum; dr, nucleus raphe dorsalis; DG, nucleus dorsalis of Gudden; SCP, superior cerebellar peduncle.
in the middle one-third of the nucleus (Fig. 4). Dendrites of these cells arise at 3 or more different locations on the perikaryon, thus giving the cells their triangular or multipolar appearance. Their dendrites are more randomly distributed, overlapping extensively with the dendrites of the ventral tegmental nucleus of Gudden and nucleus raphe medianus, particularly in the middle third of the nucleus (Fig. 4). Some of the dendritic processes of the multipolar cells of the N R D extend into M L F at the rostral and middle third of the nucleus (Fig. 4) while others come into close contact with small blood vessels dorsal to the M L F at the level of the rostral third of the nucleus (Fig. 3, double arrows). Axons were found to arise primarily from the soma in 84 ~ of the multipolar cells. The ovoid-shaped cell was the least frequently encountered neuron in the nucleus raphe dorsalis and showed the fewest differences between its major and minor axis. This cell was also found at all levels in the NRD. Dendrites appear randomly distributed and in the more midline cells they are frequently wrapped around blood vessels (Figs. 3 and 4, single arrow). The axons arise with almost equal frequency from the cell body (53 ~ ) or a proximal dendrite (47 ~).
In Tables I and II the major and minor axis of the perikaryon, number, diameter, and extent of the dendritic processes, and the density of perisomatic and dendritic spines are recorded for each cell type at 30, 90 and 220 days of age. At 30 days of age the major and minor axes of the perikarya of thefusiforrn cells were 25.79 4- 4.75 and 15.12 4- 3.33/~m, respectively, and the number of perikaryal spines was 3.21 4- 2.71. The number of primary dendrites was 3.37 4- 0.75 and secondary dendrites 2.86 4- 0.85 per cell, with respective thicknesses of 1.92 ~ 0.49 and 1.58 4- 0.38/tm. The linear extent of the primary dendrites was 313 4- 93 #m and of the secondary dendrites 192 4- 76/~m. Their respective number of spines/50/~m were 6.54 4- 2.07 and 6.00 4- 2.29. At 90 days of age there was a 20 ~/o increase in the number of spines on the primary (P < 0.001) and secondary (P < 0.05) dendrites, respectively. At 220 days there was a 12 ~ (P < 0.001) decrease in the number of these spines on the primary dendrite. All other measurements failed to show significant age-related changes. At 30 days of age the major and minor axes of the perikarya of the multipolar cells were 22.92 4- 4.16 and 17.94 4- 3.81 #m, respectively, and the number of perisomatic spines was 3.53 4- 2.71. The number of primary dendrites was 3.86 4- 0.78 and secondary dendrites 2.88 4- 0.93, with their respective diameters being 1.94 40.49 and 1.56 4- 0.34/ma. The linear extent of their primary dendrites was 291 ± 90 #m and of their secondary dendrites 186 4- 70 /~m. The respective numbers of spines/50/~m were 6.66 4- 2.14 and 5.75 4- 1.96. At 90 days of age they showed a significant 13 ~ (P < 0.05) increase in linear extent of their primary dendrites, a 24 increase in spine density on the primary dendritic processes, and a 34~o increase in spine density on their secondary dendrites (both P < 0.001). At 220 days there was an 18 ~ (P < 0.001) and 16~ (P < 0.02) decrease, respectively, in spine density in these locations together with a 6 4 ~ (P < 0.001) decrease in perisomatic spines. All other measurements failed to show significant age-related changes. At 30 days of age the major and minor axes of the perikarya of the ovoidcells were 19.66 4- 4.45 and 14.49 4- 2.74 ~m, respectively, and the number of perisomatic spines 2.16 4- 1.91. The number of primary dendrites was 2.72 4- 0.66 and secondary dendrites 2.44 4- 0.61, with respective thicknesses of 1.82 4- 0.42 and 1.40 4- 0.26/~m. The linear extent of the primary dendrite was 280 4- 83 /~m and of the secondary dendrite 171 4- 56/~m. The respective numbers of spines/50 um were 6.19 4- 2.27 and 5.30 4- 1.63. At 90 days there was a 72~o (P < 0.02) increase in the number of perisomatic spines, a 20 °/o (P < 0.05) increase in the linear extent of the primary dendrites, a 24 ~ (P < 0.01) increase in spine density on primary dendrites, and a 33 (P < 0.001) increase in spine density on the secondary dendrites accompanied by a 10~ (P < 0.05) decrease in diameter of their secondary dendritic processes. At 220 days the number of primary dendrites increased 21 ~ (P < 0.01), and the linear extent of the primary dendrite decreased 17~ (P < 0.05) and the diameter of secondary dendrites increased 9 ~ (P < 0.02). All other measurements failed to show significant age-related changes. The measurements of the major and minor axis of these 3 cell types in Nissl stained sections was essentially identical to that found in the Golgistained material (Tables I and IV).
Minor axis (t~m)
No. of peri- No. of somatic cells spines
25.794-4.75 15.124-3.33 3.214-2.71 45
Major axis (l~m)
** = P < 0 . 0 2 . §§ = P < 0.001.
220 days 56 23.33±4.53 Change 90 vs 220 days (--3.0 ~)
2.914-2.52 22 (+2~)
14.384.2.62
(+1%) (+0.3 ~)
(--7~)
17.354.3.17
14
18
(--64~)§§
1.594.1.46 22
(+25 ~)
(+4%)
3.534-2.71
(--2~) 22.56±4.05
0 void No. of per±- No. of somatic cells spines
18.644.3.86 4.404-2.50
17.94±3.81
Minor axis (l~m)
22.494-3.82
22.924,4.16
Major axis (l~m)
Multipolar
90 days 66 24.064.3.81 14.234-2.83 2.844-1.98 20 Change 30 vs 90 days (--7 ~) (--6 %) (--I 2 ~)
30 days 37
No. of cells
Fusiform
Major and minor axes of the cell body and number of perisomatic spines for each cell type at 30, 90 and 220 days
TABLE I
(--2%)
17.924.2.95
(--7%)
18.36+4.09
19.664.4.45
Major axis (l~m)
No. of perisomatic spines
(+72%)**
(+3 %)
(--30%)
15.34:k:3.10 2.594-2.34
(+2%)
14.844.4.87 3.714-1.58
14.494-2.74 2.164,1.91
Minor axis (pm)
Prim.
Sec.
Number of dendrites
(--6%)
(0%)
(4- 0.6 %)
220 days 22 % Change 90 vs 220 days
90 days 20 % Change 30 vs 90 days (--1%)
(+-1%)
(--5 %)
(--4 %)
(--3 %)
(,4,10%)
(+20%)
1.48 4- 0.31
1.89 -4- 0.38
4.00 -4- 0.69 3.13 ± 1.24
(--6 %)
1.54 -4- 0.32
1.95 -4- 0.34
1.56 -4- 0.34
3.65 -4- 0.58 2.60 -4- 0.94
1.94 4- 0.49
(+5%)
(+5%)
(--1%)
(--2 %)
1.55 4- 0.31
2.01 -4- 0.38
3.37 -4- 0.72 2.66 4- 0.90
(--5 %)
1.48 -4- 0.32
1.58 -4- 0.38
Sec.
1.92 + 0.42
1.92 4-4-0.49
Prim.
Dendritic diameter (#m)
3.39 -4- 0.74 2.71 + 0.97
Multipolar shape 30 days 45 3.86 -4- 0.78 2.88 -4- 0.93
220 days 56 % Change 90 vs 220 days
90 days 66 % Change 30 vs 90 days
Fusiform shape 30 days 37 3.37 -4- 0.75 2.86 ~ 0.85
cells
Number of
Prim. = primary dendrite; sec. = secondary dendrite. Results are ~ 4- S.D.
44
40
90
112
132
74
Number of dendrites examined
(--0.3 %)
327 -4- 92
(+13%)*
328 + 84
291 -4- 90
(+0.1%)
314 4- 97
(+0.3%)
314 4- 88
313 -4- 93
Prim.
(--7 %)
175 5= 50
(+2%)
189 4- 60
186 -I- 70
(--2%)
181 4- 67
(--4%)
185 4- 63
192 4- 76
Sec.
Sec.
(-4-20%)*
(--15%)
(+34%)§§
(--16%)**
1.96 6.50 ± 2.14 (--18%)§§
6.77 i
(+24%)§§
8.27 -- 1.82 7.70 -4- 2.18
6.66 4- 2.14 5.75 ! 1.96
(--12%)§§
6.91 -4- 1.82 6.10 -4- 1.90
(+20%)§§
7.87 -4-4-2.02 7.19 -4- 1.96
6.54 + 2.07 6.00 -4- 2.29
Prim.
Dendritic linear extent (l~m) Number of dendritic spines~50 I~m
Number, diameter and extent of dendrites and synaptic spine density for each cell type at 30, 90 and 220 days of age
TABLE II
§§ -- P < 0 . ~ 1 .
* = P < 0.05. ** = P < 0.02. § = P < 0.01.
22 % Change 90 vs 220 days
220 days
14 % Chang~ 3 0 vs 90 days
90 days
18
0 void shape 30 days
( - 4 %)
(--0.4 %)
(+8%)
(+21%)~
(--2 %)
1.89 4- 0.38
3.27 4- 0.55 2.59 4- 0.79
(+8%)
1.75 4- 0.26
1.82 4- 0.42
2.71 ± 0.72 2.64 4- 0.84
2.72 4- 0.66 2.44 4- 0.61
(+9%)**
1.37 : i 0.24
(--lO%)*
1.26 4- 0
1.40 4- 0.26
44
28
36
(--17%)*
280 4- 83
(+20%)*
336 4- 112
280 4- 83
(--8 %)
166 + 49
(+5%)
180 =t= 75
171 4- 56
( + 3 3 %)§§
(--11%)
(--11%)
6.84 4- 2.05 6.31 ± 1.81
(+24%)§
7.67 4- 2.00 7.07 ± 1.76
6.19 4- 2.27 5.30 ± 1.63
30 days
Minor axis
+31~§§ +4~ --20%*
* = P < 0.05. ** = P < 0 . 0 2 . § = P < 0.01. §§ = P < 0.001.
Dendrites
+55~§ +30~ --16%
+10~ --33% --39~*
+19~§§ --3 % --18%§§
+8~ --20%§§ --26~§§
+15%§ --19~§ --30~*
+18~ --3 ~ --17%
---4~ --3~ +2%
+0.7% --15 ~* --15%*
No. of Number of dendrites perisomatic spines Prim. Sec.
Fusiformvsmultipolar - - 3 ~ + 2 1 % § § ---45%* Fusiform vs ovoid --23 %§§ + 7 % --119/00 Multipolarvsovoid --21~§§ --12%* +39%
220 days
Fusiformvsmultipolar --7% Fusiformvsovoid --24%§§ Multipolarvsovoid --18%§
90 days
Fusiformvsmultipolar --11~§ +19~§§ Fusiform vs ovoid --31 ~§§ - - 4 ~ Multipolarvsovoid --14~** --10%§§
Major axis
Perikarya
Prim. = primary dendrite; Sec. = secondary dendrite.
--6% --6 ~ 0%
+2~ --9%* --10~
+1~ --5 % --6~
Prim.
Diameter
--5~ --12 ~ --7~
+4% --15~§§ --18~§§
+1% --11 ~* --10~*
Sec.
Statistical analysis of the differences in morphometric data of the various cell types at 30, 90 and 220 days
TABLE III
+2~ --3~ --5~
--3~ --11 ~ --8~
Sec.
+4~ --3~ --11 ~§ --8 %* --14~** - - 5 ~
+4~ +7~ +3~
--7% --11 ~o --6~
Prim.
Linear extent
--2% --1 ~ +1~
+5~ --3% --7~
+2~ --5 ~ --7%
Prim.
+7% +3 --3%
+7~ --2~ --9%
--4~ --12~ --8~
Sec.
Number of spines~50 I~m
26.69-4-5.70
25.05±4.06 (--6~)
25.64±3.91 (+2~)
30 days 44
90 days 42 ~ C h a n g e 30 vs 90 days
220 days 42 ~ C h a n g e 90 vs 220 days 12.93 ~ 1.91 (--6~)
13.73 :k2.63 (+4~)
13.21 ± 2 . 3 1
43
38
36
No. of cells
Minor axis (~m)
No. of cells
Major axis (t~m)
Multipolar
Fusiform
Results are ~ :k S.D.
22.00 ± 3.95 (+0.17o)
21.98 -4- 4.48 (--6 ~ )
23.30±4.32
Major axis (#m)
15.53 ± 3.31 (--0.8%)
15.66 ± 4.01 (--6 G)
16.59±3.08
Minor axis (l~m)
Major and minor axes of the cell body ]br each cell type at 30, 90 and 220 days of age ( Nissl-stained material)
TABLE IV
15
20
20
No. of cells
Ovoid
19.32 ± 2.11 ( - - 2 ~o)
19.78 4- 3.37 (+5%)
18.83 ± 4 . 3 1
Major axis (t~m)
17.80 + 2.17 (--2 ~)
18.08 ± 3.09 (+6~)
17.05 ± 4.24
Minor axis (t~m)
U.
14 As can be seen from these data, clear differences between these 3 cell types with age emerge. Therefore, when the measurements for each cell type are compared, one notices that they are statistically separable in all age groups by several criteria (Table III). This table contains the statistical data showing the comparisons of all 3 cell types at 3 ages for the various parameters under consideration. DISCUSSION Based on cell body shape, orientation of dendrites, origin of the axon and quantitative measurements of the soma, dendrites and dendritic spines, we have defined 3 cell types in the N R D : fusiform, multipolar and ovoid-shaped neurons. A fusiform cell in the raphe dorsalis had been previously noted by Ram6n y CajaP 4, Taber 18, Azmitia a and Felten and Cummings 4. In the present material, in agreement with Azmitia 1, it is the most frequently encountered cell in all parts of the nucleus. The fusiform cell is further characterized as showing the least age-related changes of all three cell types. Notable characteristics of this cell are marked differences in length between the major and minor axis of the cell body, an axon that arises most frequently from a proximal dendrite, and the orientation of its cell body and dendrites to adjacent fiber systems, particularly the MLF. Special orientation of raphe cells to adjacent fiber bundles had been commented on by Ram6n y CajaP 4, Taber ~8 and Felten and Cummings 4 but without reference to any particular cell type. The multipolar cell appears to correspond to Ram6n y Cajal's ~4 star-like and triangular cell and to the medium-sized multipolar cell described by Taber 18. Its characteristic features are its multipolar cell body, an axon that arises most frequently from its cell body, and randomly oriented dendrites that overlap dendrites of adjacent nuclei. Its most characteristic age-related change is a 25 °//Uincrease in the number of perisomatic spines between 30 and 90 days followed by a marked decrease of 64 % (P ~< 0.001) at 220 days. Although not achieving statistical significance, there is an increase in the number of both primary and secondary dendrites between 90 and 220 days. Ovoid-shaped cells in the nucleus raphe dorsalis have been noted by Azmitia 1 and Felten and Cummings 4. A characteristic feature of the ovoid cell is an axon that arises with equal frequency from its cell body or proximal dendrite and the random orientation of its dendrites. Its most striking age-related change is a marked increase of 72 % in perisomatic spines between 30 and 90 days (P < 0.02) followed by a less marked decrease of 30 ~ at 220 days. It shows a significant 21 ~o increase in number of primary dendrites between 90 and 220 days (P < 0.01) but not in number of secondary dendrites. It is the only cell to show significant age-related changes in secondary dendritic diameter. The extent to which these cell types are representative of cells in other raphe nuclei is not known. However, it is interesting to note that Fox et al. 8, in a Golgi study of the caudal medullary raphe nuclei (obscurus and pallidus), observed similar cell types to those described herein. These authors also noted medium fusiform, large multipolar, medium stellate, and small spherical neurons in these posterior raphe nuclei.
15 Despite their differences the cells of the N R D have many features in common. Both the multipolar and ovoid cells show a close relationship to blood vessels throughout the NRD. This relationship was first noted by Scheibel et al. 16 in the nucleus raphe pontis and nucleus linearis rostralis and, later, by Felten and Crutcher 5 in the N R D and nucleus raphe medianus. In the present study we find that at 30 days of age this relationship is established by dendrites of the ovoid-shaped and multipolar cells and at 90 days of age some cell bodies of ovoid-shaped cells are found to be closely allied to blood vessels. Other striking similarities are the patterns of age-related changes in number of dendrites, dendritic extent, and synaptic spine density, particularly for the multipolar and ovoid-shaped cells. Both the multipolar and ovoid-shaped cells show a significant increase in extent of their primary dendrites between 30 and 90 days of age. They also show a lesser (non-significant) increase in extent of their secondary dendrites. At 220 days we find a significant decrease in the linear extent in the primary dendrites on ovoid-shaped cells. However, at this time there is an increase in the number of primary dendrites that is significant only for the ovoid-shaped ceils. Additionally, there is a significant increase in the number of secondary dendrites of the multipolar, but not the ovoid-shaped cells. At 90 days both the multipolar and ovoid-shaped cells show an increase in perisomatic spines, yet this is significant only for the latter. At 220 days both show a spine loss on the cell bodies that is significant only for the multipolar cells. Ovoid-shaped, multipolar, and fusiform cell types show an increase in dendritic spine density on primary and secondary dendrites between 30 and 90 days which in all cases is statistically significant. In most cells between 30 and 90 days there is a period of linear growth of dendritic processes associated with an increase in dendritic spines. Between 90 and 220 days there is an increase in number, but not the extent, of dendritic processes and a decrease in density of dendritic spines. In the present study we find the prolonged period of dendritic growth in all 3 cell types is also reflected in the progressive increase in rostro-caudal length of the N R D from 30 to 220 days. This pattern of progressive growth of the N R D appears to be in conjunction with an overall pattern of growth of the brain stem during this period. For example, Sugita 17 has provided data from rats on the weight of the brain and brain stem at ages similar to those used in the present study. He showed that between 35 and 150 days there is a 33.5 ~ increase in the brain stem weight. We showed a 27 ~ increase in the length of the N R D between 30 and 220 days. All 3 types of cells we have identified in the nucleus raphe dorsalis are seen in the rostral, middle and posterior parts of the nucleus. However, certain cell types are more concentrated in particular divisions of the nucleus. Thus, the fusiform and multipolar cells are in relatively greater numbers in the middle one-third of the nucleus relative to the ovoid cells. In all parts of the nucleus raphe dorsalis the fusiform-shaped cells are much more numerous than both the multipolar and ovoid-shaped cells. Generally, the N R D is organized as a reticular nucleus with neurons having few, long, straight, poorly ramified dendrites11,12,15. Within the NRD, based on cell body shape, origin of axon, morphometric data on its somal and dendritic architecture and
16 age-related changes, we were able to identify 3 cell types. I n these, the dendritic a n d cell m o r p h o l o g y o f each type show v a r y i n g relations with b l o o d vessels a n d s u r r o u n d i n g nuclei a n d fiber bundles. ACKNOWLEDGEMENTS W e t h a n k M. Feinstein for his help with the histological preparations. S u p p o r t e d by G r a n t s B N S 77-16512 (NSF), H D - 0 6 3 6 4 ( N I C H H D ) an d Public H e a l t h Service I n t e r n a t i o n a l R e s e a r c h Fellowship 5 F05 T W O 2693-02. REFERENCES 1 Azmitia, E. C., The serotonin-producing neurons of the midbrain medial and dorsal raphe nuclei. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Plenum Press, New York, 1978, pp. 233-314. 2 Bok, S. T., Histonomy of the Cerebral Cortex, Elsevier, Amsterdam, 1956, pp. 60-98. 3 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 4 Felten, D. L. and Cummings, J. P., The raphe nuclei of the rabbit brain stem, J. comp. Neurol., 187 (1979) 199-244. 5 Felten, D. L. and Crutcher, K. A., Neuronal-vascular relationships in the raphe nuclei, locus coeruleus, and substantia nigra in primates, Amer. J. Anat., 155 (1979) 467-482. 6 Felten, D. L. and Harrigan, P., Serotonergic dendrite bundles in nuclei raphe dorsalis and centralis superior of the rabbit, Anat. Rec., 196 (1980) 55A. 7 Felten, D. L. and Harrigan, P., Dendrite bundles in nuclei raphe dorsalis and centralis superior of the rabbit: a possible substrate for local control of serotonergic neurons, Neurosci. Lett., 16 (1980) 275-280. 8 Fox, G. Q., Pappas, G. D. and Purpura, D. P., Morphology and fine structure of the feline neonatal medullary raphe nuclei, Brain Research, 101 (1976) 385-410. 9 Kemper, T. L., Caveness, W. F. and Yakovlev, P. I., The neuronographic and metric study of the dendritic arbours of neurons in the motor cortex of Macaca mulatta at birth and at 24 months of age, Brain, 96 (1973) 765-782. 10 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963, pp. 1-162. 11 Leontovich, T. A. and Zhukova, G. P., The specificity of the neuronal structure and topography of the reticular formation in the brain and spinal cord of carnivora, J. comp. Neurol., 121 (1963) 347-379. 12 Leontovich, T. A., Quantitative analysis and classification of subcortical forebrain neurons. In M. Santini (Ed.), Golgi Centennial Symposium, Raven Press, New York, 1975, pp. 101-122. 13 Morgane, P. J., Miller, M., Kemper, T., Stern, W., Forbes, W., Hall, R., Bronzino, J., Kissane, E., Hawrylewicz, E. and Resnick, O., The effects of protein malnutrition on the developing central nervous system in the rat, Neurosci. biobehav. Rev., 2 (1978) 137-230. 14 Ram6n y Cajal, S., Histologie du Syst~me Nerveaux de l'Homme et des Vertdbrds, Vol. 11, 2nd Impression, C.S.I.S., Instituto Ram6n y Cajal, Madrid, 1972, pp. 227-251. 15 Ram6n-Moliner, E. and Nauta, W. J. H., The isodendritic core of the brain stem, J. comp. Neurol., 126 (1966) 311-336. 16 Scheibel, M. E., Tomiyasu, U. and Scheibel, A. B., Do raphe nuclei of the reticular formation have a neurosecretory or vascular sensor function? Exp. Neurol., 47 (1975) 316-329. 17 Sugita, N., Comparative studies on the growth of the cerebral cortex. I. On the changes in the size and shape of the cerebrum during the postnatal growth of the brain. Albino rat., J. comp. Neurol., 28 (1917) 495-509. 18 Taber, E., The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat, J. comp. Neurol., 116 (1961) 27-69. 19 Valverde, F., Reticular formation of the pons and medulla oblongata. A Golgi study, J. comp. Neurol., 116 (1961) 71-99. Note added in proof Since this paper has been in press a new paper (Pfister, C. and Danner, H., Fluorescence histochemical and neurohistological investigations on the nucleus raphe dorsalis of the rat, Acta histochem., 66 (1980) 253-261) has appeared. These authors identified 3 cell types in the NRD: polygonal, fusiform and pyriform, which appear to correspond, respectively, to our multipolar, fusiform and ovoid cells.
1
Research Reports
NUCLEUS RAPHE DORSALIS: A MORPHOMETRIC GOLGI STUDY IN RATS OF T H R E E A G E G R O U P S
S. DIAZ-CINTRA,* L. CINTRA,* T. KEMPER, O. RESNICK and P. J. MORGANE Worcester Foundation for Experimental Biology, Shrewsbury, Mass. and Neurological Unit, Boston City Hospital, Boston, Mass. (U.S.A.)
(Accepted July 31st, 1980) Key words: nucleus raphe dorsalis - - reticular neurons - - Golgi morphometric study - - spine density -
-
dendritic architecture - - raphe cell-vascular relations - - raphe dorsalis cells
SUMMARY Using Rapid Golgi and Nissl techniques, three major cell types: fusiform, multipolar and ovoid-shaped were identified in the nucleus raphe dorsalis of male rats at 30, 90, and 220 days of age. We have described the orientation and dendritic architecture of raphe cells as to type and the relationships of these cells to blood vessels and surrounding structures. For each cell type, the origin of the axon is characteristic. One hundred neurons per age group were measured at their maximal linear extent and the number of spines on the somal surface was counted. Dendritic number, linear extent, diameter and the number of spines along a 50/~m segment near the mid- point of dendritic length in an equal number of primary and secondary dendrites were quantified in each age group. The most striking age-related changes in the multipolar and ovoid-shaped cells were dendritic number, diameter and spine number as well as the number of perisomatic spines. The fusiform cells showed the least age-related changes. In general, the nucleus raphe dorsalis is organized as a reticular nucleus with neurons having few, straight and poorly ramified dendrites.
INTRODUCTION In recent years great interest has developed in the anatomical, physiological and chemical organization of the nucleus raphe dorsalis (NRD). Ram6n y CajaP 4, using several Golgi methods, examined and described cells of the 'central magnocellular nucleus of the raphe' (corresponding to the nucleus raphe dorsalis) in the cat and rabbit * On leave from: Departamento de Fisiologia, Instituto de Investigaciones Biom6dicas, Universidad Nacional Aut6noma de M6xico, M6xico 20, D.F., M6xico.
as being large, fusiform, triangular, or starlike in shape with multiple diverging dendrites that were covered with spines and tended to be grouped into vertical fasciculi. Axons of these cells were described as following an ascending course which, after giving off collaterals, were followed into several brain areas, including the medial longitudinal fasciculus (MLF). Recently, Leontovich and Zhukova 11 described the cells in the raphe nuclei as typical reticular neurons based on their appearance in Golgi stained sections. In a cytoarchitectonic study of the raphe nuclei in the cat, Taber is described two cell types in the N R D : medium-sized multipolar neurons and fusiform cells. Azmitia 1, likewise, recognized in the rat two main classes of neurons in the N R D : a medium-sized fusiform type cell that comprised about three-quarters of the total number of cells and a large ovoid type cell that accounted for the remaining one-quarter. Serotoninergic neurons in the N R D were identified by Dahlstr6m and Fuxe3just above and medial to the M L F and were classified as cell group B7 in their fluorescence mapping studies. In a recent study in the rabbit Felten and Cummings 4, and Felten and Harrigan6, 7, using Golgi and cresyl violet stains and histofluorescence techniques, described serotonin-containing neurons in the N R D as medium-sized oval and fusiform cells oriented vertically in the midline, within and tangential to the MLF. In the nucleus linearis rostralis and the nucleus raphe pontis a neurovascular proximity between raphe dendrites and blood vessels was described by Scheibel et al. 16. Felten and Crutcher 5, in a combined fluorescence, histochemical, and electron microscopic study in the N R D and nucleus raphe medialis in the squirrel monkey and rhesus monkey, demonstrated direct apposition of blood vessels to both the perikarya and dendrites of the serotoninergic neurons. These particular neurovascular relationships were present in relation to 20-30 ~ of the small vessels in the NRD. Felten and Crutcher postulated that the serotonin-containing neurons of the raphe may thus be influenced directly by hormones or other substances in the blood. The objective of the present study was to define and describe the cell types in the NRD, using quantitative techniques on rapid Golgi impregnated neurons, in male rats of three different ages. MATERIALS AND METHODS The material in this study was obtained from male Charles River C.D. Sprague-Dawley rats fed a 25 ~o~ casein diet according to the paradigm used in the Worcester Foundation Protein Program Project 13. Since in that project we are carrying out ontogenetic studies at certain critical ages, we have chosen these ages for the present anatomical analyses. Accordingly, at 30, 90 and 220 days of age the animals were weighed, anesthetized with pentobarbital, perfused through the heart with 10 ~ neutral buffered formalin and, on the following day, the brains removed and weighed. Slices of midbrain tissue were cut into 3 mm thick blocks in the frontal and sagittal planes and post-fixed in a potassium dichromate 4 ~o~ solution in buffered formalin. Each block was then transferred to classical rapid Golgi fixative for 6-11 days and silvered in a 0 . 7 5 ~ silver nitrate solution for 18-48 h. For some blocks in
220-day-old animals double or triple impregnation was used according to the method of Valverde 19. Finally the blocks were embedded in low viscosity nitrocellulose and cut in serial section at a thickness of 120-180 #m. One hundred neurons from 4 well impregnated brains per age group were selected for quantitative studies at random from all parts of the nucleus raphe dorsalis, including the anterior and posterior poles and the middle third of the nucleus. This area corresponds to the region of the N R D in the stereotaxic atlas of Krnig and Klippel lo as well as the region outlined in histofluorescence studies by Dahlstrrm and FuxeL All measurements were made at 400 x using a calibrated ocular reticle. Each cell chosen was outlined in pencil with the aid of a camera lucida and the following measurements were made: (1)the major and minor axes of the cell body were measured at their maximal extent; and (2) the number of spines on the somal surface was counted by focusing through several planes. In addition, dendrites were divided into primary and secondary branches according to the method illustrated in Fig. 2. Primary dendrites were defined as those arising from the perikarya, whereas secondary dendrites were defined by their origin from primary dendrites, their thinner diameters, and a lesser density of spines. Then the linear extent per cell of two primary dendrites and their secondary dendrites was measured. The linear extent of dendrites within the focal plane was measured directly by establishing the distance from the dendritic point of origin on the perikaryon to the tip of the dendrite. Those not in the plane of section were estimated by triangulation according to the method of Bok z and Kemper et al. 9. Measurements of the diameter of primary and secondary dendrites was made at the midpoint between their origin and tip. From the same middle segment the number of spines per 5 0 / t m of dendritic length was counted. Two additional rats from each age group were perfused with 1 0 ~ neutral buffered formalin, their brains embedded in albumin-gelatin and serially sectioned in the frontal and sagittal plane, respectively, at a thickness of 30 #m. These sections were stained with cresyl violet and examined for the general morphology of cell bodies, extent of the nucleus and various cytoarchitectural details. From this material the major and minor axes of the perikarya of 100 cells, including the three types selected at random from anterior, middle and posterior parts of the N R D per age group, were also measured and compared to similar naeasures in the Golgi material. Statistical significance of all measurements was determined by using the Student's t-test. RESULTS In the rat the N R D extends rostrally to the caudal pole of the trochlear nucleus and caudally to the posterior pole of the dorsal tegmental nucleus of Gudden, with its long axis directed rostro-caudally. In this long axis, it measures 1.6, 1.9 and 2.2 mm, respectively, at 30, 90 and 220 days. Throughout the entire extent of the nucleus 3 cell types were found which, based primarily on the shape of their perikaryon, are referred to as fusiform, multipolar and ovoid-shaped cells (Fig. 1). The appearance of all 3 types of cells at 3 different rostro-caudal levels of the nucleus raphe dorsalis are shown in Figs. 3-5.
~ la a( ÷
A
I
B
I
30 p.
Fig. 1. Camera lucida drawings of the perikaryon of fnsiform (la), multipolar (1 b), and ovoid (lc) cells in 90-day-old rats. The cells in column A are the largest examples encountered and those in B the smallest. On the right the major and minor axes of each cell type are shown schematically, a, axons.
/
S
p~-
[ I00
jim
i
p
Fig. 2. Camera lucida drawing of the cellular types in the nucleus raphe dorsalis. Primary dendrites (P) were defined as the largest extention of the processes on the perikarya, secondary dendrites (S) were defined as originating from the primary dendrites. Brackets show the dendritic extent on which spines were counted. The arrows indicate the loci for thickness measurement in the primary and secondary dendrites. Abbreviations: la, fusiform cell; lb,'_multipolar cell; lc, ovoid cell; a, axons.
The fusiform cells were the most frequently encountered type in the Golgi impregnated material at all levels in the N R D but are more heavily concentrated in the middle one-third o f the nucleus. Their perikarya showed the most marked differences between the major and minor axis (Fig. 1 and Table I). The primary dendrites arise from polar regions o f the cell, thereby further emphasizing their fusiform shape. A t lhe middle one-third o f the nucleus fusiform-shaped neurons situated on the superior aspect o f the M L F and the superior cerebellar peduncle show a tangential orientation o f their perikarya to these bundles. Dendrites o f some o f these fusiform cells penetrate deeply into both o f those fiber systems and radiate widely into other parts o f the N R D (Figs. 3 and 4). In the most caudal part o f the nucleus raphe dorsalis the fusiform cells are more vertically oriented. The dendritic process o f the fusiform cells extend
Fig. 3. Camera lucida drawing of the NRD at the level of the trochlear nucleus (Nu IV). Asterisks identify ceils with special characteristics discussed in the text. The single headed arrow shows the dendrites of an ovoid cell wrapped around a blood vessel. The double headed arrows show dendritic tips of a multipolar cell in close relation to a blood vessel. The triple headed arrow shows the close relationship of a fusiform cell dendrite to the fibers of the MLF. Abbreviations: l a, fusiform cell; l b, multipolar cell; I c, ovoid cell, aq, aqueduct; CG, central gray; dr, nucleus raphe dorsalis; DSCP, superior cerebellar peduncle decussation; MLF, medial longitudinal fasciculus; mr, nucleus raphe medianus.
f' /
\
\ VG
.
RF
100 jim
~
\
z
Fig. 4. Camera lucida drawing of the NRD midway between its caudal and rostral poles. The dendrites of the ovoid cell indicated by the arrow are following the vertical course of a blood vessel. Note the overlap of dendrites of multipolar cells (1b) with those of the neurons of the ventral tegmental nucleus of Gudden (VG) and nucleus raphe medianus (mr). Abbreviations: la, fusiform cell; lb, multipolar cell; lc, ovoid cell; RF, reticular formation; SCP, superior cerebellar peduncle.
vertically between the two M L F bundles and into the dorsal and ventral parts of the N R D coming into close relation with dendrites of the nucleus raphe medianus. Other dendrites extend into the M F L at this level (Fig. 5). In the rostral third of the nucleus raphe dorsalis the fusiform cells are largely oriented similar to those at the middle third of the nucleus. Here the dendrites disperse widely into other parts of the nucleus raphe dorsalis and tend to be more horizontally oriented than in the middle third of the nucleus. The axons arise primarily from a proximal dendrite in 75 ~o of the fusiform cells while on most of the remainder the axon arises from the soma. The multipolar cell was the next most frequently encountered cell in the N R D . This cell is found frequently at all levels in the N R D but are most heavily concentrated
7
aq
....
LOC
dr
.....~:-: SCP
,,
iV
~'lltF ' 100 pro'
Fig. 5. Camera lucida drawing of the NRD near its caudal pole. Note the close relationship of the dendrites of the fusiform ceils (la) to the medial longitudinal fasciculus (MLF). Also note the vertical orientation of the dendrites of these fusiform cells in the midline. Abbreviations: la, fusiform cell; 1b, multipolar cell; lc, ovoid cell; aq, aqueduct; C, cerebellum; dr, nucleus raphe dorsalis; DG, nucleus dorsalis of Gudden; SCP, superior cerebellar peduncle.
in the middle one-third of the nucleus (Fig. 4). Dendrites of these cells arise at 3 or more different locations on the perikaryon, thus giving the cells their triangular or multipolar appearance. Their dendrites are more randomly distributed, overlapping extensively with the dendrites of the ventral tegmental nucleus of Gudden and nucleus raphe medianus, particularly in the middle third of the nucleus (Fig. 4). Some of the dendritic processes of the multipolar cells of the N R D extend into M L F at the rostral and middle third of the nucleus (Fig. 4) while others come into close contact with small blood vessels dorsal to the M L F at the level of the rostral third of the nucleus (Fig. 3, double arrows). Axons were found to arise primarily from the soma in 84 ~ of the multipolar cells. The ovoid-shaped cell was the least frequently encountered neuron in the nucleus raphe dorsalis and showed the fewest differences between its major and minor axis. This cell was also found at all levels in the NRD. Dendrites appear randomly distributed and in the more midline cells they are frequently wrapped around blood vessels (Figs. 3 and 4, single arrow). The axons arise with almost equal frequency from the cell body (53 ~ ) or a proximal dendrite (47 ~).
In Tables I and II the major and minor axis of the perikaryon, number, diameter, and extent of the dendritic processes, and the density of perisomatic and dendritic spines are recorded for each cell type at 30, 90 and 220 days of age. At 30 days of age the major and minor axes of the perikarya of thefusiforrn cells were 25.79 4- 4.75 and 15.12 4- 3.33/~m, respectively, and the number of perikaryal spines was 3.21 4- 2.71. The number of primary dendrites was 3.37 4- 0.75 and secondary dendrites 2.86 4- 0.85 per cell, with respective thicknesses of 1.92 ~ 0.49 and 1.58 4- 0.38/tm. The linear extent of the primary dendrites was 313 4- 93 #m and of the secondary dendrites 192 4- 76/~m. Their respective number of spines/50/~m were 6.54 4- 2.07 and 6.00 4- 2.29. At 90 days of age there was a 20 ~/o increase in the number of spines on the primary (P < 0.001) and secondary (P < 0.05) dendrites, respectively. At 220 days there was a 12 ~ (P < 0.001) decrease in the number of these spines on the primary dendrite. All other measurements failed to show significant age-related changes. At 30 days of age the major and minor axes of the perikarya of the multipolar cells were 22.92 4- 4.16 and 17.94 4- 3.81 #m, respectively, and the number of perisomatic spines was 3.53 4- 2.71. The number of primary dendrites was 3.86 4- 0.78 and secondary dendrites 2.88 4- 0.93, with their respective diameters being 1.94 40.49 and 1.56 4- 0.34/ma. The linear extent of their primary dendrites was 291 ± 90 #m and of their secondary dendrites 186 4- 70 /~m. The respective numbers of spines/50/~m were 6.66 4- 2.14 and 5.75 4- 1.96. At 90 days of age they showed a significant 13 ~ (P < 0.05) increase in linear extent of their primary dendrites, a 24 increase in spine density on the primary dendritic processes, and a 34~o increase in spine density on their secondary dendrites (both P < 0.001). At 220 days there was an 18 ~ (P < 0.001) and 16~ (P < 0.02) decrease, respectively, in spine density in these locations together with a 6 4 ~ (P < 0.001) decrease in perisomatic spines. All other measurements failed to show significant age-related changes. At 30 days of age the major and minor axes of the perikarya of the ovoidcells were 19.66 4- 4.45 and 14.49 4- 2.74 ~m, respectively, and the number of perisomatic spines 2.16 4- 1.91. The number of primary dendrites was 2.72 4- 0.66 and secondary dendrites 2.44 4- 0.61, with respective thicknesses of 1.82 4- 0.42 and 1.40 4- 0.26/~m. The linear extent of the primary dendrite was 280 4- 83 /~m and of the secondary dendrite 171 4- 56/~m. The respective numbers of spines/50 um were 6.19 4- 2.27 and 5.30 4- 1.63. At 90 days there was a 72~o (P < 0.02) increase in the number of perisomatic spines, a 20 °/o (P < 0.05) increase in the linear extent of the primary dendrites, a 24 ~ (P < 0.01) increase in spine density on primary dendrites, and a 33 (P < 0.001) increase in spine density on the secondary dendrites accompanied by a 10~ (P < 0.05) decrease in diameter of their secondary dendritic processes. At 220 days the number of primary dendrites increased 21 ~ (P < 0.01), and the linear extent of the primary dendrite decreased 17~ (P < 0.05) and the diameter of secondary dendrites increased 9 ~ (P < 0.02). All other measurements failed to show significant age-related changes. The measurements of the major and minor axis of these 3 cell types in Nissl stained sections was essentially identical to that found in the Golgistained material (Tables I and IV).
Minor axis (t~m)
No. of peri- No. of somatic cells spines
25.794-4.75 15.124-3.33 3.214-2.71 45
Major axis (l~m)
** = P < 0 . 0 2 . §§ = P < 0.001.
220 days 56 23.33±4.53 Change 90 vs 220 days (--3.0 ~)
2.914-2.52 22 (+2~)
14.384.2.62
(+1%) (+0.3 ~)
(--7~)
17.354.3.17
14
18
(--64~)§§
1.594.1.46 22
(+25 ~)
(+4%)
3.534-2.71
(--2~) 22.56±4.05
0 void No. of per±- No. of somatic cells spines
18.644.3.86 4.404-2.50
17.94±3.81
Minor axis (l~m)
22.494-3.82
22.924,4.16
Major axis (l~m)
Multipolar
90 days 66 24.064.3.81 14.234-2.83 2.844-1.98 20 Change 30 vs 90 days (--7 ~) (--6 %) (--I 2 ~)
30 days 37
No. of cells
Fusiform
Major and minor axes of the cell body and number of perisomatic spines for each cell type at 30, 90 and 220 days
TABLE I
(--2%)
17.924.2.95
(--7%)
18.36+4.09
19.664.4.45
Major axis (l~m)
No. of perisomatic spines
(+72%)**
(+3 %)
(--30%)
15.34:k:3.10 2.594-2.34
(+2%)
14.844.4.87 3.714-1.58
14.494-2.74 2.164,1.91
Minor axis (pm)
Prim.
Sec.
Number of dendrites
(--6%)
(0%)
(4- 0.6 %)
220 days 22 % Change 90 vs 220 days
90 days 20 % Change 30 vs 90 days (--1%)
(+-1%)
(--5 %)
(--4 %)
(--3 %)
(,4,10%)
(+20%)
1.48 4- 0.31
1.89 -4- 0.38
4.00 -4- 0.69 3.13 ± 1.24
(--6 %)
1.54 -4- 0.32
1.95 -4- 0.34
1.56 -4- 0.34
3.65 -4- 0.58 2.60 -4- 0.94
1.94 4- 0.49
(+5%)
(+5%)
(--1%)
(--2 %)
1.55 4- 0.31
2.01 -4- 0.38
3.37 -4- 0.72 2.66 4- 0.90
(--5 %)
1.48 -4- 0.32
1.58 -4- 0.38
Sec.
1.92 + 0.42
1.92 4-4-0.49
Prim.
Dendritic diameter (#m)
3.39 -4- 0.74 2.71 + 0.97
Multipolar shape 30 days 45 3.86 -4- 0.78 2.88 -4- 0.93
220 days 56 % Change 90 vs 220 days
90 days 66 % Change 30 vs 90 days
Fusiform shape 30 days 37 3.37 -4- 0.75 2.86 ~ 0.85
cells
Number of
Prim. = primary dendrite; sec. = secondary dendrite. Results are ~ 4- S.D.
44
40
90
112
132
74
Number of dendrites examined
(--0.3 %)
327 -4- 92
(+13%)*
328 + 84
291 -4- 90
(+0.1%)
314 4- 97
(+0.3%)
314 4- 88
313 -4- 93
Prim.
(--7 %)
175 5= 50
(+2%)
189 4- 60
186 -I- 70
(--2%)
181 4- 67
(--4%)
185 4- 63
192 4- 76
Sec.
Sec.
(-4-20%)*
(--15%)
(+34%)§§
(--16%)**
1.96 6.50 ± 2.14 (--18%)§§
6.77 i
(+24%)§§
8.27 -- 1.82 7.70 -4- 2.18
6.66 4- 2.14 5.75 ! 1.96
(--12%)§§
6.91 -4- 1.82 6.10 -4- 1.90
(+20%)§§
7.87 -4-4-2.02 7.19 -4- 1.96
6.54 + 2.07 6.00 -4- 2.29
Prim.
Dendritic linear extent (l~m) Number of dendritic spines~50 I~m
Number, diameter and extent of dendrites and synaptic spine density for each cell type at 30, 90 and 220 days of age
TABLE II
§§ -- P < 0 . ~ 1 .
* = P < 0.05. ** = P < 0.02. § = P < 0.01.
22 % Change 90 vs 220 days
220 days
14 % Chang~ 3 0 vs 90 days
90 days
18
0 void shape 30 days
( - 4 %)
(--0.4 %)
(+8%)
(+21%)~
(--2 %)
1.89 4- 0.38
3.27 4- 0.55 2.59 4- 0.79
(+8%)
1.75 4- 0.26
1.82 4- 0.42
2.71 ± 0.72 2.64 4- 0.84
2.72 4- 0.66 2.44 4- 0.61
(+9%)**
1.37 : i 0.24
(--lO%)*
1.26 4- 0
1.40 4- 0.26
44
28
36
(--17%)*
280 4- 83
(+20%)*
336 4- 112
280 4- 83
(--8 %)
166 + 49
(+5%)
180 =t= 75
171 4- 56
( + 3 3 %)§§
(--11%)
(--11%)
6.84 4- 2.05 6.31 ± 1.81
(+24%)§
7.67 4- 2.00 7.07 ± 1.76
6.19 4- 2.27 5.30 ± 1.63
30 days
Minor axis
+31~§§ +4~ --20%*
* = P < 0.05. ** = P < 0 . 0 2 . § = P < 0.01. §§ = P < 0.001.
Dendrites
+55~§ +30~ --16%
+10~ --33% --39~*
+19~§§ --3 % --18%§§
+8~ --20%§§ --26~§§
+15%§ --19~§ --30~*
+18~ --3 ~ --17%
---4~ --3~ +2%
+0.7% --15 ~* --15%*
No. of Number of dendrites perisomatic spines Prim. Sec.
Fusiformvsmultipolar - - 3 ~ + 2 1 % § § ---45%* Fusiform vs ovoid --23 %§§ + 7 % --119/00 Multipolarvsovoid --21~§§ --12%* +39%
220 days
Fusiformvsmultipolar --7% Fusiformvsovoid --24%§§ Multipolarvsovoid --18%§
90 days
Fusiformvsmultipolar --11~§ +19~§§ Fusiform vs ovoid --31 ~§§ - - 4 ~ Multipolarvsovoid --14~** --10%§§
Major axis
Perikarya
Prim. = primary dendrite; Sec. = secondary dendrite.
--6% --6 ~ 0%
+2~ --9%* --10~
+1~ --5 % --6~
Prim.
Diameter
--5~ --12 ~ --7~
+4% --15~§§ --18~§§
+1% --11 ~* --10~*
Sec.
Statistical analysis of the differences in morphometric data of the various cell types at 30, 90 and 220 days
TABLE III
+2~ --3~ --5~
--3~ --11 ~ --8~
Sec.
+4~ --3~ --11 ~§ --8 %* --14~** - - 5 ~
+4~ +7~ +3~
--7% --11 ~o --6~
Prim.
Linear extent
--2% --1 ~ +1~
+5~ --3% --7~
+2~ --5 ~ --7%
Prim.
+7% +3 --3%
+7~ --2~ --9%
--4~ --12~ --8~
Sec.
Number of spines~50 I~m
26.69-4-5.70
25.05±4.06 (--6~)
25.64±3.91 (+2~)
30 days 44
90 days 42 ~ C h a n g e 30 vs 90 days
220 days 42 ~ C h a n g e 90 vs 220 days 12.93 ~ 1.91 (--6~)
13.73 :k2.63 (+4~)
13.21 ± 2 . 3 1
43
38
36
No. of cells
Minor axis (~m)
No. of cells
Major axis (t~m)
Multipolar
Fusiform
Results are ~ :k S.D.
22.00 ± 3.95 (+0.17o)
21.98 -4- 4.48 (--6 ~ )
23.30±4.32
Major axis (#m)
15.53 ± 3.31 (--0.8%)
15.66 ± 4.01 (--6 G)
16.59±3.08
Minor axis (l~m)
Major and minor axes of the cell body ]br each cell type at 30, 90 and 220 days of age ( Nissl-stained material)
TABLE IV
15
20
20
No. of cells
Ovoid
19.32 ± 2.11 ( - - 2 ~o)
19.78 4- 3.37 (+5%)
18.83 ± 4 . 3 1
Major axis (t~m)
17.80 + 2.17 (--2 ~)
18.08 ± 3.09 (+6~)
17.05 ± 4.24
Minor axis (t~m)
U.
14 As can be seen from these data, clear differences between these 3 cell types with age emerge. Therefore, when the measurements for each cell type are compared, one notices that they are statistically separable in all age groups by several criteria (Table III). This table contains the statistical data showing the comparisons of all 3 cell types at 3 ages for the various parameters under consideration. DISCUSSION Based on cell body shape, orientation of dendrites, origin of the axon and quantitative measurements of the soma, dendrites and dendritic spines, we have defined 3 cell types in the N R D : fusiform, multipolar and ovoid-shaped neurons. A fusiform cell in the raphe dorsalis had been previously noted by Ram6n y CajaP 4, Taber 18, Azmitia a and Felten and Cummings 4. In the present material, in agreement with Azmitia 1, it is the most frequently encountered cell in all parts of the nucleus. The fusiform cell is further characterized as showing the least age-related changes of all three cell types. Notable characteristics of this cell are marked differences in length between the major and minor axis of the cell body, an axon that arises most frequently from a proximal dendrite, and the orientation of its cell body and dendrites to adjacent fiber systems, particularly the MLF. Special orientation of raphe cells to adjacent fiber bundles had been commented on by Ram6n y CajaP 4, Taber ~8 and Felten and Cummings 4 but without reference to any particular cell type. The multipolar cell appears to correspond to Ram6n y Cajal's ~4 star-like and triangular cell and to the medium-sized multipolar cell described by Taber 18. Its characteristic features are its multipolar cell body, an axon that arises most frequently from its cell body, and randomly oriented dendrites that overlap dendrites of adjacent nuclei. Its most characteristic age-related change is a 25 °//Uincrease in the number of perisomatic spines between 30 and 90 days followed by a marked decrease of 64 % (P ~< 0.001) at 220 days. Although not achieving statistical significance, there is an increase in the number of both primary and secondary dendrites between 90 and 220 days. Ovoid-shaped cells in the nucleus raphe dorsalis have been noted by Azmitia 1 and Felten and Cummings 4. A characteristic feature of the ovoid cell is an axon that arises with equal frequency from its cell body or proximal dendrite and the random orientation of its dendrites. Its most striking age-related change is a marked increase of 72 % in perisomatic spines between 30 and 90 days (P < 0.02) followed by a less marked decrease of 30 ~ at 220 days. It shows a significant 21 ~o increase in number of primary dendrites between 90 and 220 days (P < 0.01) but not in number of secondary dendrites. It is the only cell to show significant age-related changes in secondary dendritic diameter. The extent to which these cell types are representative of cells in other raphe nuclei is not known. However, it is interesting to note that Fox et al. 8, in a Golgi study of the caudal medullary raphe nuclei (obscurus and pallidus), observed similar cell types to those described herein. These authors also noted medium fusiform, large multipolar, medium stellate, and small spherical neurons in these posterior raphe nuclei.
15 Despite their differences the cells of the N R D have many features in common. Both the multipolar and ovoid cells show a close relationship to blood vessels throughout the NRD. This relationship was first noted by Scheibel et al. 16 in the nucleus raphe pontis and nucleus linearis rostralis and, later, by Felten and Crutcher 5 in the N R D and nucleus raphe medianus. In the present study we find that at 30 days of age this relationship is established by dendrites of the ovoid-shaped and multipolar cells and at 90 days of age some cell bodies of ovoid-shaped cells are found to be closely allied to blood vessels. Other striking similarities are the patterns of age-related changes in number of dendrites, dendritic extent, and synaptic spine density, particularly for the multipolar and ovoid-shaped cells. Both the multipolar and ovoid-shaped cells show a significant increase in extent of their primary dendrites between 30 and 90 days of age. They also show a lesser (non-significant) increase in extent of their secondary dendrites. At 220 days we find a significant decrease in the linear extent in the primary dendrites on ovoid-shaped cells. However, at this time there is an increase in the number of primary dendrites that is significant only for the ovoid-shaped ceils. Additionally, there is a significant increase in the number of secondary dendrites of the multipolar, but not the ovoid-shaped cells. At 90 days both the multipolar and ovoid-shaped cells show an increase in perisomatic spines, yet this is significant only for the latter. At 220 days both show a spine loss on the cell bodies that is significant only for the multipolar cells. Ovoid-shaped, multipolar, and fusiform cell types show an increase in dendritic spine density on primary and secondary dendrites between 30 and 90 days which in all cases is statistically significant. In most cells between 30 and 90 days there is a period of linear growth of dendritic processes associated with an increase in dendritic spines. Between 90 and 220 days there is an increase in number, but not the extent, of dendritic processes and a decrease in density of dendritic spines. In the present study we find the prolonged period of dendritic growth in all 3 cell types is also reflected in the progressive increase in rostro-caudal length of the N R D from 30 to 220 days. This pattern of progressive growth of the N R D appears to be in conjunction with an overall pattern of growth of the brain stem during this period. For example, Sugita 17 has provided data from rats on the weight of the brain and brain stem at ages similar to those used in the present study. He showed that between 35 and 150 days there is a 33.5 ~ increase in the brain stem weight. We showed a 27 ~ increase in the length of the N R D between 30 and 220 days. All 3 types of cells we have identified in the nucleus raphe dorsalis are seen in the rostral, middle and posterior parts of the nucleus. However, certain cell types are more concentrated in particular divisions of the nucleus. Thus, the fusiform and multipolar cells are in relatively greater numbers in the middle one-third of the nucleus relative to the ovoid cells. In all parts of the nucleus raphe dorsalis the fusiform-shaped cells are much more numerous than both the multipolar and ovoid-shaped cells. Generally, the N R D is organized as a reticular nucleus with neurons having few, long, straight, poorly ramified dendrites11,12,15. Within the NRD, based on cell body shape, origin of axon, morphometric data on its somal and dendritic architecture and
16 age-related changes, we were able to identify 3 cell types. I n these, the dendritic a n d cell m o r p h o l o g y o f each type show v a r y i n g relations with b l o o d vessels a n d s u r r o u n d i n g nuclei a n d fiber bundles. ACKNOWLEDGEMENTS W e t h a n k M. Feinstein for his help with the histological preparations. S u p p o r t e d by G r a n t s B N S 77-16512 (NSF), H D - 0 6 3 6 4 ( N I C H H D ) an d Public H e a l t h Service I n t e r n a t i o n a l R e s e a r c h Fellowship 5 F05 T W O 2693-02. REFERENCES 1 Azmitia, E. C., The serotonin-producing neurons of the midbrain medial and dorsal raphe nuclei. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Plenum Press, New York, 1978, pp. 233-314. 2 Bok, S. T., Histonomy of the Cerebral Cortex, Elsevier, Amsterdam, 1956, pp. 60-98. 3 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 4 Felten, D. L. and Cummings, J. P., The raphe nuclei of the rabbit brain stem, J. comp. Neurol., 187 (1979) 199-244. 5 Felten, D. L. and Crutcher, K. A., Neuronal-vascular relationships in the raphe nuclei, locus coeruleus, and substantia nigra in primates, Amer. J. Anat., 155 (1979) 467-482. 6 Felten, D. L. and Harrigan, P., Serotonergic dendrite bundles in nuclei raphe dorsalis and centralis superior of the rabbit, Anat. Rec., 196 (1980) 55A. 7 Felten, D. L. and Harrigan, P., Dendrite bundles in nuclei raphe dorsalis and centralis superior of the rabbit: a possible substrate for local control of serotonergic neurons, Neurosci. Lett., 16 (1980) 275-280. 8 Fox, G. Q., Pappas, G. D. and Purpura, D. P., Morphology and fine structure of the feline neonatal medullary raphe nuclei, Brain Research, 101 (1976) 385-410. 9 Kemper, T. L., Caveness, W. F. and Yakovlev, P. I., The neuronographic and metric study of the dendritic arbours of neurons in the motor cortex of Macaca mulatta at birth and at 24 months of age, Brain, 96 (1973) 765-782. 10 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963, pp. 1-162. 11 Leontovich, T. A. and Zhukova, G. P., The specificity of the neuronal structure and topography of the reticular formation in the brain and spinal cord of carnivora, J. comp. Neurol., 121 (1963) 347-379. 12 Leontovich, T. A., Quantitative analysis and classification of subcortical forebrain neurons. In M. Santini (Ed.), Golgi Centennial Symposium, Raven Press, New York, 1975, pp. 101-122. 13 Morgane, P. J., Miller, M., Kemper, T., Stern, W., Forbes, W., Hall, R., Bronzino, J., Kissane, E., Hawrylewicz, E. and Resnick, O., The effects of protein malnutrition on the developing central nervous system in the rat, Neurosci. biobehav. Rev., 2 (1978) 137-230. 14 Ram6n y Cajal, S., Histologie du Syst~me Nerveaux de l'Homme et des Vertdbrds, Vol. 11, 2nd Impression, C.S.I.S., Instituto Ram6n y Cajal, Madrid, 1972, pp. 227-251. 15 Ram6n-Moliner, E. and Nauta, W. J. H., The isodendritic core of the brain stem, J. comp. Neurol., 126 (1966) 311-336. 16 Scheibel, M. E., Tomiyasu, U. and Scheibel, A. B., Do raphe nuclei of the reticular formation have a neurosecretory or vascular sensor function? Exp. Neurol., 47 (1975) 316-329. 17 Sugita, N., Comparative studies on the growth of the cerebral cortex. I. On the changes in the size and shape of the cerebrum during the postnatal growth of the brain. Albino rat., J. comp. Neurol., 28 (1917) 495-509. 18 Taber, E., The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat, J. comp. Neurol., 116 (1961) 27-69. 19 Valverde, F., Reticular formation of the pons and medulla oblongata. A Golgi study, J. comp. Neurol., 116 (1961) 71-99. Note added in proof Since this paper has been in press a new paper (Pfister, C. and Danner, H., Fluorescence histochemical and neurohistological investigations on the nucleus raphe dorsalis of the rat, Acta histochem., 66 (1980) 253-261) has appeared. These authors identified 3 cell types in the NRD: polygonal, fusiform and pyriform, which appear to correspond, respectively, to our multipolar, fusiform and ovoid cells.