Brain Research, 107 (1976)221-237 © ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
221
Research Reports
THE EFFECT O F A LOW PROTEIN DIET ON THE ANATOMICAL DEVELOPMENT OF THE RAT BRAIN
CHRISTOPHER D. WEST ANt) THOMAS L. KEMPER Harvard Medical School, Department of Neurology and Worcester Foundation for Experimental Biology, Boston, Mass. (U.S.A.)
(Accepted September24th, 1975)
SUMMARY Albino rats were conceived and suckled by mothers maintained on a 8 % or an isocaloric 25 % casein diet. After weaning, pups were maintained on their respective diets ad libitum. By most parameters of CNS maturation, rats on the low protein diet closely resembled their age-matched controls. Only by the parameter of the ratio of brain weight to body weight did they resemble rats of a younger age. Camera lucida drawings of comparable Rapid Golgi-impregnated cortical neurons of 10-day-old control and experimental rats were nearly identical to each other. A similar finding was also noted in 30-day-old rats. However, with quantitative studies, the 30-day-old experimental animals showed a decrease in synaptic spine density and reduced dendritic length for some but not all dendritic processes. At all ages, experimental animals closely resembled age matched controls in the proportion of their brain weight that was neocortex, archicortex, and cerebellum. In these results we concur with Dobbing and Sands e that developmental timetables for the brain are not affected by undernutrition, though extent of development may be.
INTRODUCTION Neuroanatomical indices of maturation such as myelination, cell packing density, Nissl substance differentiation, dendritic elaboration and spine density have been found to be retarded or curtailed in certain conditions of mental retardation in man 1, 12,15,19. Although mental retardation in man or its possible analogues in rats has not been conclusively demonstrated as an effect of malnutrition per sele, 21, the onset of
222
Fig. 1. The effect of the 25 % and 8 3/00protein diets on the brain size and on the body size of the rat at 30 days of age.
certain developmental milestones such as eye opening, ear opening, auditory and righting reflexes have been found to be delayed in undernourished rats2, 21. It has been well established in rats and other species that early undernutrition retards the growth of the body more than the growth of the brain z~ (see Fig. 1). As noted by Eayrs and Horn 9, the brain-body weight ratio of an undernourished animal at certain ages resembles that of a younger control animal. To what extent this holds true for other criteria of growth and maturation is the subject of this investigation. The proportions of the total brain which are forebrain, neocortex, cerebellum, and other structures have been shown to change with age in the albino rat 7,22,24 as has the dendritic elaboration of cortical neurons s. This study proposes to examine these 3 levels of development in rats raised on low and high protein diets; (1) brain weight and body weight, (2) relative proportions of the brain which are neocortex, hippocampus and cerebellum, and (3) development of individual cerebral cortical and cerebellar neurons. In the majority of morphological studies reported by others, undernutrition has been confined to the period of lactation and has been produced by increasing litter size or by removing the pups from the dam for part of the day. In the present paradigm, experimental rats were conceived, suckled, and weaned in the presence of a low protein diet, a situation similar to that of humans in certain parts of the world.
223 MATERIALS A N D METHODS
Virgin female Charles River CD (outbred albino) Sprague-Dawley descended rats were placed on either an 8 % or an isocaloric 25 % casein diet 5 weeks prior to conception* and were maintained on these diets during gestation and lactation. It has been shown by Miller is that the quality of milk produced by mothers on the 8 % casein diet is the same as the quality of milk produced by mothers on the 25 % casein diet, but the quantity of milk is reduced. In this experimental model, protein availability rather than caloric restriction is considered to be the limiting factor in pup growth. Both experimental and control litters were randomized and adjusted at birth to a total of 8 pups. The pups were left undisturbed until weaning at 21 days. They were then segregated by sex (4 to a cage), maintained ad libitum on their respective diets, and left undisturbed until needed for experimental observations. The composition of the experimental and control diets, which were developed by Dr. S. Miller of the Massachusetts Institute of Technology, has been compared with laboratory chow by Stern et al. la (see Table I). For neuroanatomical study the rats were weighed, anesthetized with ether, and perfused with either Karnovsky's solution or 10 % formalin. The brains were allowed to harden overnight in situ; the following day they were weighed and their volume was determined. Fixed brains from experimental and control rats at 13, 30 and 90
TABLE I SUMMARY OF COMPOSITION OF DIETS
The percentages, by weight, of the components of the laboratory chow (pellet form, formula 4RF, Charles River Co., Wilmington, Mass.), 25%, and 8% casein (dry mash; made by General Biochemicals, Chagrin Falls, Ohio) diets are listed below. Approximately 80% of the protein of the laboratory chow was metabolically usable. Casein consisted of 87% protein (fully utilizable), 1.5 % fat and ll.5 % water. Thus, the amounts of utilizable protein in the laboratory chow and the 25 % casein diets were comparable. The casein diets contained a supplement of L-methionine (0.4%) since casein lacks this amino acid. Per cent nutritional composition Normal protein diet
Protein Fat Carbohydrate Salt mix Vitamin mix Water Non-nutritive filler
25 % Casein
Laboratory chow
21.8 15.4 50.9 4.7 1.0 2.2 4.2
26.3 7.1 42.9 3.5 0.3 9.6 10.3
Low protein diet 8 % Casein
7.0 15.1 67.4 4.7 1.0 0.9 4.2
* Thirteen-day-old experimental rats found in Tables II and III and all 13-day-old controls were born of dams placed on diets 2 weeks prior to conception.
224
postnatal days were embedded whole in an albumin gelatin and cut in gapless serial frozen sections at a thickness of 30 #m in either coronal, sagittal or horizontal plane of section. Every 10th section was stained with cresyl violet for nerve cells and glial nuclei, and the adjacent section was stained with modified Loyez stain for myelinated fibers. From the series of mounted Nissl stained sections, every second or third slide throughout the entire brain was projected onto a flat surface with a fixed magnification projector. The cross-sectional area of the neocortex, hippocampus, cerebellum and whole brain was measured with a compensating polar planimeter. The proportion of total brain tissue in each of these 3 regions was calculated by dividing the total of the cross-sectional area measurements for each region by the total of the crosssectional area measured for the whole brain. Weight estimates for neocortex, hippocampus, and cerebellum could be calculated by multiplying the wet weight of the whole brain by the proportion - - or percentage - - of total brain comprised by each region. These proportions and the means of these proportions are recorded in Tables II and II1 for ages 13, 30 and 90 days. Also recorded in Table Ili are the proportions of hippocampus and cerebellum calculated from means of regional weights and whole brain weights found in the literature for ages 6, 8, 10, 14, 17, 18, 21 and 210 days 3,sa°. Linear shrinkage in preparing Nissl sections was essentially the same for both experimental and control brains. The length and width of the cerebra and width of the cerebellum were reduced on the mounted sections an average of 18.2~ in experimental animals and an average of 18.9"/,, in controls. In Fig. 2, 7 points on the graph are the mean weights of perfusion fixed tissue. All the other points on this graph are mean weights of fresh tissue. For the study of individual neurons in the cerebral and cerebellar cortex an additional series of experimental and control rats at 10 and 30 days of age were sacrificed and perfused using the same procedure described above. Blocks of visual cortex and of cerebellum were post-fixed in Rapid Golgi solution and silvered with 0.75 ~; AgNOa. These were embedded in low viscosity nitrocellulose and cut in serial section at a thickness of 150 /tm. From the mounted histological sections representative neurons at varying depths within the visual cortex of experimental and control rats were drawn at a magnification of 400 x with the aid of a camera lucida. Drawings were similarly made of Purkinje cells, stellate cells and granule cells of the cerebellum of the 30-day-old rats at 1000 ×. in sampling neurons for camera lucida drawings from the various layers of visual cortex in the 10-day-old rats, all cells drawn for both the undernourished and control animals, respectively, lay on single sections. From the 30-day-old rats, drawings were limited as much as possible to single sections containing well impregnated neurons at several cortical depths. Although this procedure minimized neuron sampling bias, it did not control for individual variation or slight differences in age (Figs. 3 and 4). In the 30-day-old control and experimental rats, quantitative measurements were also made on readily identifiable cell types (Tables IV and V, and Figs. 9-11). For these a greater number of animals were sampled. In the visual cortex the lengths of basal and oblique dendrites and the spine density on the main apical dendritic shaft, on its terminal ramifications, and on the mid-part of the basal and oblique
225 dendrites of layer IIIb pyramidal neurons were measured. In the cerebellum, the dendritic length and cell body diameter of the granule cells were measured in the rostral bank of the primary fissure. The dendritic length and spine density of the less numerous basket cells were measured throughout the cerebellar vermis. The diameters of Purkinje cell bodies and the number of dendrites on granule cells were measmed throughout the cerebellum. Measurements were made with an ocular reticle that was calibrated with a stage micrometer after the methods of Kemper et al. 14. Dendritic lengths in the plane of section were measured directly. Those not in the plane of section were estimated by assuming that the dendrite could be represented as the hypotenuse of the right triangle formed by the measured distance between the root of the dendrite and its tip and the measured difference in depth between the two. Spine density measures were made on dendrites that were within one focal plane by counting the number of spine profiles falling between two points on the ocular grid. Cortical plate thickness measures were made from Rapid Golgi sections cut parallel to apical dendritic shafts of the pyramidal cells in the occipital cortex, and parallel to Purkinje dendritic trees in the cerebellar cortex. RESULTS
The mean brain weights and the log mean body weights of experimental and control rats of various ages are graphed in Fig. 2. At one postnatal day there is no significant difference between experimental and control rats in brain weight, body weight or brain-body weight ratio. From 11 days on, both brain weight and body weight are smaller in experimental rats than in controls, and the brain-body weight ratios of experimental rats are greater than in controls. When brain weight is plotted against log body weight, the locus of points of control rats appears linear. In contrast,
RELATIONSHIP OF BRAIN WEIGHT TO BODY WEIGHT
2.5
A
2.0 ~6
n,. z
0
0
1.5
II
AA a o
1.0
o Q
o
z
,,*
o
0.5
o A
5
I0
20
50
I00
LOG BODY WEIGHT IN GRAMS
200
500
= CONTROL o UNDERNOURISHED
Fig. 2. The relationship of mean brain weight plotted against log mean body weight for rats on 8 % casein and 25% casein diets. Open circles, experimental animals; open triangles, control animals.
226 TABLE 11 BRAIN WEIGHTS AND
PROPORTIONS OF HIPPOCAMPUS, CEREBELLUM AND NEOCORTEX EXPRESSED AS
PERCENTAGE OF WHOLE BRAIN
Animal
Age (days)
Brain weight
Regional proportion o f total brain (%) ............................. Hippocampus Cerebellum Neoeortex Prepyriform cortex, entorhinal cortex and neocortex
Exp.-3 Exp.-I Exp.-2 Exp.-6 Contl.-4 Contl.-5 Contl.-7 Contl.-1
13 13 13 13 13 13 13 13
1.02 1.27 1.28 1.28 1.29 1.29 1.35 1.36
5.5 3.2 4.6 4.6 3.4 5.1 5.3 3.6
11.4 10.0 9.2 8.8 10.0 11.9 11.4 8.8
28.0 29.1 26.3 31.8 29.1 26.9 29.1 29.4
40.0 38.8 34.6 39.0 34.7 35.4 37.7 38.0
Exp.-39 Exp.-38 Contl.-41 Contl.-37
30 30 30 30
1.19 1.34 1.97 1.98
4.7 5.5 6.5 5.6
14.5 13.5 12.8 12.9
26.8 28.3 25.4 26.4
33.9 35.5 36.5 36.4
Exp.-49 Exp.-47 Contl.-50 Contl.-45
90 90 90 90
1.48 1.69 2.27 2.51
5.8 6.0 5.5 5.1
14.0 16.0 14.5 14.3
26.3 24.5 23.2 24.4
34.0 32.4 33.5 32.6
that of experimental rats appears to describe a monotonic increasing, negatively accelerating curve. Under 40 g of body weight, experimental rats have slightly larger brains for their body size than controls; over 100 g of body weight, the reverse is true. The relative proportions of individual brains which are neocortex, hippocampus, and cerebellum in experimental and control rats at 3 ages, are found in Table II. The means of these data and proportions calculated from mean regional and mean whole brain weights found in Fish and Winick 10, Chase et al. 3 and Dobbing et al. ~, are found in Table III. In both experimental and control rats, similar tendencies are noted. The cerebellar percentage of total brain size increases with increasing age. The hippocampal percentage appears constant, while there is a decrease in neocortical percentage. Although the neocortical percentage appears somewhat larger in the experimental animals, a pooled measure of neocortex, entorhinal cortex, and prepyriform cortex showed no consistent difference (Table II). Rapid Golgi drawings of representative cortical neurons found at increasing depths from the pial surface of visual cortex from 10-day-old experimental and control rats are illustrated in Figs. 3 and 4. Representative neurons similarly arranged from Rapid Golgi preparations of 30-day-old experimental and control rats are found in Figs. 5 and 6. A great increase in spine density and dendritic elaboration can be seen between the 10-day-old and 30-day-old rats. However, when experimental and
227 TABLE 1II MEAN BRAIN WEIGHTS AND PROPORTIONS OF H1PPOCAMPUS~ CEREBELLUM AND NEOCORTEX EXPRESSED AS PERCENTAGE OF TOTAL BRAIN
Percentages at 13, 30, and 90 days are means from data of Table II.
Animal
Age (days)
Mean Regional proportion of total brain (%) brain weight Hippocampus Cerebellum
Neocortex
Exp. Contl.
6* 6*
0.4580 0.5472
6.3 6.0
5.1 6.1
---
Exp. Contl.
8* 8*
0.6489 0.7121
6.5 6.2
5.1 7.2
---
Exp. Contl.
10" 10"
0.7564 0.9102
5.9 4.8
7.2 8.6
---
Exp. Contl.
13 13
1.21 1.32
4.4 4.5
9.8 10.5
28.8 28.6
Exp. Contl.
14" 14"
0.8944 1.0931
5.4 4.9
10.2 11.4
---
Exp. Contl.
17" 17'
0.9871 1.2637
5.1 5.7
10.4 12.6
---
Exp. Contl.
18'* 18"*
1.340 1.500
---
11.5 12.5
---
Exp. Contl.
21 * 21 *
1.0521 1.3792
5.0 5.1
10.9 14.1
---
Exp. Contl.
30 30
1.26 1.98
5.1 6.0
14.0 12.8
27.6 25.9
Exp. Contl.
90 90
1.58 2.39
5.9 5.3
15.0 14.4
25.4 23.8
1.664 1.849
---
15.7 16.5
---
Exp. Contl.
210"** 210"**
* Percentages at 6, 10, 14, and 21 days were calculated from the data of Fish and Winick 10. ** Percentages at 18 days of age were calculated from data of Chase et al.3. *** Percentages at 210 days of age were calculated from data of Dobbing et aLL c o n t r o l r a t s o f t h e s a m e age are c o m p a r e d , p r o n o u n c e d q u a l i t a t i v e differences are n o t seen. D e n d r i t i c t h i c k n e s s d i d a p p e a r slightly r e d u c e d in 3 0 - d a y - o l d e x p e r i m e n t a l rats as c o m p a r e d t o t h e i r c o n t r o l s . S p i n e - p o o r stellate cells w e r e n o t e n c o u n t e r e d in e i t h e r e x p e r i m e n t a l o r c o n t r o l 1 0 - d a y - o l d rats, a l t h o u g h t h e y w e r e f o u n d in all c o r t i c a l cell layers in b o t h t h e e x p e r i m e n t a l a n d c o n t r o l 3 0 - d a y - o l d rats (Figs. 7 a n d 8). N o differences b e t w e e n t h e s e t w o g r o u p s are a p p a r e n t . Figs. 9, 10 a n d 11 s h o w e x a m p l e s o f P u r k i n j e cells, b a s k e t cells a n d g r a n u l e cells o f t h e c e r e b e l l u m f o r e x p e r i m e n t a l a n d c o n t r o l rats at 30 days. W h i l e d i m e n s i o n s o f t h e s e cells differ b e t w e e n e x p e r i m e n t a l a n d c o n t r o l a n i m a l s , t h e i r g e n e r a l c o n f i g u r a t i o n is similar. Q u a n t i t a t i v e m e a s u r e m e n t s o f t h e R a p i d G o l g i i m p r e g n a t e d n e u r o n s f r o m 30-
228
7
N
/
VISUAL CORTEX I 0 DAY OLD RAT - CONTROL
I 0 0 .u
/
Fig. 3. Visual cortex of 10-day,old control rat. Camera lucida drawings of representative neurons found at increasing depths from the pial surface.
f I00
J
Fig. 4. Visual cortex of 10-day-old experimental rat. Camera !ucida drawings of representative neurons found at increasing depths from the pial surface.
day-old animals reveal differences between experimental and control rats which often cannot be appreciated by visual inspection alone. In the experimental rats the thickness of the visual cortex is reduced by 11% as compared to controls. The length of the oblique and basal dendrites of the pyramidal cells of layer IIIb are not significantly affected. Since Nissl preparations reveal a uniform reduction in all cortical layers, the apical dendrites, are presumably reduced by an amount approximately equal to the
229
/ f f
VISUAL CORTEX 30
DAY OLD
RAT - CONTROL I00
Fig. 5. Visual cortex of 30-day-old control rats. Camera lucida drawings of representative neurons found at increasing depths from the pial surface.
VISUAL CORTEX 30
DAY OLD
RAT - UNDERNOURISHED
ioY. /
J
\ Fig. 6. Visual cortex of 30-day-old experimental rats. Camera lucida drawings of representative neurons found at increasing depths from the pial surface.
230
,/
\"i
I '
/
!
/
i
)/f'
SPINE-POOR STELL VISUAL CORTEX 30
,
/
\
DAY OLD
RAT-
CONTROL
/
loT.
~.
Fig. 7. Visual cortex of 30-day-old control rats. Camera lucida drawings of representative spine-poor stellate neurons found at increasing depths from the pial surface.
,,
j/
'i / /J
)
I
SPINE-POOR
STELLATES
J
~/
i\-,
VISUAL CORTEX 50
DAY OLD
RAT-
UNDERNOURISHED IO0 ,u
Fig. 8. Visual cortex of 30-day-old experimental rats. Camera lueida drawings of representative spinepoor stellate neurons found at increasing depths from the pial surface.
231 TABLE IV QUANTITATIVE MEASURES OF RAPID GOLGI "IMPREGNATED PYRAMIDAL NEURONS OF 30-DAY-OLD RATS' LAYER I n b OCCIPITAL CORTEX
Mean
S.D.
Contl. Exp.
1.51/~m 1.34/~m Difference --11%
0.056 0.084
5* 7*
Apical terminal branches Contl. Exp.
0.42 spines//~m 0.35 spines//~m Difference --17 % 0.94 spines//tm 0.85 spines/#m Difference --9 % 0.68 spines/#m 0.61 spines//tm Difference --10 % 0.74 spines/#m 0.58 spines/#m Difference --22 %
0.061 0.060
9** 8**
0.087 0.058
10"* 10"*
0.146 0.046
10"* 8**
0.156 0.054
10"* 10"*
17.0 17.8
8** 6**
13.1 12.4
10'* 10'*
Cortical thickness
N
Spine density
Apical main shaft
Contl. Exp.
Oblique branch
Contl. Exp.
Basal branch
Contl. Exp.
Dendritic length
Oblique branch
Contl. Exp.
Basal branch
Contl. Exp.
71.8/tm 74.3/~m Difference + 3 % 103.7/~m 98.3 #m Difference --5 %
* Number of animals. ** Number of cells. reduction in cortical thickness. Spine density is reduced by from 9 to 22 % on the 4 kinds of dendritic processes measured (see Table IV). As in the cerebrum, the thickness of the eerebellar cortical plate is affected by the low protein diet. The molecular layer in experimental rats is 16 % smaller than in control rats, while the granular layer is not significantly reduced. Reductions in dendritic length are found in the cerebellum for all cell types measured. Since the dendritic arbors of Purkinje cells and the combined ascending and descending vertical dendrites of basket cells extend across the entire thickness of the molecular layer (see Figs. 9 and 10), they are also reduced by 16% in this dimension. The measured length of basket cell oblique branches are reduced by 16% and the length of granule cell dendrites is reduced by 19 %. Cell bodies of both granule cells and Purkinje cells are reduced in diameter by 5 %. Granule cell dendritic number is essentially the same in experimental and control rats. DISCUSSION
Observers agree that the most conspicuous effect of undernutrition is on the somatic tissues of the body and that the brain appears relatively spared. According to
232
CONTROL
UNDERNOURISHED
Fig. 9. Examples of Purkinje cells from control (left) and experimental (right) 30-day-old rats.
'1 I
/ F
/
!
I
CONTROL
UNINIIII~tlIItD
Fig. 10. Examples of basket cells from control (left) and experimental (right) 30-day-old rats. Jackson la, it affects the growth of the spinal cord less than the brain, and the growth of the eye least of all. It is because of the disproportionately greater effect on the body size than on brain size that the undernourished rat resembles younger controls in outward appearance more than age-matched controls. Eayrs and H o r n 9 noted that when this effect was expressed in terms of the brain-body weight ratio, their experimental rats could be matched with younger controls. While our experimental rats from 60 days
233 onward can be matched in their brain-body weight ratio with younger controls, this ratio in experimental rats between 5 days and 30 days of age is actually larger than in any postnatal controls. In Fig. 2, where mean brain weights are plotted against log mean body weights, the curve described by experimental animals is clearly different from that of controls. Unlike control rats, the growth pattern of experimental rats is not linear on loglinear co-ordinates. Below 40 g body weight experimental rats have larger brains for a given body weight than controls. Above 100 g the reverse is true. This supports the early observation of Sugita~5 that young undernourished rats have brains somewhat larger than controls at any given body weight, and the more recent observations of Dobbing and Sands 6 that, at older ages, undernourished rats have smaller brains for a given body weight than do controls. Although in relative proportion of brain weight to body weight, undernourished rats may resemble younger controls, this resemblance does not hold for the relative proportions of the various regions within the brain. Certain brain regions show age related changes in their relative proportions. Donaldson and Hatai 7 found that in the albino rat the percent of the total brain weight which is cerebellum increased with age, and that following an initial early postnatal increase the relative proportion of forebrain decreased with age. Smithz2 likewise found an initial increase in the relative proportion of neocortex followed by a decrease to 3 years of age. Our own data show a progressive decrease in proportion of neocortex for both undernourished and control rats from 13 to 30 to 90 days of age. Our data along with calculations from the data of Fish and Winick10 show relatively constant hippocampal proportions with age, while our material, that of Fish and Winick10, Chase et al. 8 and Dobbing et al. 5 show an increase in cerebellar proportions in both experimental and control groups with increasing age. These increasing and decreasing proportional trends in the percentages of whole brain made up by neocortex, hippocampus, and cerebellum appear rather constant, despite weight difference among the different strains of rats used in the above investigations, and despite diet-induced weight differences within a given strain (see Table III). Thus, the age-related changes in neocortical, hippocampal, and cerebellar proportions appear to occur on schedule in experimental rats and support the contention of Dobbing and Sands6 that developmental timetables are not affected by undernutrition though extent of development may be. Of their material, they state t h a t . . . 'those aspects of brain growth which have been examined are organized as part of the growth program to occur at a fixed chronological age, in spite of the experimental manipulation of growth rate'. This statement may also be made in reference to our material of Rapid Golgi impregnated pyramidal neurons of the visual cortex in which the 10-day-old and 30day-old experimental rats closely resemble their respective controls. This is true despite a measured decrease in cortical thickness and spine density in 30-day-old experimental rats, and despite the fact that a 30-day-old experimental rat is closest in brain weight to a 16-day-old control. According to the Golgi-Cox measures of Eayrs and Goodheads, pyramidal cell dendrites in layer V should more than double in branching between 16 and 30 days of age. Yet from examination of Figs. 3-6, den-
234 dritic branching is similar for 30-day-old experimental and control rats. It can be seen from these figures that the pyramidal neurons of the visual cortex in both experimental and control rats undergo unmistakable changes between 10 and 30 days. Spine numbers increase many fold and there is a great elaboration of dendritic processes. Similar dendritic patterns are seen for similar cell types found at the same relative cortical depths. This is also true of the spine-poor stellate cells. Although these cells were not recognized in the Rapid Golgi preparations of I 0-day-old rats, at 30 days of age spinepoor stellate cells of similar configuration were found in layers II-VI in both experimental and control rats (Figs. 7 and 8). In the present study, spine density counts show a decrease of 9 to 22 % at the 4 counting sites on layer IIIb pyramidal cells of visual cortex in experimental rats (Table IV). These findings are consistent with the findings of Cragg 4. Although Cragg TABLE V QUANTITATIVE MEASURES OF RAPID GOLGI IMPREGNATED CEREBELLAR NEURONS OF 30-DAY-OLD RATS
Mean
S.D.
N
Cerebellar cortical thickness
Molecular (and Purkinje) layer Contl. Exp. Granular layer
Contl. Exp.
219/~m 183/tm Difference --16 % 150/~m 143 ktm Difference --5 %
9.02 8.36
5* 4*
8.72 4.06
5* 4*
0.059 0.072
10"* 10"*
Basket cells
Spine density
Contl. Exp.
Length of oblique dendrite
Contl. Exp.
0.26 spines/4 0.21 spines/4 Difference - - 19 % 100.3/zm 84.0/~m Difference --16
29.2 21.3
t0'* 10"*
Purkinje cells
(same as molecular layer, see above) Contl. 27.4 ktm 3.39 Exp. 25.9/~m 2.0l Difference --5
40** 40**
Dendritic number
Contl. Exp.
0.543 0.524
40** 40**
Dendritic length
Contl. Exp.
4.40 3.90
30** 30**
Cell body diameter
Contl. Exp.
0.841 0.651
30** 30**
Vertical dendritic length Cell body diameter
Granule cells
* Number of animals. ** Number of cells.
3.59 3.56 Difference --1% 22.3/~m 17.9 #m Difference --19 % 8.67 #m 8.22/~m Difference --5 %
235 found no significant difference in the size and density of presynaptic endings seen in electron microscopical preparations of visual cortex in undernourished rats, he did find a greater cell packing density in the undernourished rats. From this he calculated that there were fewer presynaptic endings per neuron in the cortex of the undernourished rats. More recently, Salas et al. z° in a Rapid Golgi study have also reported a reduced spine density on layer V pyramids in frontal and occipital areas of undernourished rats. Dendritic length reduction, when it occurs, is selective. Since the cortical plate is reduced in undernourished rats by 11 ~o and since in Nissl preparations all cortical layers appear uniformly reduced, apical dendritic length would be similarly reduced. Measures of oblique and basal dendritic length however, show no reduction. Our visual impression of generally reduced dendritic thickness agrees with the measures of Salas et al. ~°. The only measure of dendritic number in our study, that of cerebellar granule cells, showed essentially no difference between experimental and control animals (Table V). Our data do not reveal the differentially greater reduction in cerebellar size seen in the data of other investigators (see Table III). There are, however, differential effects of the low protein diet on individual neurons within the cerebellum. The vertical length of Purkinje cell dendrites is reduced by 16 ~, the length of oblique dendrites of basket cells is reduced by 16 ~, and the length of granule cell dendrites is reduced by 19 ~. Purkinje and granule cell bodies, on the other hand, are reduced in diameter only by 5 ~. Likewise, the width reduction of the cerebellar cortical plate is significantly higher for the molecular layer, which is rich in axons and dendrites and poor in neuron cell bodies, than it is in the cell body dense granule cell layer (Table V, Fig. 11). PIA
MOLECULAR LAYER
6RANULAR LAYER
ALBA CONTROL
UNDERNOURISHED
Fig. 11. Granule cells in relation to granular and molecular layers of cerebellar cortex in control (left) and experimental (right) 30-day-oldrats.
236 A l t h o u g h there are n e u r o a n a t o m i c a l differences between the e x p e r i m e n t a l a n d c o n t r o l rats in this study, it is not clear w h a t the consequences o f these differences are. F r o m the clinical studies of children recently reviewed by L a t h a m TM, it has n o t been resolved that p r o t e i n - c a l o r i e m a l n u t r i t i o n w i t h o u t c o n c o m i t a n t social d e p r i v a t i o n results in irreversible intellectual i m p a i r m e n t . Similarly, Slob et al. ~1 f o u n d t h a t early u n d e r n u t r i t i o n in the rat h a d no effect on tests o f m o t o r c o - o r d i n a t i o n , open field behavior, or learning when it was p r o d u c e d w i t h o u t o v e r c r o w d i n g o f litters or s e p a r a t i n g pups f r o m a d u l t females. In rats f r o m our own b r e e d i n g p o p u l a t i o n , M c K e a r n e y a n d Stitzer 17 f o u n d no difference in sensory d i s c r i m i n a t i o n or schedule cont r o l l e d behavior. Both M c K e a r n e y a n d Stitzer 17 and Slob et al. 21 described their e x p e r i m e n t a l animals as m o r e active t h a n controls. Similarly, H e d l e y - W h y t e a n d M e u s e r 11, despite evidence o f r e d u c e d m y e l i n a t i o n in the sciatic nerve, described the u n d e r n o u r i s h e d rats used in their electron m i c r o s c o p i c a l study as 'very lively'. W h e t h e r or n o t the n e u r o a n a t o m i c a l differences f o u n d in this study are in some w a y d e t r i m e n t a l to the b e h a v i o r a l o r h o m e o s t a t i c f u n c t i o n i n g o f the a n i m a l has yet to be d e t e r m i n e d . ACKNOWLEDGEMENTS
S u p p o r t e d in p a r t by G r a n t s N . I . C . H . D . 06364 a n d N o . 04-147-04.
REFERENCES 1 BAUMAN,M. L., AND KEMPER, T. L., Curtailed histoanatomic development of the brain in phenylketonuria, J. Neuropath. exp. Neurol., 30 (1974) 181. 2 BIEL, W. C., The effect of early inanition on a developmental schedule in the albino rat, J. comp. Psychol., 28 (1939) 1-15. 3 CHASE,H. P., LINDSEY,W. F. B., AND O'BRIEN, D., Undernutrition and cerebellar development, Nature (Lend.), 221 (1969) 554-555. 4 CRAGG,B. G., The development of cortical synapses during starvation in the rat, Brain, 95 (1972)
143-150. 5 DOBBING,J., HOPEWELL,J. W., AND LYNCH, A., Vulnerability of developing brain. VII. Permanent deficit of neurons in cerebral and cerebellar cortex following early mild undernutrition, Exp. Neurok, 32 (1971) 439-2147. 6 DOBBING,J., AND SANDS,J., Vulnerability of developing brain. IX. The effect of nutrition growth retardation on the timing of the brain growth-spurt, BioL Neonat. (Basel), 19 (1971) 363-378. 7 DONALDSON,H. H., AND HATAI, S., On the weight of the parts of the brain and on the percentage
8
9 10 11 12
of water in them according to brain weight and to age, in albino and in wild Norway rats, J. comp. Neurol., 53 (1931) 263-307. EAYRS, J. T., AND GOODHEAD, B., Postnatal development of the cerebral cortex in the rat, J. Anat. (Lend.), 93 (1955) 385--402. EAYRS,J. T., AND HORN, G., The development of cerebral cortex in hypothyroid and starved rats, Anat. Rec., 121 (1955) 53-61. FlSH, I., AND WINICK,M., Effect of malnutrition on regional growth of the developing rat brain, Exp. NeuroL, 25 (1969) 534-540. HEDLEY-WHYTE,E. T., AND MEUSER, C. S., The effect of undernutrition on myelination of rat sciatic nerve, Lab. Invest., 24 (1971) 156-161. HUTTENLOCHER,P. R., Dendritic development in neocortex of children with mental defect and infantile spasms, Neurology (Minneap.), 24 (1974) 203-210.
237
13 JACKSON,C. M., Changes in the relative weights of the various parts, systems and organs of young 14
15 16 17 18 19
20 21 22 23
24 25
albino rats held at a constant body weight by underfeeding for various periods, J. exp. ZooL, 19 (1915) 99-106. KEMPER, T. L., CAVENESS,W. F., AND YAKOVLEV,P. I., The neuronographic and metric study of dendritic arbours of neurones in the motor cortex of Macaca mulatta at birth and 24 months of age, Brain, 96 (1973) 765-782. KEMPER, T. L., LECOURS,A. R., GATES, M. J., AND YAKOVLEV,P. I., Delayed maturation of the brain in the congenital rubella encephalopathy, Res. Publ. Ass. nerv. ment. Dis., 51 (1972) 23-61. LATHAM,M. C., Protein-calorie malnutrition in children and its relation to psychological development and behavior, PhysioL Rev., 54 (1974) 541-565. McKEARNEY, J., AND STITZER, M., Unpublished. MILLER, S., Nutrition in the neonatal development of protein metabolism, Fed. Proc., 29 (1970) 1497-1502. POGOCAR, S., DYCKMAN, J., AND KEMPER, T. L., Neuropathology of the Rubinstein-Taybi syndrome, J. Neuropath. exp. Neurol., 34 (1975) 110. SALAS,i . , DIAZ, S., AND NIETO, A., Effects of neonatal food deprivation on cortical spines and dendritic development of the rat, Brain Research, 23 (1974) 139-144. SLOB,A. K., SNOW, C. E., AND NATRIS-MATHOT,E., Absence of behavioral deficits following neonatal undernutrition in the rat, Develop. PsychobioL, 6 (1973) 177-186. SMITH,C. G., The volume of the neocortex of the albino rat and the changes it undergoes with age after birth, J. comp. NeuroL, 60 (1934) 319-347. STERN, W., FORBES, W. B., RESNICK, O., AND MORGANE, P. J., Seizure susceptibility and brain amine levels following protein malnutrition during development in the rat, Brain Research, 89 (1974) 375-384. 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 (1918) 495-509. SUGITA,N., Comparative studies on the growth of the cerebral cortex. VII. On the influence of starvation at an early age upon the development of the cerebral cortex. Albino rat, J. comp. NeuroL, 29 (1918) 199-240.
221
Research Reports
THE EFFECT O F A LOW PROTEIN DIET ON THE ANATOMICAL DEVELOPMENT OF THE RAT BRAIN
CHRISTOPHER D. WEST ANt) THOMAS L. KEMPER Harvard Medical School, Department of Neurology and Worcester Foundation for Experimental Biology, Boston, Mass. (U.S.A.)
(Accepted September24th, 1975)
SUMMARY Albino rats were conceived and suckled by mothers maintained on a 8 % or an isocaloric 25 % casein diet. After weaning, pups were maintained on their respective diets ad libitum. By most parameters of CNS maturation, rats on the low protein diet closely resembled their age-matched controls. Only by the parameter of the ratio of brain weight to body weight did they resemble rats of a younger age. Camera lucida drawings of comparable Rapid Golgi-impregnated cortical neurons of 10-day-old control and experimental rats were nearly identical to each other. A similar finding was also noted in 30-day-old rats. However, with quantitative studies, the 30-day-old experimental animals showed a decrease in synaptic spine density and reduced dendritic length for some but not all dendritic processes. At all ages, experimental animals closely resembled age matched controls in the proportion of their brain weight that was neocortex, archicortex, and cerebellum. In these results we concur with Dobbing and Sands e that developmental timetables for the brain are not affected by undernutrition, though extent of development may be.
INTRODUCTION Neuroanatomical indices of maturation such as myelination, cell packing density, Nissl substance differentiation, dendritic elaboration and spine density have been found to be retarded or curtailed in certain conditions of mental retardation in man 1, 12,15,19. Although mental retardation in man or its possible analogues in rats has not been conclusively demonstrated as an effect of malnutrition per sele, 21, the onset of
222
Fig. 1. The effect of the 25 % and 8 3/00protein diets on the brain size and on the body size of the rat at 30 days of age.
certain developmental milestones such as eye opening, ear opening, auditory and righting reflexes have been found to be delayed in undernourished rats2, 21. It has been well established in rats and other species that early undernutrition retards the growth of the body more than the growth of the brain z~ (see Fig. 1). As noted by Eayrs and Horn 9, the brain-body weight ratio of an undernourished animal at certain ages resembles that of a younger control animal. To what extent this holds true for other criteria of growth and maturation is the subject of this investigation. The proportions of the total brain which are forebrain, neocortex, cerebellum, and other structures have been shown to change with age in the albino rat 7,22,24 as has the dendritic elaboration of cortical neurons s. This study proposes to examine these 3 levels of development in rats raised on low and high protein diets; (1) brain weight and body weight, (2) relative proportions of the brain which are neocortex, hippocampus and cerebellum, and (3) development of individual cerebral cortical and cerebellar neurons. In the majority of morphological studies reported by others, undernutrition has been confined to the period of lactation and has been produced by increasing litter size or by removing the pups from the dam for part of the day. In the present paradigm, experimental rats were conceived, suckled, and weaned in the presence of a low protein diet, a situation similar to that of humans in certain parts of the world.
223 MATERIALS A N D METHODS
Virgin female Charles River CD (outbred albino) Sprague-Dawley descended rats were placed on either an 8 % or an isocaloric 25 % casein diet 5 weeks prior to conception* and were maintained on these diets during gestation and lactation. It has been shown by Miller is that the quality of milk produced by mothers on the 8 % casein diet is the same as the quality of milk produced by mothers on the 25 % casein diet, but the quantity of milk is reduced. In this experimental model, protein availability rather than caloric restriction is considered to be the limiting factor in pup growth. Both experimental and control litters were randomized and adjusted at birth to a total of 8 pups. The pups were left undisturbed until weaning at 21 days. They were then segregated by sex (4 to a cage), maintained ad libitum on their respective diets, and left undisturbed until needed for experimental observations. The composition of the experimental and control diets, which were developed by Dr. S. Miller of the Massachusetts Institute of Technology, has been compared with laboratory chow by Stern et al. la (see Table I). For neuroanatomical study the rats were weighed, anesthetized with ether, and perfused with either Karnovsky's solution or 10 % formalin. The brains were allowed to harden overnight in situ; the following day they were weighed and their volume was determined. Fixed brains from experimental and control rats at 13, 30 and 90
TABLE I SUMMARY OF COMPOSITION OF DIETS
The percentages, by weight, of the components of the laboratory chow (pellet form, formula 4RF, Charles River Co., Wilmington, Mass.), 25%, and 8% casein (dry mash; made by General Biochemicals, Chagrin Falls, Ohio) diets are listed below. Approximately 80% of the protein of the laboratory chow was metabolically usable. Casein consisted of 87% protein (fully utilizable), 1.5 % fat and ll.5 % water. Thus, the amounts of utilizable protein in the laboratory chow and the 25 % casein diets were comparable. The casein diets contained a supplement of L-methionine (0.4%) since casein lacks this amino acid. Per cent nutritional composition Normal protein diet
Protein Fat Carbohydrate Salt mix Vitamin mix Water Non-nutritive filler
25 % Casein
Laboratory chow
21.8 15.4 50.9 4.7 1.0 2.2 4.2
26.3 7.1 42.9 3.5 0.3 9.6 10.3
Low protein diet 8 % Casein
7.0 15.1 67.4 4.7 1.0 0.9 4.2
* Thirteen-day-old experimental rats found in Tables II and III and all 13-day-old controls were born of dams placed on diets 2 weeks prior to conception.
224
postnatal days were embedded whole in an albumin gelatin and cut in gapless serial frozen sections at a thickness of 30 #m in either coronal, sagittal or horizontal plane of section. Every 10th section was stained with cresyl violet for nerve cells and glial nuclei, and the adjacent section was stained with modified Loyez stain for myelinated fibers. From the series of mounted Nissl stained sections, every second or third slide throughout the entire brain was projected onto a flat surface with a fixed magnification projector. The cross-sectional area of the neocortex, hippocampus, cerebellum and whole brain was measured with a compensating polar planimeter. The proportion of total brain tissue in each of these 3 regions was calculated by dividing the total of the cross-sectional area measurements for each region by the total of the crosssectional area measured for the whole brain. Weight estimates for neocortex, hippocampus, and cerebellum could be calculated by multiplying the wet weight of the whole brain by the proportion - - or percentage - - of total brain comprised by each region. These proportions and the means of these proportions are recorded in Tables II and II1 for ages 13, 30 and 90 days. Also recorded in Table Ili are the proportions of hippocampus and cerebellum calculated from means of regional weights and whole brain weights found in the literature for ages 6, 8, 10, 14, 17, 18, 21 and 210 days 3,sa°. Linear shrinkage in preparing Nissl sections was essentially the same for both experimental and control brains. The length and width of the cerebra and width of the cerebellum were reduced on the mounted sections an average of 18.2~ in experimental animals and an average of 18.9"/,, in controls. In Fig. 2, 7 points on the graph are the mean weights of perfusion fixed tissue. All the other points on this graph are mean weights of fresh tissue. For the study of individual neurons in the cerebral and cerebellar cortex an additional series of experimental and control rats at 10 and 30 days of age were sacrificed and perfused using the same procedure described above. Blocks of visual cortex and of cerebellum were post-fixed in Rapid Golgi solution and silvered with 0.75 ~; AgNOa. These were embedded in low viscosity nitrocellulose and cut in serial section at a thickness of 150 /tm. From the mounted histological sections representative neurons at varying depths within the visual cortex of experimental and control rats were drawn at a magnification of 400 x with the aid of a camera lucida. Drawings were similarly made of Purkinje cells, stellate cells and granule cells of the cerebellum of the 30-day-old rats at 1000 ×. in sampling neurons for camera lucida drawings from the various layers of visual cortex in the 10-day-old rats, all cells drawn for both the undernourished and control animals, respectively, lay on single sections. From the 30-day-old rats, drawings were limited as much as possible to single sections containing well impregnated neurons at several cortical depths. Although this procedure minimized neuron sampling bias, it did not control for individual variation or slight differences in age (Figs. 3 and 4). In the 30-day-old control and experimental rats, quantitative measurements were also made on readily identifiable cell types (Tables IV and V, and Figs. 9-11). For these a greater number of animals were sampled. In the visual cortex the lengths of basal and oblique dendrites and the spine density on the main apical dendritic shaft, on its terminal ramifications, and on the mid-part of the basal and oblique
225 dendrites of layer IIIb pyramidal neurons were measured. In the cerebellum, the dendritic length and cell body diameter of the granule cells were measured in the rostral bank of the primary fissure. The dendritic length and spine density of the less numerous basket cells were measured throughout the cerebellar vermis. The diameters of Purkinje cell bodies and the number of dendrites on granule cells were measmed throughout the cerebellum. Measurements were made with an ocular reticle that was calibrated with a stage micrometer after the methods of Kemper et al. 14. Dendritic lengths in the plane of section were measured directly. Those not in the plane of section were estimated by assuming that the dendrite could be represented as the hypotenuse of the right triangle formed by the measured distance between the root of the dendrite and its tip and the measured difference in depth between the two. Spine density measures were made on dendrites that were within one focal plane by counting the number of spine profiles falling between two points on the ocular grid. Cortical plate thickness measures were made from Rapid Golgi sections cut parallel to apical dendritic shafts of the pyramidal cells in the occipital cortex, and parallel to Purkinje dendritic trees in the cerebellar cortex. RESULTS
The mean brain weights and the log mean body weights of experimental and control rats of various ages are graphed in Fig. 2. At one postnatal day there is no significant difference between experimental and control rats in brain weight, body weight or brain-body weight ratio. From 11 days on, both brain weight and body weight are smaller in experimental rats than in controls, and the brain-body weight ratios of experimental rats are greater than in controls. When brain weight is plotted against log body weight, the locus of points of control rats appears linear. In contrast,
RELATIONSHIP OF BRAIN WEIGHT TO BODY WEIGHT
2.5
A
2.0 ~6
n,. z
0
0
1.5
II
AA a o
1.0
o Q
o
z
,,*
o
0.5
o A
5
I0
20
50
I00
LOG BODY WEIGHT IN GRAMS
200
500
= CONTROL o UNDERNOURISHED
Fig. 2. The relationship of mean brain weight plotted against log mean body weight for rats on 8 % casein and 25% casein diets. Open circles, experimental animals; open triangles, control animals.
226 TABLE 11 BRAIN WEIGHTS AND
PROPORTIONS OF HIPPOCAMPUS, CEREBELLUM AND NEOCORTEX EXPRESSED AS
PERCENTAGE OF WHOLE BRAIN
Animal
Age (days)
Brain weight
Regional proportion o f total brain (%) ............................. Hippocampus Cerebellum Neoeortex Prepyriform cortex, entorhinal cortex and neocortex
Exp.-3 Exp.-I Exp.-2 Exp.-6 Contl.-4 Contl.-5 Contl.-7 Contl.-1
13 13 13 13 13 13 13 13
1.02 1.27 1.28 1.28 1.29 1.29 1.35 1.36
5.5 3.2 4.6 4.6 3.4 5.1 5.3 3.6
11.4 10.0 9.2 8.8 10.0 11.9 11.4 8.8
28.0 29.1 26.3 31.8 29.1 26.9 29.1 29.4
40.0 38.8 34.6 39.0 34.7 35.4 37.7 38.0
Exp.-39 Exp.-38 Contl.-41 Contl.-37
30 30 30 30
1.19 1.34 1.97 1.98
4.7 5.5 6.5 5.6
14.5 13.5 12.8 12.9
26.8 28.3 25.4 26.4
33.9 35.5 36.5 36.4
Exp.-49 Exp.-47 Contl.-50 Contl.-45
90 90 90 90
1.48 1.69 2.27 2.51
5.8 6.0 5.5 5.1
14.0 16.0 14.5 14.3
26.3 24.5 23.2 24.4
34.0 32.4 33.5 32.6
that of experimental rats appears to describe a monotonic increasing, negatively accelerating curve. Under 40 g of body weight, experimental rats have slightly larger brains for their body size than controls; over 100 g of body weight, the reverse is true. The relative proportions of individual brains which are neocortex, hippocampus, and cerebellum in experimental and control rats at 3 ages, are found in Table II. The means of these data and proportions calculated from mean regional and mean whole brain weights found in Fish and Winick 10, Chase et al. 3 and Dobbing et al. ~, are found in Table III. In both experimental and control rats, similar tendencies are noted. The cerebellar percentage of total brain size increases with increasing age. The hippocampal percentage appears constant, while there is a decrease in neocortical percentage. Although the neocortical percentage appears somewhat larger in the experimental animals, a pooled measure of neocortex, entorhinal cortex, and prepyriform cortex showed no consistent difference (Table II). Rapid Golgi drawings of representative cortical neurons found at increasing depths from the pial surface of visual cortex from 10-day-old experimental and control rats are illustrated in Figs. 3 and 4. Representative neurons similarly arranged from Rapid Golgi preparations of 30-day-old experimental and control rats are found in Figs. 5 and 6. A great increase in spine density and dendritic elaboration can be seen between the 10-day-old and 30-day-old rats. However, when experimental and
227 TABLE 1II MEAN BRAIN WEIGHTS AND PROPORTIONS OF H1PPOCAMPUS~ CEREBELLUM AND NEOCORTEX EXPRESSED AS PERCENTAGE OF TOTAL BRAIN
Percentages at 13, 30, and 90 days are means from data of Table II.
Animal
Age (days)
Mean Regional proportion of total brain (%) brain weight Hippocampus Cerebellum
Neocortex
Exp. Contl.
6* 6*
0.4580 0.5472
6.3 6.0
5.1 6.1
---
Exp. Contl.
8* 8*
0.6489 0.7121
6.5 6.2
5.1 7.2
---
Exp. Contl.
10" 10"
0.7564 0.9102
5.9 4.8
7.2 8.6
---
Exp. Contl.
13 13
1.21 1.32
4.4 4.5
9.8 10.5
28.8 28.6
Exp. Contl.
14" 14"
0.8944 1.0931
5.4 4.9
10.2 11.4
---
Exp. Contl.
17" 17'
0.9871 1.2637
5.1 5.7
10.4 12.6
---
Exp. Contl.
18'* 18"*
1.340 1.500
---
11.5 12.5
---
Exp. Contl.
21 * 21 *
1.0521 1.3792
5.0 5.1
10.9 14.1
---
Exp. Contl.
30 30
1.26 1.98
5.1 6.0
14.0 12.8
27.6 25.9
Exp. Contl.
90 90
1.58 2.39
5.9 5.3
15.0 14.4
25.4 23.8
1.664 1.849
---
15.7 16.5
---
Exp. Contl.
210"** 210"**
* Percentages at 6, 10, 14, and 21 days were calculated from the data of Fish and Winick 10. ** Percentages at 18 days of age were calculated from data of Chase et al.3. *** Percentages at 210 days of age were calculated from data of Dobbing et aLL c o n t r o l r a t s o f t h e s a m e age are c o m p a r e d , p r o n o u n c e d q u a l i t a t i v e differences are n o t seen. D e n d r i t i c t h i c k n e s s d i d a p p e a r slightly r e d u c e d in 3 0 - d a y - o l d e x p e r i m e n t a l rats as c o m p a r e d t o t h e i r c o n t r o l s . S p i n e - p o o r stellate cells w e r e n o t e n c o u n t e r e d in e i t h e r e x p e r i m e n t a l o r c o n t r o l 1 0 - d a y - o l d rats, a l t h o u g h t h e y w e r e f o u n d in all c o r t i c a l cell layers in b o t h t h e e x p e r i m e n t a l a n d c o n t r o l 3 0 - d a y - o l d rats (Figs. 7 a n d 8). N o differences b e t w e e n t h e s e t w o g r o u p s are a p p a r e n t . Figs. 9, 10 a n d 11 s h o w e x a m p l e s o f P u r k i n j e cells, b a s k e t cells a n d g r a n u l e cells o f t h e c e r e b e l l u m f o r e x p e r i m e n t a l a n d c o n t r o l rats at 30 days. W h i l e d i m e n s i o n s o f t h e s e cells differ b e t w e e n e x p e r i m e n t a l a n d c o n t r o l a n i m a l s , t h e i r g e n e r a l c o n f i g u r a t i o n is similar. Q u a n t i t a t i v e m e a s u r e m e n t s o f t h e R a p i d G o l g i i m p r e g n a t e d n e u r o n s f r o m 30-
228
7
N
/
VISUAL CORTEX I 0 DAY OLD RAT - CONTROL
I 0 0 .u
/
Fig. 3. Visual cortex of 10-day,old control rat. Camera lucida drawings of representative neurons found at increasing depths from the pial surface.
f I00
J
Fig. 4. Visual cortex of 10-day-old experimental rat. Camera !ucida drawings of representative neurons found at increasing depths from the pial surface.
day-old animals reveal differences between experimental and control rats which often cannot be appreciated by visual inspection alone. In the experimental rats the thickness of the visual cortex is reduced by 11% as compared to controls. The length of the oblique and basal dendrites of the pyramidal cells of layer IIIb are not significantly affected. Since Nissl preparations reveal a uniform reduction in all cortical layers, the apical dendrites, are presumably reduced by an amount approximately equal to the
229
/ f f
VISUAL CORTEX 30
DAY OLD
RAT - CONTROL I00
Fig. 5. Visual cortex of 30-day-old control rats. Camera lucida drawings of representative neurons found at increasing depths from the pial surface.
VISUAL CORTEX 30
DAY OLD
RAT - UNDERNOURISHED
ioY. /
J
\ Fig. 6. Visual cortex of 30-day-old experimental rats. Camera lucida drawings of representative neurons found at increasing depths from the pial surface.
230
,/
\"i
I '
/
!
/
i
)/f'
SPINE-POOR STELL VISUAL CORTEX 30
,
/
\
DAY OLD
RAT-
CONTROL
/
loT.
~.
Fig. 7. Visual cortex of 30-day-old control rats. Camera lucida drawings of representative spine-poor stellate neurons found at increasing depths from the pial surface.
,,
j/
'i / /J
)
I
SPINE-POOR
STELLATES
J
~/
i\-,
VISUAL CORTEX 50
DAY OLD
RAT-
UNDERNOURISHED IO0 ,u
Fig. 8. Visual cortex of 30-day-old experimental rats. Camera lueida drawings of representative spinepoor stellate neurons found at increasing depths from the pial surface.
231 TABLE IV QUANTITATIVE MEASURES OF RAPID GOLGI "IMPREGNATED PYRAMIDAL NEURONS OF 30-DAY-OLD RATS' LAYER I n b OCCIPITAL CORTEX
Mean
S.D.
Contl. Exp.
1.51/~m 1.34/~m Difference --11%
0.056 0.084
5* 7*
Apical terminal branches Contl. Exp.
0.42 spines//~m 0.35 spines//~m Difference --17 % 0.94 spines//tm 0.85 spines/#m Difference --9 % 0.68 spines/#m 0.61 spines//tm Difference --10 % 0.74 spines/#m 0.58 spines/#m Difference --22 %
0.061 0.060
9** 8**
0.087 0.058
10"* 10"*
0.146 0.046
10"* 8**
0.156 0.054
10"* 10"*
17.0 17.8
8** 6**
13.1 12.4
10'* 10'*
Cortical thickness
N
Spine density
Apical main shaft
Contl. Exp.
Oblique branch
Contl. Exp.
Basal branch
Contl. Exp.
Dendritic length
Oblique branch
Contl. Exp.
Basal branch
Contl. Exp.
71.8/tm 74.3/~m Difference + 3 % 103.7/~m 98.3 #m Difference --5 %
* Number of animals. ** Number of cells. reduction in cortical thickness. Spine density is reduced by from 9 to 22 % on the 4 kinds of dendritic processes measured (see Table IV). As in the cerebrum, the thickness of the eerebellar cortical plate is affected by the low protein diet. The molecular layer in experimental rats is 16 % smaller than in control rats, while the granular layer is not significantly reduced. Reductions in dendritic length are found in the cerebellum for all cell types measured. Since the dendritic arbors of Purkinje cells and the combined ascending and descending vertical dendrites of basket cells extend across the entire thickness of the molecular layer (see Figs. 9 and 10), they are also reduced by 16% in this dimension. The measured length of basket cell oblique branches are reduced by 16% and the length of granule cell dendrites is reduced by 19 %. Cell bodies of both granule cells and Purkinje cells are reduced in diameter by 5 %. Granule cell dendritic number is essentially the same in experimental and control rats. DISCUSSION
Observers agree that the most conspicuous effect of undernutrition is on the somatic tissues of the body and that the brain appears relatively spared. According to
232
CONTROL
UNDERNOURISHED
Fig. 9. Examples of Purkinje cells from control (left) and experimental (right) 30-day-old rats.
'1 I
/ F
/
!
I
CONTROL
UNINIIII~tlIItD
Fig. 10. Examples of basket cells from control (left) and experimental (right) 30-day-old rats. Jackson la, it affects the growth of the spinal cord less than the brain, and the growth of the eye least of all. It is because of the disproportionately greater effect on the body size than on brain size that the undernourished rat resembles younger controls in outward appearance more than age-matched controls. Eayrs and H o r n 9 noted that when this effect was expressed in terms of the brain-body weight ratio, their experimental rats could be matched with younger controls. While our experimental rats from 60 days
233 onward can be matched in their brain-body weight ratio with younger controls, this ratio in experimental rats between 5 days and 30 days of age is actually larger than in any postnatal controls. In Fig. 2, where mean brain weights are plotted against log mean body weights, the curve described by experimental animals is clearly different from that of controls. Unlike control rats, the growth pattern of experimental rats is not linear on loglinear co-ordinates. Below 40 g body weight experimental rats have larger brains for a given body weight than controls. Above 100 g the reverse is true. This supports the early observation of Sugita~5 that young undernourished rats have brains somewhat larger than controls at any given body weight, and the more recent observations of Dobbing and Sands 6 that, at older ages, undernourished rats have smaller brains for a given body weight than do controls. Although in relative proportion of brain weight to body weight, undernourished rats may resemble younger controls, this resemblance does not hold for the relative proportions of the various regions within the brain. Certain brain regions show age related changes in their relative proportions. Donaldson and Hatai 7 found that in the albino rat the percent of the total brain weight which is cerebellum increased with age, and that following an initial early postnatal increase the relative proportion of forebrain decreased with age. Smithz2 likewise found an initial increase in the relative proportion of neocortex followed by a decrease to 3 years of age. Our own data show a progressive decrease in proportion of neocortex for both undernourished and control rats from 13 to 30 to 90 days of age. Our data along with calculations from the data of Fish and Winick10 show relatively constant hippocampal proportions with age, while our material, that of Fish and Winick10, Chase et al. 8 and Dobbing et al. 5 show an increase in cerebellar proportions in both experimental and control groups with increasing age. These increasing and decreasing proportional trends in the percentages of whole brain made up by neocortex, hippocampus, and cerebellum appear rather constant, despite weight difference among the different strains of rats used in the above investigations, and despite diet-induced weight differences within a given strain (see Table III). Thus, the age-related changes in neocortical, hippocampal, and cerebellar proportions appear to occur on schedule in experimental rats and support the contention of Dobbing and Sands6 that developmental timetables are not affected by undernutrition though extent of development may be. Of their material, they state t h a t . . . 'those aspects of brain growth which have been examined are organized as part of the growth program to occur at a fixed chronological age, in spite of the experimental manipulation of growth rate'. This statement may also be made in reference to our material of Rapid Golgi impregnated pyramidal neurons of the visual cortex in which the 10-day-old and 30day-old experimental rats closely resemble their respective controls. This is true despite a measured decrease in cortical thickness and spine density in 30-day-old experimental rats, and despite the fact that a 30-day-old experimental rat is closest in brain weight to a 16-day-old control. According to the Golgi-Cox measures of Eayrs and Goodheads, pyramidal cell dendrites in layer V should more than double in branching between 16 and 30 days of age. Yet from examination of Figs. 3-6, den-
234 dritic branching is similar for 30-day-old experimental and control rats. It can be seen from these figures that the pyramidal neurons of the visual cortex in both experimental and control rats undergo unmistakable changes between 10 and 30 days. Spine numbers increase many fold and there is a great elaboration of dendritic processes. Similar dendritic patterns are seen for similar cell types found at the same relative cortical depths. This is also true of the spine-poor stellate cells. Although these cells were not recognized in the Rapid Golgi preparations of I 0-day-old rats, at 30 days of age spinepoor stellate cells of similar configuration were found in layers II-VI in both experimental and control rats (Figs. 7 and 8). In the present study, spine density counts show a decrease of 9 to 22 % at the 4 counting sites on layer IIIb pyramidal cells of visual cortex in experimental rats (Table IV). These findings are consistent with the findings of Cragg 4. Although Cragg TABLE V QUANTITATIVE MEASURES OF RAPID GOLGI IMPREGNATED CEREBELLAR NEURONS OF 30-DAY-OLD RATS
Mean
S.D.
N
Cerebellar cortical thickness
Molecular (and Purkinje) layer Contl. Exp. Granular layer
Contl. Exp.
219/~m 183/tm Difference --16 % 150/~m 143 ktm Difference --5 %
9.02 8.36
5* 4*
8.72 4.06
5* 4*
0.059 0.072
10"* 10"*
Basket cells
Spine density
Contl. Exp.
Length of oblique dendrite
Contl. Exp.
0.26 spines/4 0.21 spines/4 Difference - - 19 % 100.3/zm 84.0/~m Difference --16
29.2 21.3
t0'* 10"*
Purkinje cells
(same as molecular layer, see above) Contl. 27.4 ktm 3.39 Exp. 25.9/~m 2.0l Difference --5
40** 40**
Dendritic number
Contl. Exp.
0.543 0.524
40** 40**
Dendritic length
Contl. Exp.
4.40 3.90
30** 30**
Cell body diameter
Contl. Exp.
0.841 0.651
30** 30**
Vertical dendritic length Cell body diameter
Granule cells
* Number of animals. ** Number of cells.
3.59 3.56 Difference --1% 22.3/~m 17.9 #m Difference --19 % 8.67 #m 8.22/~m Difference --5 %
235 found no significant difference in the size and density of presynaptic endings seen in electron microscopical preparations of visual cortex in undernourished rats, he did find a greater cell packing density in the undernourished rats. From this he calculated that there were fewer presynaptic endings per neuron in the cortex of the undernourished rats. More recently, Salas et al. z° in a Rapid Golgi study have also reported a reduced spine density on layer V pyramids in frontal and occipital areas of undernourished rats. Dendritic length reduction, when it occurs, is selective. Since the cortical plate is reduced in undernourished rats by 11 ~o and since in Nissl preparations all cortical layers appear uniformly reduced, apical dendritic length would be similarly reduced. Measures of oblique and basal dendritic length however, show no reduction. Our visual impression of generally reduced dendritic thickness agrees with the measures of Salas et al. ~°. The only measure of dendritic number in our study, that of cerebellar granule cells, showed essentially no difference between experimental and control animals (Table V). Our data do not reveal the differentially greater reduction in cerebellar size seen in the data of other investigators (see Table III). There are, however, differential effects of the low protein diet on individual neurons within the cerebellum. The vertical length of Purkinje cell dendrites is reduced by 16 ~, the length of oblique dendrites of basket cells is reduced by 16 ~, and the length of granule cell dendrites is reduced by 19 ~. Purkinje and granule cell bodies, on the other hand, are reduced in diameter only by 5 ~. Likewise, the width reduction of the cerebellar cortical plate is significantly higher for the molecular layer, which is rich in axons and dendrites and poor in neuron cell bodies, than it is in the cell body dense granule cell layer (Table V, Fig. 11). PIA
MOLECULAR LAYER
6RANULAR LAYER
ALBA CONTROL
UNDERNOURISHED
Fig. 11. Granule cells in relation to granular and molecular layers of cerebellar cortex in control (left) and experimental (right) 30-day-oldrats.
236 A l t h o u g h there are n e u r o a n a t o m i c a l differences between the e x p e r i m e n t a l a n d c o n t r o l rats in this study, it is not clear w h a t the consequences o f these differences are. F r o m the clinical studies of children recently reviewed by L a t h a m TM, it has n o t been resolved that p r o t e i n - c a l o r i e m a l n u t r i t i o n w i t h o u t c o n c o m i t a n t social d e p r i v a t i o n results in irreversible intellectual i m p a i r m e n t . Similarly, Slob et al. ~1 f o u n d t h a t early u n d e r n u t r i t i o n in the rat h a d no effect on tests o f m o t o r c o - o r d i n a t i o n , open field behavior, or learning when it was p r o d u c e d w i t h o u t o v e r c r o w d i n g o f litters or s e p a r a t i n g pups f r o m a d u l t females. In rats f r o m our own b r e e d i n g p o p u l a t i o n , M c K e a r n e y a n d Stitzer 17 f o u n d no difference in sensory d i s c r i m i n a t i o n or schedule cont r o l l e d behavior. Both M c K e a r n e y a n d Stitzer 17 and Slob et al. 21 described their e x p e r i m e n t a l animals as m o r e active t h a n controls. Similarly, H e d l e y - W h y t e a n d M e u s e r 11, despite evidence o f r e d u c e d m y e l i n a t i o n in the sciatic nerve, described the u n d e r n o u r i s h e d rats used in their electron m i c r o s c o p i c a l study as 'very lively'. W h e t h e r or n o t the n e u r o a n a t o m i c a l differences f o u n d in this study are in some w a y d e t r i m e n t a l to the b e h a v i o r a l o r h o m e o s t a t i c f u n c t i o n i n g o f the a n i m a l has yet to be d e t e r m i n e d . ACKNOWLEDGEMENTS
S u p p o r t e d in p a r t by G r a n t s N . I . C . H . D . 06364 a n d N o . 04-147-04.
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