- Email: [email protected]
Effects of prtein undernutrition on the dentate gyrus in rats of three] age groups
Brain Research, 532 (1990) 271-277 Elsevier
271
BRES 16017
Effects of protein undernutrition on the dentate gyrus in rats of three age groups L. Cintra 1, S. Diaz-Cintra 1, A. Galvfin 1, T. Kemper 2 and P.J. Morgane 3 I Department of Physiology, Instituto de lnvestigaciones Biomddicas, UNAM, Ciudad Universitaria, MOxico 04510, D.E (Mexico), 2Neurological Unit, Boston City Hospital, Boston, MA 02118 (U.S.A.) and 3Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 (U.S.A.) (Accepted 22 May 1990)
Key words: Protein malnutrition; Hippocampus; Dentate gyrus; Dentate granule cell; Dendritic development; Morphometric Golgi study; Malnutrition and hippocampal formation
The effect of an 8% casein and a control 25% casein diet on the granule cells in the dorsal blade of the dentate gyrus of the rat hippocampal formation was studied at 30, 90 and 220 days of age. Female rats were fed either an 8% or 25% casein diet 5 weeks prior to conception and the litters were maintained on these respective diets until killed. In rapid-Golgi-impregnated cells, we measured major and minor axes of the soma of the dentate granule neurons, the number of spines on 50-¢tmsegments of proximal, middle and terminal regions of the largest dendrite per granule cell and the number of dendrites intersecting 8 concentric rings 38/~m apart. At all 3 ages studied undernourished rats showed, when compared to controls, significant reductions of the major and minor axes of the somata and significant reductions in the number of spines on dendrites in the middle and terminal dendritic segments. Dendritic branching was significantly reduced in undernourished rats compared to controls in all but the 4th concentric rings, with the greatest effect being seen on the outer 3 concentric rings at 90 and 220 days of age. The location of the deficit in dendritic synaptic spines and the greatest deficit in dendritic branching correspond to the sites of termination of the lateral and medial perforant pathway projection to the dentate gyrus on the terminal and middle dendritic segments of the granule cells. The deficits noted in the granule cells of the dentate gyrus in this study were more severe than those found in our previous studies on the effect of the low protein diet in these same rats on visual cortical pyramidal cells and on the 3 cell types in the nucleus raphe dorsalis and nucleus locus coeruleus.
INTRODUCTION In the present study we have investigated the effect of a low protein diet on the postnatal development of individual granule cells in the dentate gyrus of rats at 30, 90 and 220 days of age. In order to mimic the h u m a n condition in which u n d e r n u t r i t i o n most frequently occurs, female rats were adapted to their diets prior to conception and then maintained on the same diet as dams. The granule cells were selected for several reasons. O n e of these is that they are an essential link in the hippocampal trisynaptic circuit that has been implicated as a substrate for memory 4"15'27"41. The input to the trisynaptic circuit is via the perforant pathway projection from the entorhinal cortex to the granule cell dendrites in the outer two-thirds of the molecular layer of the dentate gyrus. The granule cells then, via their mossy fiber axonal projections, complete the next limb of the circuit with their projection to the CA3 pyramidal neurons. These
neurons, in turn via their Schaffer axonal collaterals, project to the CA1 pyramidal cell n e u r o n s which complete the trisynaptic circuit. The granule cells are also of interest in that they permit us to study the effect of protein deprivation on cells generated primarily after birth 5-7"1°'36. Only 15% of the adult population of granule cells is present at the time of birth with approximately 45% of the adult population generated by the seventh postnatal day 6. These n e u r o n s have been shown to continue to be generated in the early postnatal period 2°'21 as well as throughout postnatal life of the rat 1°'38'39. In contrast the pyramidal cell n e u r o n s in the hippocampal formation are generated prior to birth in the rat 6'18'33'36'43. Further, a n u m b e r of studies have shown an effect of undernutrition on the d e v e l o p m e n t of the hippocampal formation 1'9'16'23'24"26"28"29'30'31'32'42.m comparison of two of these studies suggests that the effect of nutritional deprivation on the fascia dentata may be greater than that shown by the hippocampal complex as a whole. Fish
Correspondence: P.J. Morgane, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
272 and Winick 16 studied the effect of increased litter size
not show a deficit in the hippocampus until day 17, with
during the period of lactation on total D N A content of
deficits in D N A content of the cerebellum noted on day
various parts of the brain. This index of cell n u m b e r did
6 and in the cerebral cortex on day 14. In contrast, the
FT3 ~f'3
12
Fig. 1. A. Diagram showing the level in the rat brain in which a 4-mm block was taken, stained with the rapid-Golgi technique and then sectioned at 120/~m. The square shows the area of the dentate gyrus studied. Photomicrograph B shows the part of the dorsal blade (DB) of the dentate gyrus from where granule cells (GC) were sampled in the inferior part of the granule cell layer (GL). C: higher magnification showing spinous dentate granule cell and its apical dendrites extending into the molecular layer (ML). Perforant path (pp) axons are shown crossing the apical dendrite of granule cells, a, axon.
273
TABLE I
Effects o f pre- and postnatal undernutrition on granule cells in denate gyrus of rats at 30, 90 and 200 days old a F-values
Perikarya major axis ~um) minor axis (/tin) Dendrites number of spines per 50-/xm segments: proximal middle terminal intersections at: 38/2m 76/~m 114/~m 152ktm 190k~m 228Bm 266/~m 304/xm
Diet (dff 1,24)
Age (df2,24)
12.79 . . . . 9.70* *
3.95 *'d 4.72"*
0.17 NS 15.43"** 32.11"**
Interaction (df2,24)
Comparisons between ages 30 vs. 90 (df l,24)
90 vs. 220 (df l,24)
30 vs. 220 (df l,24)
0.31 NS 2.39 NS
4.14 NS 7.65*
0.44 NS 0.05 NS
7.26* 6.46 NS
8.17" * 6.75"* 2.12 NS
0.12 NS 0.80 NS 0.93 NS
1.17 NS 0.03 N _e
8.02* 9.52* _
13.46"* 15.40"** 7.51 ** 2.37 NS 15.00"**
2.53 NS 2.21 NS 0.13 NS 0.79 NS 2.81 NS
0.08 NS 2.21 NS 0.04 NS 0.47 NS 0.89 NS
44.32*** 90.99*** 83.49***
14.99"** 14.69"** 12.26"**
13.37"** 26.36*** 33.00***
Diet effect compared among agesf 5.84 NS 7.56 NS 31.78"** 1.31 NS 13.30"* 19.92"**
15.33"* 10.68"* _ 26.70*** 45.98*** 65.77***
Analysis of variance (ANOVA) with nutritional status and age as the two between subjects factors. Also provided are comparisons between ages (n = 10 animals/age: 5 control and 5 undernourished) where appropriate. b df = degrees of freedom. c Probability values (undernourished vs. controls): **P < 0.01, ***P < 0.001. d Probability values (across ages): *P < 0.05, **P < 0.01, ***P < 0.001. e When the main effect of age was not significant, no comparisons between age groups were made. f Analysis of variance (ANOVA) for morphometric measures showing a significant interaction effect between diet and age. The effect of diet was compared among ages by comparing the magnitude of the differences between control and undernourished animals. a
a u t o r a d i o g r a p h i c s t u d y o f L e w i s et al. 26 s u g g e s t s a m o r e s t r i k i n g e f f e c t o n t h e fascia d e n t a t a t h a n t h a t s h o w n b y t h e h i p p o c a m p u s as a w h o l e . T h e y n o t e d in rats u n d e r n o u r i s h e d f r o m t h e 6th d a y o f p r e g n a n c y until killing o n p o s t n a t a l days 1, 6 a n d 12, a p r o l o n g e d cell cycle t i m e a n d a m a r k e d l y c u r t a i l e d r a t e o f a c c u m u l a t i o n o f g r a n u l e cell n e u r o n s o n d a y s 1 a n d 6. Finally, in s e v e r a l s t u d i e s w e h a v e s h o w n s t r i k i n g a l t e r a t i o n s in n e u r o n a l plasticity o f t h e h i p p o c a m p a l f o r m a t i o n in m a l n o u r i s h e d rats as m e a s u r e d b y c h a n g e s in l o n g - t e r m p o t e n t i a t i o n a n d k i n d l i n g 2's. C h a n g e s in v a r i o u s b e h a v i o r s t h a t m e a s u r e h i p p o c a m p a l f u n c t i o n 17"25 have
also b e e n
shown following prenatal nutritional
deprivation.
MATERIALS AND METHODS
Animals The rats used for this investigation are the same rats used for our previously published studies of the effect of the 8% casein diet on selected nuclei in the brainstem ~2"~3 and the visual cortex TM.
Methods The specific details of the dietary paradigm and breeding procedures have been previously published by our group 28. To summarize, virgin female Charles River C.D., Sprague-Dawley descended rats were fed a 25% or 8% casein diet 5 weeks prior to conception and mated with males fed a standard rat chow diet. The dams remained on these diets all during gestation. Following delivery litters born the same day from each diet group were randomized and culled to 8 pups. The dams were continued on their diets during the lactation period following which the pups were weaned and then maintained on the same diets as those fed their mothers during gestation and lactation. At 30, 90 and 200 days of age a total of 30 male rats, 15 on the 25% diet and 15 on the 8% diet, were anesthetized with pentobarbital, perfused through the heart with 10% neutral buffered formalin and the brains removed the following day. Thus, we obtained 5 rats from each diet group in each of 3 age groups. In each rat a 4 mm wide block of hippocampal formation, including the dentate gyrus, was prepared using the rapid-Golgi technique (Fig. 1) following the modification of DiazCintra et al. 11. These blocks were embedded in low viscosity nitrocellulose, serially cut at a thickness of 120 pm and 5 sections from each brain containing the dentate gyrus were mounted in serial order. Each slide was assigned a random number to insure that all observations were blind with respect to age and diet. From these slides in each tissue section, one complete, well-impregnated granule cell was selected from the inferior part of the granule cell layer in the dorsal blade of the dentate gyrus. From these slides in
274
I
TERMINAL
[ 25• % casein
i
58~um
4O
~ zo
~
50 DAYS
MIDDLE I ,v- 0 i~.
~ 40 f
PROXIMAL[
u~ ao
I
2
5 4
5
6 7
8 ~
Fig. 2. Camera 1,ucida drawing of granule cell from the inferior part of the granule cell layer of the dentate gyrus of the rat showing 50 pm sectors (proximal, middle and terminal) of apical dendrites. Spine counts were made from the largest, best impregnated apical dendrite. Degree of branching of dendrites was established by the number of dendrites crossing the concentric ring.
~ ~0
~ each tissue section, one complete, well-impregnated granule cell was selected from the deep part of the granule cell layer in the dorsal blade of the dentate gyrus. A specific depth within the granule layer was selected in order to sample neurons generated at approximately the same time. The cells in the depth of this layer are late generated cells36 and were selected to provide comparable data for our ongoing study of the effect of prenatal undernutrition on postnatally generated granule cell neurons. Thus, we obtained a total of 150 dentate granule cells, 25 in each age and diet group. All measures were made by means of a 40x planapochromatic or 100x planapochromatic objective using the micrometer adjustment of the microscope in order to follow the cellular structures with a calibrated ocular reticle. Measurements of the number of dendrites crossing each concentric ring were made according to the method of Shol137, using a camera lucida by which the neuron is projected onto
~ 25% CASEIN o..-o 8% CASEIN
~.=.= -==-=~'~*~
A 2O
~ ....
E
~~ ~
I'-''-~
~ ~-. . . . . -~
MAJORAXIS
~
hi N
~ i~'
~
~.. . . . .
~
MINORAXIS
~ IO i-
I
I
50
90
~1
~
~ 40
0 PROXIMAL
MIDOLE
TERMINAL
Fig. 4. Bar graphs showing the effects of 8% and 25% casein diets on the number of dendritic spines on the proximal, middle and terminal 50-pm dendritic segments measured in the best impregnated and largest apical dendrite per granule cell. There is a significant overall decrease (ANOVA program across all 3 ages) of spines in the middle and terminal dendritic segments at all 3 ages in undernourished animals (striped bars) as compared to controls (white bars). Also see Table I for data and probability values.
an enlarged drawing of the 8 concentric rings 38 #m apart (Fig. 2). Using this procedure, the following measures were made: (1) major and minor axes of the granule cell body, (2) spine density per 50-#m segments of proximal, middle and terminal sectors of the largest dendrite within the plane of section (Fig. 2), and (3) number of interactions of dendrites crossing each of the 8 concentric rings.
Statistics Statistical analyses were conducted using the General Linear Model procedures of the SAS data analysis system 35. Analysis of the neural measures was made using a repeated-measures design with 2 between-subjects factors (diet and age) and 5 replicates (neurons) for each subject. Significant ANOVA effects of diet, age or interaction were further examined with three types of additional comparisons. The effect of diet was compared at each age between the control and undernourished rats (n = 5/diet), age comparisons were made across the age groups (n = 10/age) and the effect of diet was compared between the age groups after significant interaction. Probability values of these comparisons were adjusted upward using the Bonferroni method 34.
I
RESULTS 220
AGE(days) Fig. 3. Effects of 8% and 25% casein diets on somal size (major and minor axes) of dentate granule cells in the 3 age groups. The length of both major and minor axes shows significant overall decreases across all 3 age groups in undernourished rats (see Table I for data and probability values). Mean + S.E.M.
The results of the analysis of variance (ANOVA) shown in Table I with the measurements
are
graphically
s h o w n in Figs. 3 - 5 . T h e r e is a s i g n i f i c a n t d e c r e a s e in t h e major a n d minor axes o f t h e cell b o d y ( T a b l e I a n d Fig. 3) in t h e 8 % c a s e i n
275 30
DAYS
d2
[ ] 2 5 % cosein
[]
8 % casein
8 I~
4
E~
0
o
90
0 rO ~0 0 h.I I~
DAYS
42 8
.~_ I/~ I/~
4
9 0 ~
0
-~ -~ ¢"
.~2
2_20 DAYS
.
g 0 d
R
:5
4.
.5
6
'7
8
R i n g s C58jJm apart) Fig. 5. Effects of 8% and 25% casein diets on the dendritic branches crossing 8 concentric rings in rats 30, 90 and 220 days of age. Note the significant overall reduction of dendritic branching in undernourished animals (striped bars) in rings 1, 2, 3 and 5 at all 3 ages studied and at 90 and 220 days in rings 6, 7 and 8 compared to controls (white bars). Also see Table I for data and probability values.
diet rats at all 3 ages, with the greatest deficit in the m a j o r axis at 30 days ( - 1 4 % ) and in the minor axis at 220 days ( - 1 9 % ) . Comparisons across the ages show in both diet groups a significant increase in the m a j o r axis between 30 and 220 days and for the minor axis a significant increase b e t w e e n 30 and 90 days. T h e r e is a significant decrease in the n u m b e r of dendritic spines (Table I and Fig. 4) in the 8% casein diet rats at all 3 ages on the middle and distal dendritic segments, with the most m a r k e d effect at 30 days ( - 1 4 % and - 1 6 % , respectively). T h e r e is no significant diet effect on the proximal dendritic spines. Comparisons across the ages in both diet groups show significant increases in spine density on proximal dendritic segments between 30 and 90 days and for the middle dendritic segment between 30 and 90 days and 90 and 220 days. In each case the percent increase is greater in the 8% than in the 25% casein diet rats. The respective increases for the 8% and 25% casein diet rats for each of these 3 comparisons are: + 1 4 % and 18%, + 1 5 % and 8%, and + 1 7 % and 7%. There is no significant age effect on the
spines on the terminal dendritic segment. There is a significant decrease in dendritic branching (Table I and Fig. 5) in the 8% casein diet rats on all but the 4th concentric ring. In rings 1 through 3 and in ring 5 this is present at all 3 ages. In these rings the deficit varies from - 3 % to - 1 7 % . In the o u t e r 3 rings, rings 6 through 8, the deficit is n o t e d only at 90 and 220 days and varies from - 7 % to - 7 7 % with the most severe deficit in the outer 2 rings. In this latter location the deficit varies from - 4 6 % to - 7 7 % * . Significant age and interaction effects are confined to these o u t e r 3 rings. In these rings the 25% casein diet rats show an increase in n u m b e r of dendritic intersections while the 8% casein diet rats show a decrease. In ring 6 this is n o t e d when 30 and 220 days are c o m p a r e d , with the respective percent changes for the 8% and 25% casein diet rats o f - 2 % and + 4 4 % . In ring 7 this difference is seen when 30 and 90 and 30 and 220 days are c o m p a r e d with respective percent differences o f - 8 % , + 8 9 % ; - 2 5 % and + 8 4 % . In the 8th ring this difference is seen in all possible comparisons. The respective percents b e t w e e n 30 and 90, 90 and 220 and 30 and 220 days a r e - 3 9 % , + 5 0 % ; - 7 % , + 7 1 % ; - 4 3 % and +150%. DISCUSSION We have previously published c o m p a r a b l e data from these same rats on the effect of protein deprivation on pyramidal cells in the visual cortex t4 and on the 3 types of neurons in the nucleus r a p h e dorsalis 11 and nucleus locus coeruleus 13 in the brainstem. The effect of the low protein diet on the granule cells of the dentate gyrus was greater than that found in any of these previous studies. In the 3 studies noted a b o v e and in the present study, measurements are available for comparing the effect of 8% casein diet on cell size, dendritic complexity and density of dendritic spines. In the brainstem nuclei, taken together, there were 28 such comparisons between control and e x p e r i m e n t a l rats at each age, with a significant difference n o t e d at one or m o r e ages in 18 of these 28 comparisons. In the visual cortex the comparable numbers were 9 of 24 comparisons. In the present study all but 2 of 11 m o r p h o m e t r i c comparisons showed a significant diet effect. All significant findings in the present study indicated a deficit in the 8% casein diet rats. This was the result of the significant deficits p r e s e n t at 30 days persisting to 220 days and an increasing n u m b e r of distal dendritic intersections in the control rats at a time when experimental rats showed a decrease. T h e f o r m e r was associated with
* Ring 6 at 90 days -18% ( F 1 , 2 4 = 13.60, P < 0.01) and at 220 days -18% (F1.25 = 57.39, P < 0.001), ring 7 at 90 days -46% (F1,24 = 58.14, P < 0.001) and at 220 days -46% ( F 1 , 2 4 = 85.43, P < 0.001) and ring 8 at 90 days -57% (Fl,a4 = 23.91, P < 0.001) and at 220 days -77% (F1.24 = 125.50, P < 0.001).
276 significant deficits in cell size, n u m b e r of synaptic spines
rats at 30 days. Th e least frequently identified mecha-
on the middle and terminal dendritic segments and
nisms of effect of the low-protein diet were failure of
n u m b e r of dendritic intersections in all but one of the first
occurrence in the 25% casein diet rats of a significant
5 concentric rings. T h e latter was associated withdeficits
age-related change n o t e d in the 8 % casein diet rats and
in the n u m b e r of dendritic intersections in the o u t er 3
for significant differences n o t ed at 30 days to persist to
rings at 90 and 220 days. In contrast, in the visual cortical
220 days. These, each, were n o t e d 3 times. O f these 6
neurons and in the neurons in the brainstem nuclei, both
significant differences, 3 reflected a decreased measure-
control and experimental rats showed significant age-
ment in the 8% casein diet rats. A s can be seen (above),
related increases and decreases in m o r p h o m e t r i c mea-
in the present study only 2 of these mechanisms ac-
surements with resultant increases and decreases in these
counted for all the deficits in the 8% casein diet rats.
m e a s u r e m e n t s in the 8% casein diet rats. In these latter
Significant findings n o t ed at 30 days persisted to 220 days
studies the most frequent mechanism for a significant
and there was failure of the p r o t e i n - d e p r i v e d rats to show
difference at any one age was failure of occurrence of an
the age-related change n o t ed in controls.
age-related change n o t e d in controls to occur in the 8% casein diet rats or a less marked age-related change in the protein-deprived rats than that found in controls. Th er e were 15 examples of this which, in all but 2, resulted in a deficit in the undernourished rats. The next most frequent mechanism of effect of the low-protein diet was that a significant difference noted at 30 days did not persist to 90 days, suggesting a delayed d e v e l o p m e n t in the 8% casein diet rats. T h e r e were 12 examples of this, 8 of which r ep r es en t e d a deficit in the undernourished
REFERENCES 1 Ahmed, M.G.E., Bedi, K.S., Warren, M.A. and Kamel, M.M., Effects of a lengthy period of undernutrition from birth and subsequent nutritional rehabilitation on the synapse: granule cell neuron ratio in the rat dentate gyms, J. Comp. Neurol., 263 (1987) 146-158. 2 Austin, K.B., Bronzino, J. and Morgane, P.J., Prenatal protein malnutrition affects synaptic potentiation in the dentate gyrus of rats in adulthood, Dev. Brain Res., 29 (1986) 267-273. 3 Austin, K.B., Bronzino, J.D. and Morgane, P.J., Paired-pulse facilitation and inhibition in the dentate gyrus is dependent on behavioral state, Exp. Brain Res., 77 (1989) 594-604. 4 Barnes, C.A., Spatial learning and memory processes: the search for their morphological mechanisms in the rat, Trends Neurosci., 11 (1988) 163-169. 5 Bayer, S.A., Changes in the total number of dentate granule cells in juvenile and adult rats: a correlated volumetric and 3H-thymidine autoradiographic study, Exp. Brain Res., 46 (1982) 315-323. 6 Bayer, S.A. and Altman, J., Hippocampal development in the rat: cytogenesis and morphogenesis examined with autoradiography and low-level X-irradiation, J. Comp. Neurol., 158 (1974) 55-80. 7 Bayer, S.A., Yackel, J.W. and Puri, ES., Neurons in the rat dentate gyrus granular layer substantially increase during juvenile and adult life, Science, 216 (1982) 890-892. 8 Bronzino, J.D., Austin-LaFrance, R.J., Siok, C.J. and Morgane, P.J., Effect of protein malnutrition on hippocampal kindling: electrographic and behavioral measures, Brain Research, 384 (1986) 348-354. 9 Cordero, M.E., Zvaighaft, A., Muzzo, S. and Brunser, O., Histological maturation of astroglial cells in the archicortex of early malnourished rats, Pediatr. Res., 16 (1982) 187-191. 10 Crespo, D., Stanfield, B.B. and Cowan, W.M., Evidence that late-generated granule cells do not simply replace earlier formed neurons in the rat dentate gyrus, Exp. Brain Res., 62 (1986) 541-548.
Acknowledgements. Preliminary data was presented at the 17th Society for Neuroscience Meeting, New Orleans, LA, November, 1987. This study was supported by Grant PSCACNA-751969 and fellowships 20518 and 27234 from CONACyT Mrxico, and by Grants HD-22539 and HD-23338, NICHHD, NIH. We would like to thank technicians Juan Romero and Gabriela Padua for histological assistance. The facilities of Dr. R. O'Connell's laboratory at the Worcester Foundation for Experimental Biology related to statistical programs is appreciated. We are indebted to Dr. R. Patrick Akers for his help in the use of the SAS Program and Dr. David S. Reasner for his assistance in designing the ANOVA Programs.
11 Dfaz-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and Morgane, P.J., Nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups, Brain Research, 207 (1981) 1-16. 12 Dfaz-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and Morgane, P.J., The effects of protein deprivation on the nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups, Brain Research, 221 (1981) 243-255. 13 D/az-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and Morgane, P.J., The effects of protein deprivation on the nucleus locus coeruleus: a morphometric Golgi study in rats of three age groups, Brain Research, 304 (1984) 243-253. 14 Dfaz-Cintra, S., Cintra, L., Ortega, A., Kemper, T. and Morgane, P.J., Effects of protein deprivation on pyramidal cells of the visual cortex in rats of three age groups, J. Comp. NeuroL, 292 (1990) 117-126. 15 Eichenbaum, H. and Cohen, N.J., Representation in the hippocampus: what do hippocampal neurons code?, Trends Neurosci., 11 (1988) 244-248. 16 Fish, I. and Winick, M., Effect of malnutrition on regional growth of the developing rat brain, Exp. Neurol., 25 (1969) 534-540. 17 Goodlett, C.R., Valentino, M.L., Morgane, P.J. and Resnick, O., Spatial cue utilization in chronically malnourished rats: task-specific learning deficits, Dev. Psychobiol., 19 (1986) 1-15. 18 Hicks, S.P. and D'Amato, C.J., Cell migration to the isocortex in the rat, Anat. Rec., 160 (1968) 629-634. 19 Jordan, T.C., Howells, K.F., McNaughton, N. and Heatlie, P.L., Effects of early undernutrition on hippocampal development and function, Res. Exp. Med. (Berl.), 180 (1982) 201-207. 20 Kaplan, M.S. and Bell, D.H., Neuronal proliferation in the 9-month-old rodent - - Radioautographic study of granule cells in the hippocampus, Exp. Brain Res., 52 (1983) 1-5. 21 Kaplan, M.S. and Hinds, J.W., Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs, Science, 197 (1977) 1092-1094. 22 Katz, H.B. and Davies, C.A., The effects of early-life undernutrition and subsequent environment on morphological param-
277 eters of the rat brain, Behav. Brain Res., 5 (1982) 53-64. 23 Katz, H.B. and Davies, C.A., The separate and combined effects of early undernutrition and environmental complexity at different ages on cerebral measures in rats, Dev. Psychobiol., 16 (1983) 47-58. 24 Katz, H.B., Davies, C.A. and Dobbing, J., Effects of undernutrition at different ages early in life and later environmental complexity on parameters of the cerebrum and hippocampus in rats, J. Nutr., 112 (1982) 1362-1368. 25 Laughlin, N.K., Stanley, F. and Bell, J., Early undernutrition and later hippocampal damage: effects on spontaneous behaviors and reversal learning, Physiol. Psychol., 11 (1984) 269-277. 26 Lewis, P.D., Patel, A.J. and Bal~izs, R., Effect of undernutrition on cell generation in the rat hippocampus, Brain Research, 168 (1979) 186-189. 27 Lynch, G., Muller, D., Seubert, P. and Larson, J., Long-term potentiation: persisting problems and recent results, Brain Res. Bull., 21 (1988) 363-372. 28 Morgane, P.J., Miller, M., Kemper, T., Stern, W., Forbes, W., Hall, R., Bronzino, J., Kissane, J., 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. 29 Noback, C.R. and Eisenman, L.M., Some effect of proteincalorie undernutrition on the developing central nervous system of the rat, Anat. Rec., 201 (1981) 67-73. 30 Paula-Barbpsa, M.M., Andrade, J.P., Castedo, J.L., Azevedo, F.P., Camoes, I., Volk, B. and Tavares, M.A., Cell loss in the cerebellum and hippocampal formation of adult rats after long-term low-protein diet, Exp. Neurol., 103 (1989) 186-193. 31 Plaschke, M., Wenzei, J., Dorner, G. and Tonjes, R., Der Einfluss einer friihpostnatalen, sozialen und nutritiven Deprivation und einer gleichzeitigen Behandlung mit Pyridostigmin auf die Synaptogenese im Hippocampus der Ratte. Elektronenmikroskopische, morphometrische und stereologische Untersuchungen, J. Hirnforsch., 27 (1986) 145-158. 32 Plaschke, M., Dorner, G., Krause, E., Krause, M., Kuhlmey,
33
34 35 36
37 38
39
40
41
42
43
H.M., Schuster, T. and Wenzel, J., Untersuchungen zur Vesikelpopulation von Synapsen im Hippocampus der Ratte nach frfihpostnataler Deprivation und Verabreichung von Pyridostigmin, J. Hirnforsch., 28 (1987) 1-11. Pokorny, J. and Yamamoto, T., Postnatal ontogenesis of hippocampal CA 4 area in rats, I. Development of dendritic arborizations in pyramidal neurons, Brain Res. Bull., 7 (1981) 113-120. Ryan, T.A., Multiple comparisons in psychological research, Psychol. Bull., 56 (1959) 26-47. SAS Institute, Inc., SAS User's Guide: Statistics, Version 5 edn., SAS Institute Inc., Cary, NC, 1985. Schlessinger, A.R., Cowan, W.H. and Gottlieb, D.I., An autoradiographic study of the time and the pattern of granule cell migration in the dentate gyrus of the rat, J. Comp. Neurol., 159 (1975) 149-176. Sholl, D.A., The Organization of the Neurons of the Cerebral Cortex, Hafner, New York, 1956. Stanfield, B.B. and Cowan, W.M., The development of the hippocampal region. In A. Peters and E.G. Jones (Eds.), Development and Maturation of Cerebral Cortex, (Cerebral Cortex, Vol. 7), Plenum Press, New York, 1988, pp. 91-131. Stanfield, B.B. and Trice, J.E., Evidence that granule cells generated in dentate gyrus of adult rats extend axonal projections, Exp. Brain Res., 72 (1988) 399-406. Stewart, R.J.C., Merat, A. and Dickerson, J.W.T., Effect of a low protein diet in mother rats on the structure of the brains of the offspring, Biol. Neonate, 25 (1974) 125-134. Teyler, T.J. and DiScenna, P., The topological anatomy of the hippocampus: a clue to its function, Brain Res. Bull., 12 (1984) 711-719. West, C.D. and Kemper, T.L., The effect of a low protein diet on the anatomical development of the rat brain, Brain Research, 107 (1976) 221-237. Zimmer, J., Development of the hippocampus and fascia dentata: morphological and histochemical aspects, Prog. Brain Res., 48 (1978) 171-189.
271
BRES 16017
Effects of protein undernutrition on the dentate gyrus in rats of three age groups L. Cintra 1, S. Diaz-Cintra 1, A. Galvfin 1, T. Kemper 2 and P.J. Morgane 3 I Department of Physiology, Instituto de lnvestigaciones Biomddicas, UNAM, Ciudad Universitaria, MOxico 04510, D.E (Mexico), 2Neurological Unit, Boston City Hospital, Boston, MA 02118 (U.S.A.) and 3Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 (U.S.A.) (Accepted 22 May 1990)
Key words: Protein malnutrition; Hippocampus; Dentate gyrus; Dentate granule cell; Dendritic development; Morphometric Golgi study; Malnutrition and hippocampal formation
The effect of an 8% casein and a control 25% casein diet on the granule cells in the dorsal blade of the dentate gyrus of the rat hippocampal formation was studied at 30, 90 and 220 days of age. Female rats were fed either an 8% or 25% casein diet 5 weeks prior to conception and the litters were maintained on these respective diets until killed. In rapid-Golgi-impregnated cells, we measured major and minor axes of the soma of the dentate granule neurons, the number of spines on 50-¢tmsegments of proximal, middle and terminal regions of the largest dendrite per granule cell and the number of dendrites intersecting 8 concentric rings 38/~m apart. At all 3 ages studied undernourished rats showed, when compared to controls, significant reductions of the major and minor axes of the somata and significant reductions in the number of spines on dendrites in the middle and terminal dendritic segments. Dendritic branching was significantly reduced in undernourished rats compared to controls in all but the 4th concentric rings, with the greatest effect being seen on the outer 3 concentric rings at 90 and 220 days of age. The location of the deficit in dendritic synaptic spines and the greatest deficit in dendritic branching correspond to the sites of termination of the lateral and medial perforant pathway projection to the dentate gyrus on the terminal and middle dendritic segments of the granule cells. The deficits noted in the granule cells of the dentate gyrus in this study were more severe than those found in our previous studies on the effect of the low protein diet in these same rats on visual cortical pyramidal cells and on the 3 cell types in the nucleus raphe dorsalis and nucleus locus coeruleus.
INTRODUCTION In the present study we have investigated the effect of a low protein diet on the postnatal development of individual granule cells in the dentate gyrus of rats at 30, 90 and 220 days of age. In order to mimic the h u m a n condition in which u n d e r n u t r i t i o n most frequently occurs, female rats were adapted to their diets prior to conception and then maintained on the same diet as dams. The granule cells were selected for several reasons. O n e of these is that they are an essential link in the hippocampal trisynaptic circuit that has been implicated as a substrate for memory 4"15'27"41. The input to the trisynaptic circuit is via the perforant pathway projection from the entorhinal cortex to the granule cell dendrites in the outer two-thirds of the molecular layer of the dentate gyrus. The granule cells then, via their mossy fiber axonal projections, complete the next limb of the circuit with their projection to the CA3 pyramidal neurons. These
neurons, in turn via their Schaffer axonal collaterals, project to the CA1 pyramidal cell n e u r o n s which complete the trisynaptic circuit. The granule cells are also of interest in that they permit us to study the effect of protein deprivation on cells generated primarily after birth 5-7"1°'36. Only 15% of the adult population of granule cells is present at the time of birth with approximately 45% of the adult population generated by the seventh postnatal day 6. These n e u r o n s have been shown to continue to be generated in the early postnatal period 2°'21 as well as throughout postnatal life of the rat 1°'38'39. In contrast the pyramidal cell n e u r o n s in the hippocampal formation are generated prior to birth in the rat 6'18'33'36'43. Further, a n u m b e r of studies have shown an effect of undernutrition on the d e v e l o p m e n t of the hippocampal formation 1'9'16'23'24"26"28"29'30'31'32'42.m comparison of two of these studies suggests that the effect of nutritional deprivation on the fascia dentata may be greater than that shown by the hippocampal complex as a whole. Fish
Correspondence: P.J. Morgane, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
272 and Winick 16 studied the effect of increased litter size
not show a deficit in the hippocampus until day 17, with
during the period of lactation on total D N A content of
deficits in D N A content of the cerebellum noted on day
various parts of the brain. This index of cell n u m b e r did
6 and in the cerebral cortex on day 14. In contrast, the
FT3 ~f'3
12
Fig. 1. A. Diagram showing the level in the rat brain in which a 4-mm block was taken, stained with the rapid-Golgi technique and then sectioned at 120/~m. The square shows the area of the dentate gyrus studied. Photomicrograph B shows the part of the dorsal blade (DB) of the dentate gyrus from where granule cells (GC) were sampled in the inferior part of the granule cell layer (GL). C: higher magnification showing spinous dentate granule cell and its apical dendrites extending into the molecular layer (ML). Perforant path (pp) axons are shown crossing the apical dendrite of granule cells, a, axon.
273
TABLE I
Effects o f pre- and postnatal undernutrition on granule cells in denate gyrus of rats at 30, 90 and 200 days old a F-values
Perikarya major axis ~um) minor axis (/tin) Dendrites number of spines per 50-/xm segments: proximal middle terminal intersections at: 38/2m 76/~m 114/~m 152ktm 190k~m 228Bm 266/~m 304/xm
Diet (dff 1,24)
Age (df2,24)
12.79 . . . . 9.70* *
3.95 *'d 4.72"*
0.17 NS 15.43"** 32.11"**
Interaction (df2,24)
Comparisons between ages 30 vs. 90 (df l,24)
90 vs. 220 (df l,24)
30 vs. 220 (df l,24)
0.31 NS 2.39 NS
4.14 NS 7.65*
0.44 NS 0.05 NS
7.26* 6.46 NS
8.17" * 6.75"* 2.12 NS
0.12 NS 0.80 NS 0.93 NS
1.17 NS 0.03 N _e
8.02* 9.52* _
13.46"* 15.40"** 7.51 ** 2.37 NS 15.00"**
2.53 NS 2.21 NS 0.13 NS 0.79 NS 2.81 NS
0.08 NS 2.21 NS 0.04 NS 0.47 NS 0.89 NS
44.32*** 90.99*** 83.49***
14.99"** 14.69"** 12.26"**
13.37"** 26.36*** 33.00***
Diet effect compared among agesf 5.84 NS 7.56 NS 31.78"** 1.31 NS 13.30"* 19.92"**
15.33"* 10.68"* _ 26.70*** 45.98*** 65.77***
Analysis of variance (ANOVA) with nutritional status and age as the two between subjects factors. Also provided are comparisons between ages (n = 10 animals/age: 5 control and 5 undernourished) where appropriate. b df = degrees of freedom. c Probability values (undernourished vs. controls): **P < 0.01, ***P < 0.001. d Probability values (across ages): *P < 0.05, **P < 0.01, ***P < 0.001. e When the main effect of age was not significant, no comparisons between age groups were made. f Analysis of variance (ANOVA) for morphometric measures showing a significant interaction effect between diet and age. The effect of diet was compared among ages by comparing the magnitude of the differences between control and undernourished animals. a
a u t o r a d i o g r a p h i c s t u d y o f L e w i s et al. 26 s u g g e s t s a m o r e s t r i k i n g e f f e c t o n t h e fascia d e n t a t a t h a n t h a t s h o w n b y t h e h i p p o c a m p u s as a w h o l e . T h e y n o t e d in rats u n d e r n o u r i s h e d f r o m t h e 6th d a y o f p r e g n a n c y until killing o n p o s t n a t a l days 1, 6 a n d 12, a p r o l o n g e d cell cycle t i m e a n d a m a r k e d l y c u r t a i l e d r a t e o f a c c u m u l a t i o n o f g r a n u l e cell n e u r o n s o n d a y s 1 a n d 6. Finally, in s e v e r a l s t u d i e s w e h a v e s h o w n s t r i k i n g a l t e r a t i o n s in n e u r o n a l plasticity o f t h e h i p p o c a m p a l f o r m a t i o n in m a l n o u r i s h e d rats as m e a s u r e d b y c h a n g e s in l o n g - t e r m p o t e n t i a t i o n a n d k i n d l i n g 2's. C h a n g e s in v a r i o u s b e h a v i o r s t h a t m e a s u r e h i p p o c a m p a l f u n c t i o n 17"25 have
also b e e n
shown following prenatal nutritional
deprivation.
MATERIALS AND METHODS
Animals The rats used for this investigation are the same rats used for our previously published studies of the effect of the 8% casein diet on selected nuclei in the brainstem ~2"~3 and the visual cortex TM.
Methods The specific details of the dietary paradigm and breeding procedures have been previously published by our group 28. To summarize, virgin female Charles River C.D., Sprague-Dawley descended rats were fed a 25% or 8% casein diet 5 weeks prior to conception and mated with males fed a standard rat chow diet. The dams remained on these diets all during gestation. Following delivery litters born the same day from each diet group were randomized and culled to 8 pups. The dams were continued on their diets during the lactation period following which the pups were weaned and then maintained on the same diets as those fed their mothers during gestation and lactation. At 30, 90 and 200 days of age a total of 30 male rats, 15 on the 25% diet and 15 on the 8% diet, were anesthetized with pentobarbital, perfused through the heart with 10% neutral buffered formalin and the brains removed the following day. Thus, we obtained 5 rats from each diet group in each of 3 age groups. In each rat a 4 mm wide block of hippocampal formation, including the dentate gyrus, was prepared using the rapid-Golgi technique (Fig. 1) following the modification of DiazCintra et al. 11. These blocks were embedded in low viscosity nitrocellulose, serially cut at a thickness of 120 pm and 5 sections from each brain containing the dentate gyrus were mounted in serial order. Each slide was assigned a random number to insure that all observations were blind with respect to age and diet. From these slides in each tissue section, one complete, well-impregnated granule cell was selected from the inferior part of the granule cell layer in the dorsal blade of the dentate gyrus. From these slides in
274
I
TERMINAL
[ 25• % casein
i
58~um
4O
~ zo
~
50 DAYS
MIDDLE I ,v- 0 i~.
~ 40 f
PROXIMAL[
u~ ao
I
2
5 4
5
6 7
8 ~
Fig. 2. Camera 1,ucida drawing of granule cell from the inferior part of the granule cell layer of the dentate gyrus of the rat showing 50 pm sectors (proximal, middle and terminal) of apical dendrites. Spine counts were made from the largest, best impregnated apical dendrite. Degree of branching of dendrites was established by the number of dendrites crossing the concentric ring.
~ ~0
~ each tissue section, one complete, well-impregnated granule cell was selected from the deep part of the granule cell layer in the dorsal blade of the dentate gyrus. A specific depth within the granule layer was selected in order to sample neurons generated at approximately the same time. The cells in the depth of this layer are late generated cells36 and were selected to provide comparable data for our ongoing study of the effect of prenatal undernutrition on postnatally generated granule cell neurons. Thus, we obtained a total of 150 dentate granule cells, 25 in each age and diet group. All measures were made by means of a 40x planapochromatic or 100x planapochromatic objective using the micrometer adjustment of the microscope in order to follow the cellular structures with a calibrated ocular reticle. Measurements of the number of dendrites crossing each concentric ring were made according to the method of Shol137, using a camera lucida by which the neuron is projected onto
~ 25% CASEIN o..-o 8% CASEIN
~.=.= -==-=~'~*~
A 2O
~ ....
E
~~ ~
I'-''-~
~ ~-. . . . . -~
MAJORAXIS
~
hi N
~ i~'
~
~.. . . . .
~
MINORAXIS
~ IO i-
I
I
50
90
~1
~
~ 40
0 PROXIMAL
MIDOLE
TERMINAL
Fig. 4. Bar graphs showing the effects of 8% and 25% casein diets on the number of dendritic spines on the proximal, middle and terminal 50-pm dendritic segments measured in the best impregnated and largest apical dendrite per granule cell. There is a significant overall decrease (ANOVA program across all 3 ages) of spines in the middle and terminal dendritic segments at all 3 ages in undernourished animals (striped bars) as compared to controls (white bars). Also see Table I for data and probability values.
an enlarged drawing of the 8 concentric rings 38 #m apart (Fig. 2). Using this procedure, the following measures were made: (1) major and minor axes of the granule cell body, (2) spine density per 50-#m segments of proximal, middle and terminal sectors of the largest dendrite within the plane of section (Fig. 2), and (3) number of interactions of dendrites crossing each of the 8 concentric rings.
Statistics Statistical analyses were conducted using the General Linear Model procedures of the SAS data analysis system 35. Analysis of the neural measures was made using a repeated-measures design with 2 between-subjects factors (diet and age) and 5 replicates (neurons) for each subject. Significant ANOVA effects of diet, age or interaction were further examined with three types of additional comparisons. The effect of diet was compared at each age between the control and undernourished rats (n = 5/diet), age comparisons were made across the age groups (n = 10/age) and the effect of diet was compared between the age groups after significant interaction. Probability values of these comparisons were adjusted upward using the Bonferroni method 34.
I
RESULTS 220
AGE(days) Fig. 3. Effects of 8% and 25% casein diets on somal size (major and minor axes) of dentate granule cells in the 3 age groups. The length of both major and minor axes shows significant overall decreases across all 3 age groups in undernourished rats (see Table I for data and probability values). Mean + S.E.M.
The results of the analysis of variance (ANOVA) shown in Table I with the measurements
are
graphically
s h o w n in Figs. 3 - 5 . T h e r e is a s i g n i f i c a n t d e c r e a s e in t h e major a n d minor axes o f t h e cell b o d y ( T a b l e I a n d Fig. 3) in t h e 8 % c a s e i n
275 30
DAYS
d2
[ ] 2 5 % cosein
[]
8 % casein
8 I~
4
E~
0
o
90
0 rO ~0 0 h.I I~
DAYS
42 8
.~_ I/~ I/~
4
9 0 ~
0
-~ -~ ¢"
.~2
2_20 DAYS
.
g 0 d
R
:5
4.
.5
6
'7
8
R i n g s C58jJm apart) Fig. 5. Effects of 8% and 25% casein diets on the dendritic branches crossing 8 concentric rings in rats 30, 90 and 220 days of age. Note the significant overall reduction of dendritic branching in undernourished animals (striped bars) in rings 1, 2, 3 and 5 at all 3 ages studied and at 90 and 220 days in rings 6, 7 and 8 compared to controls (white bars). Also see Table I for data and probability values.
diet rats at all 3 ages, with the greatest deficit in the m a j o r axis at 30 days ( - 1 4 % ) and in the minor axis at 220 days ( - 1 9 % ) . Comparisons across the ages show in both diet groups a significant increase in the m a j o r axis between 30 and 220 days and for the minor axis a significant increase b e t w e e n 30 and 90 days. T h e r e is a significant decrease in the n u m b e r of dendritic spines (Table I and Fig. 4) in the 8% casein diet rats at all 3 ages on the middle and distal dendritic segments, with the most m a r k e d effect at 30 days ( - 1 4 % and - 1 6 % , respectively). T h e r e is no significant diet effect on the proximal dendritic spines. Comparisons across the ages in both diet groups show significant increases in spine density on proximal dendritic segments between 30 and 90 days and for the middle dendritic segment between 30 and 90 days and 90 and 220 days. In each case the percent increase is greater in the 8% than in the 25% casein diet rats. The respective increases for the 8% and 25% casein diet rats for each of these 3 comparisons are: + 1 4 % and 18%, + 1 5 % and 8%, and + 1 7 % and 7%. There is no significant age effect on the
spines on the terminal dendritic segment. There is a significant decrease in dendritic branching (Table I and Fig. 5) in the 8% casein diet rats on all but the 4th concentric ring. In rings 1 through 3 and in ring 5 this is present at all 3 ages. In these rings the deficit varies from - 3 % to - 1 7 % . In the o u t e r 3 rings, rings 6 through 8, the deficit is n o t e d only at 90 and 220 days and varies from - 7 % to - 7 7 % with the most severe deficit in the outer 2 rings. In this latter location the deficit varies from - 4 6 % to - 7 7 % * . Significant age and interaction effects are confined to these o u t e r 3 rings. In these rings the 25% casein diet rats show an increase in n u m b e r of dendritic intersections while the 8% casein diet rats show a decrease. In ring 6 this is n o t e d when 30 and 220 days are c o m p a r e d , with the respective percent changes for the 8% and 25% casein diet rats o f - 2 % and + 4 4 % . In ring 7 this difference is seen when 30 and 90 and 30 and 220 days are c o m p a r e d with respective percent differences o f - 8 % , + 8 9 % ; - 2 5 % and + 8 4 % . In the 8th ring this difference is seen in all possible comparisons. The respective percents b e t w e e n 30 and 90, 90 and 220 and 30 and 220 days a r e - 3 9 % , + 5 0 % ; - 7 % , + 7 1 % ; - 4 3 % and +150%. DISCUSSION We have previously published c o m p a r a b l e data from these same rats on the effect of protein deprivation on pyramidal cells in the visual cortex t4 and on the 3 types of neurons in the nucleus r a p h e dorsalis 11 and nucleus locus coeruleus 13 in the brainstem. The effect of the low protein diet on the granule cells of the dentate gyrus was greater than that found in any of these previous studies. In the 3 studies noted a b o v e and in the present study, measurements are available for comparing the effect of 8% casein diet on cell size, dendritic complexity and density of dendritic spines. In the brainstem nuclei, taken together, there were 28 such comparisons between control and e x p e r i m e n t a l rats at each age, with a significant difference n o t e d at one or m o r e ages in 18 of these 28 comparisons. In the visual cortex the comparable numbers were 9 of 24 comparisons. In the present study all but 2 of 11 m o r p h o m e t r i c comparisons showed a significant diet effect. All significant findings in the present study indicated a deficit in the 8% casein diet rats. This was the result of the significant deficits p r e s e n t at 30 days persisting to 220 days and an increasing n u m b e r of distal dendritic intersections in the control rats at a time when experimental rats showed a decrease. T h e f o r m e r was associated with
* Ring 6 at 90 days -18% ( F 1 , 2 4 = 13.60, P < 0.01) and at 220 days -18% (F1.25 = 57.39, P < 0.001), ring 7 at 90 days -46% (F1,24 = 58.14, P < 0.001) and at 220 days -46% ( F 1 , 2 4 = 85.43, P < 0.001) and ring 8 at 90 days -57% (Fl,a4 = 23.91, P < 0.001) and at 220 days -77% (F1.24 = 125.50, P < 0.001).
276 significant deficits in cell size, n u m b e r of synaptic spines
rats at 30 days. Th e least frequently identified mecha-
on the middle and terminal dendritic segments and
nisms of effect of the low-protein diet were failure of
n u m b e r of dendritic intersections in all but one of the first
occurrence in the 25% casein diet rats of a significant
5 concentric rings. T h e latter was associated withdeficits
age-related change n o t e d in the 8 % casein diet rats and
in the n u m b e r of dendritic intersections in the o u t er 3
for significant differences n o t ed at 30 days to persist to
rings at 90 and 220 days. In contrast, in the visual cortical
220 days. These, each, were n o t e d 3 times. O f these 6
neurons and in the neurons in the brainstem nuclei, both
significant differences, 3 reflected a decreased measure-
control and experimental rats showed significant age-
ment in the 8% casein diet rats. A s can be seen (above),
related increases and decreases in m o r p h o m e t r i c mea-
in the present study only 2 of these mechanisms ac-
surements with resultant increases and decreases in these
counted for all the deficits in the 8% casein diet rats.
m e a s u r e m e n t s in the 8% casein diet rats. In these latter
Significant findings n o t ed at 30 days persisted to 220 days
studies the most frequent mechanism for a significant
and there was failure of the p r o t e i n - d e p r i v e d rats to show
difference at any one age was failure of occurrence of an
the age-related change n o t ed in controls.
age-related change n o t e d in controls to occur in the 8% casein diet rats or a less marked age-related change in the protein-deprived rats than that found in controls. Th er e were 15 examples of this which, in all but 2, resulted in a deficit in the undernourished rats. The next most frequent mechanism of effect of the low-protein diet was that a significant difference noted at 30 days did not persist to 90 days, suggesting a delayed d e v e l o p m e n t in the 8% casein diet rats. T h e r e were 12 examples of this, 8 of which r ep r es en t e d a deficit in the undernourished
REFERENCES 1 Ahmed, M.G.E., Bedi, K.S., Warren, M.A. and Kamel, M.M., Effects of a lengthy period of undernutrition from birth and subsequent nutritional rehabilitation on the synapse: granule cell neuron ratio in the rat dentate gyms, J. Comp. Neurol., 263 (1987) 146-158. 2 Austin, K.B., Bronzino, J. and Morgane, P.J., Prenatal protein malnutrition affects synaptic potentiation in the dentate gyrus of rats in adulthood, Dev. Brain Res., 29 (1986) 267-273. 3 Austin, K.B., Bronzino, J.D. and Morgane, P.J., Paired-pulse facilitation and inhibition in the dentate gyrus is dependent on behavioral state, Exp. Brain Res., 77 (1989) 594-604. 4 Barnes, C.A., Spatial learning and memory processes: the search for their morphological mechanisms in the rat, Trends Neurosci., 11 (1988) 163-169. 5 Bayer, S.A., Changes in the total number of dentate granule cells in juvenile and adult rats: a correlated volumetric and 3H-thymidine autoradiographic study, Exp. Brain Res., 46 (1982) 315-323. 6 Bayer, S.A. and Altman, J., Hippocampal development in the rat: cytogenesis and morphogenesis examined with autoradiography and low-level X-irradiation, J. Comp. Neurol., 158 (1974) 55-80. 7 Bayer, S.A., Yackel, J.W. and Puri, ES., Neurons in the rat dentate gyrus granular layer substantially increase during juvenile and adult life, Science, 216 (1982) 890-892. 8 Bronzino, J.D., Austin-LaFrance, R.J., Siok, C.J. and Morgane, P.J., Effect of protein malnutrition on hippocampal kindling: electrographic and behavioral measures, Brain Research, 384 (1986) 348-354. 9 Cordero, M.E., Zvaighaft, A., Muzzo, S. and Brunser, O., Histological maturation of astroglial cells in the archicortex of early malnourished rats, Pediatr. Res., 16 (1982) 187-191. 10 Crespo, D., Stanfield, B.B. and Cowan, W.M., Evidence that late-generated granule cells do not simply replace earlier formed neurons in the rat dentate gyrus, Exp. Brain Res., 62 (1986) 541-548.
Acknowledgements. Preliminary data was presented at the 17th Society for Neuroscience Meeting, New Orleans, LA, November, 1987. This study was supported by Grant PSCACNA-751969 and fellowships 20518 and 27234 from CONACyT Mrxico, and by Grants HD-22539 and HD-23338, NICHHD, NIH. We would like to thank technicians Juan Romero and Gabriela Padua for histological assistance. The facilities of Dr. R. O'Connell's laboratory at the Worcester Foundation for Experimental Biology related to statistical programs is appreciated. We are indebted to Dr. R. Patrick Akers for his help in the use of the SAS Program and Dr. David S. Reasner for his assistance in designing the ANOVA Programs.
11 Dfaz-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and Morgane, P.J., Nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups, Brain Research, 207 (1981) 1-16. 12 Dfaz-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and Morgane, P.J., The effects of protein deprivation on the nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups, Brain Research, 221 (1981) 243-255. 13 D/az-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and Morgane, P.J., The effects of protein deprivation on the nucleus locus coeruleus: a morphometric Golgi study in rats of three age groups, Brain Research, 304 (1984) 243-253. 14 Dfaz-Cintra, S., Cintra, L., Ortega, A., Kemper, T. and Morgane, P.J., Effects of protein deprivation on pyramidal cells of the visual cortex in rats of three age groups, J. Comp. NeuroL, 292 (1990) 117-126. 15 Eichenbaum, H. and Cohen, N.J., Representation in the hippocampus: what do hippocampal neurons code?, Trends Neurosci., 11 (1988) 244-248. 16 Fish, I. and Winick, M., Effect of malnutrition on regional growth of the developing rat brain, Exp. Neurol., 25 (1969) 534-540. 17 Goodlett, C.R., Valentino, M.L., Morgane, P.J. and Resnick, O., Spatial cue utilization in chronically malnourished rats: task-specific learning deficits, Dev. Psychobiol., 19 (1986) 1-15. 18 Hicks, S.P. and D'Amato, C.J., Cell migration to the isocortex in the rat, Anat. Rec., 160 (1968) 629-634. 19 Jordan, T.C., Howells, K.F., McNaughton, N. and Heatlie, P.L., Effects of early undernutrition on hippocampal development and function, Res. Exp. Med. (Berl.), 180 (1982) 201-207. 20 Kaplan, M.S. and Bell, D.H., Neuronal proliferation in the 9-month-old rodent - - Radioautographic study of granule cells in the hippocampus, Exp. Brain Res., 52 (1983) 1-5. 21 Kaplan, M.S. and Hinds, J.W., Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs, Science, 197 (1977) 1092-1094. 22 Katz, H.B. and Davies, C.A., The effects of early-life undernutrition and subsequent environment on morphological param-
277 eters of the rat brain, Behav. Brain Res., 5 (1982) 53-64. 23 Katz, H.B. and Davies, C.A., The separate and combined effects of early undernutrition and environmental complexity at different ages on cerebral measures in rats, Dev. Psychobiol., 16 (1983) 47-58. 24 Katz, H.B., Davies, C.A. and Dobbing, J., Effects of undernutrition at different ages early in life and later environmental complexity on parameters of the cerebrum and hippocampus in rats, J. Nutr., 112 (1982) 1362-1368. 25 Laughlin, N.K., Stanley, F. and Bell, J., Early undernutrition and later hippocampal damage: effects on spontaneous behaviors and reversal learning, Physiol. Psychol., 11 (1984) 269-277. 26 Lewis, P.D., Patel, A.J. and Bal~izs, R., Effect of undernutrition on cell generation in the rat hippocampus, Brain Research, 168 (1979) 186-189. 27 Lynch, G., Muller, D., Seubert, P. and Larson, J., Long-term potentiation: persisting problems and recent results, Brain Res. Bull., 21 (1988) 363-372. 28 Morgane, P.J., Miller, M., Kemper, T., Stern, W., Forbes, W., Hall, R., Bronzino, J., Kissane, J., 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. 29 Noback, C.R. and Eisenman, L.M., Some effect of proteincalorie undernutrition on the developing central nervous system of the rat, Anat. Rec., 201 (1981) 67-73. 30 Paula-Barbpsa, M.M., Andrade, J.P., Castedo, J.L., Azevedo, F.P., Camoes, I., Volk, B. and Tavares, M.A., Cell loss in the cerebellum and hippocampal formation of adult rats after long-term low-protein diet, Exp. Neurol., 103 (1989) 186-193. 31 Plaschke, M., Wenzei, J., Dorner, G. and Tonjes, R., Der Einfluss einer friihpostnatalen, sozialen und nutritiven Deprivation und einer gleichzeitigen Behandlung mit Pyridostigmin auf die Synaptogenese im Hippocampus der Ratte. Elektronenmikroskopische, morphometrische und stereologische Untersuchungen, J. Hirnforsch., 27 (1986) 145-158. 32 Plaschke, M., Dorner, G., Krause, E., Krause, M., Kuhlmey,
33
34 35 36
37 38
39
40
41
42
43
H.M., Schuster, T. and Wenzel, J., Untersuchungen zur Vesikelpopulation von Synapsen im Hippocampus der Ratte nach frfihpostnataler Deprivation und Verabreichung von Pyridostigmin, J. Hirnforsch., 28 (1987) 1-11. Pokorny, J. and Yamamoto, T., Postnatal ontogenesis of hippocampal CA 4 area in rats, I. Development of dendritic arborizations in pyramidal neurons, Brain Res. Bull., 7 (1981) 113-120. Ryan, T.A., Multiple comparisons in psychological research, Psychol. Bull., 56 (1959) 26-47. SAS Institute, Inc., SAS User's Guide: Statistics, Version 5 edn., SAS Institute Inc., Cary, NC, 1985. Schlessinger, A.R., Cowan, W.H. and Gottlieb, D.I., An autoradiographic study of the time and the pattern of granule cell migration in the dentate gyrus of the rat, J. Comp. Neurol., 159 (1975) 149-176. Sholl, D.A., The Organization of the Neurons of the Cerebral Cortex, Hafner, New York, 1956. Stanfield, B.B. and Cowan, W.M., The development of the hippocampal region. In A. Peters and E.G. Jones (Eds.), Development and Maturation of Cerebral Cortex, (Cerebral Cortex, Vol. 7), Plenum Press, New York, 1988, pp. 91-131. Stanfield, B.B. and Trice, J.E., Evidence that granule cells generated in dentate gyrus of adult rats extend axonal projections, Exp. Brain Res., 72 (1988) 399-406. Stewart, R.J.C., Merat, A. and Dickerson, J.W.T., Effect of a low protein diet in mother rats on the structure of the brains of the offspring, Biol. Neonate, 25 (1974) 125-134. Teyler, T.J. and DiScenna, P., The topological anatomy of the hippocampus: a clue to its function, Brain Res. Bull., 12 (1984) 711-719. West, C.D. and Kemper, T.L., The effect of a low protein diet on the anatomical development of the rat brain, Brain Research, 107 (1976) 221-237. Zimmer, J., Development of the hippocampus and fascia dentata: morphological and histochemical aspects, Prog. Brain Res., 48 (1978) 171-189.