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The effects of intrauterine growth retardation on the development of the Purkinje cell dendritic tree in the cerebellar cortex of fetal sheep: A note on the ontogeny of the Purkinje cell
0736--5748/88 $03.00+0.00 Pergamon Press pie. ~) 1988 ISDN
Int. J. Devl. Neuroscience, Vol. 6, No. 5, pp. 461-469, 1988. Printed in Great Britain.
T H E EFFECTS OF I N T R A U T E R I N E G R O W T H R E T A R D A T I O N ON T H E D E V E L O P M E N T OF T H E P U R K I N J E CELL D E N D R I T I C T R E E IN T H E C E R E B E L L A R C O R T E X OF FETAL SHEEP: A NOTE ON T H E O N T O G E N Y OF T H E PURKINJE CELL SANDRA REES* a n d RICHARD HARDING Department of Physiology, Monash University, Clayton, Victoria, 3168, Australia (Received 18 March 1988; in revised form 4 May 1988; accepted 5 May 1988) Al~ln~et--The development of the fetal sheep cerebellum at 80, 100, 120 and 140 days gestation (term = 146 days) and 3 months postnatally was studied using Nissl stained sections and rapid Golgi preparations. The most rapid expansion of the Purkinje cell dendritic tree occurred between 100 and 120 days of gestation (5--6 fold increase in area). By 140 days it had acquired its adult form after which time growth continued mainly in the vertical direction. The effects of intrauterine growth retardation on the growth of granule and Purkinje cell dendrites in the cerebellar cortex of fetal sheep (140 days) were investigated in Golgi preparations. Compared with control cerebella the length (but not the number) of granule cell dendrites was reduced by 14% (P<0.01); the area of the Purkinje cell dendritic field was reduced by 20% (P < 0.01 ); the branching density was reduced by 8% (P < 0.01); the total branch length was reduced by 27% (P < 0.002); the density of dendritic spines per row was not affected. These factors resulted in a decrease of 26% (P<0.002) in the total number of dendritic spines per row per Purkinje cell. These findings show that the growth of granule cell dendrites and the Purkinje cell dendritic tree have been significantly affected by chronic intrauterine deprivation. Such structural abnormalities could affect the pattern of neuronal connectivity and could be associated with functional deficits. Key words: Intrauterine growth retardation, Dendritic growth, Cerebellum, Purkinje cell development.
Infants that are growth-retarded, or small-for-gestationai age, have smaller brains than normal although the brain is spared relative to other organs, m4.2oPersistent neurological deficits are more likely to be present in these infants than in children of normal birthweight. 9""~:3'22 For example, deficits in cerebellar function are suggested by a poorer than normal performance in tests of motor skills, 22 poor motor co-ordination t° and clumsiness.9 At present it is not clear how chronic intrauterine deprivation might compromise the structural development of the brain. For this reason we have been interested in studying aspects of brain growth in a model of chronic placental insufficiency which involves the reduction of placental size in the sheep. ~'-~With this technique, placental size at term must be approximately 50% of normal for there to have been effective growth retardation during fetal development. 3° These placentas have been shown to transport less nutrient than normal to the fetus, t5 The growth-retarded fetus which results is hypoxic and hypoglycaemic~ and the brain weight is reduced by approximately 21% .29 In a previous quantitative study29 of the cerebellum in these animals we reported that the areal density of Purkinje cells in the Purkinje cell layer was higher in intrauterine growth retardation (IUGR); that the area of the molecular layer, of which Purkinje cell dendrites and granule cell axons are the major components, was reduced; and that the granular layer was reduced in area. From these results we deduced that the growth of the neuropil in the cerebellar cortex in IUGR is retarded. In order to study the effects of IUGR on Purkinje and granule cell dendritic growth more directly, we undertook the present quantitative study of rapid Golgi preparations of the fetal sheep cerebellum in growth retardation. Since the normal development of the Purkinje cell in the sheep has not been studied, we made an ontogenetic study before commencing the main project.
Ontogeny study
EXPERIMENTAL PROCEDURES
The ewe and the fetus were anaesthetized with pentobarbitone (30 mg/kg i.v.) and fetuses were delivered by Caesarian section at 80 days (n = 2), 100 days (n = 1) and 120 days (n = 3) (term 146 * Author to whom correspondence should be addressed. Abbreviations: EGL, external granular layer; IGL, internal granular layer; IUGR, intrauterine growth retardation.
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days). Fetuses were perfused through the aorta with 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.3. Two hours after perfusion, the brains were removed, weighed and immersed in fixative for 3-7 days. A 3-month-old lamb brain was fixed by immersion in a similar fixative. A midline incision was made through the cerebellar vermis and blocks of tissue (2-4 mm thick) were taken from Iobule 6 at the midline and placed in a Golgi fixative based on Stensaas '32 modification of the Rio-Hortega technique. 2~ Blocks were impregnated for approximately 60 hr in a solution of aldehydes containing 5% potassium dichromate and then placed in 1.5% AgNO3 for 5-6 days. The tissue was dehydrated in graded alcohols and embedded in low viscosity nitrocellulose. Parasagittal sections (90 p,m) were cut, cleared in Histoclea~ (National Diagnostics) and mounted. From the other half of the cerebellum frozen parasagittal sections (30 ~,m) were cut at the midline, mounted on gelatinized slides and stained with 0.01% thionin in acetate buffer (pH 4.5).
IUGR study Surgery. Endometriai caruncles (potential placental attachment sites) were removed from nonpregnant cross-bred ewes (Border Leicester/Merino). t..~ All surgical procedures were performed under halothane anaesthesia (1.5% in O2/N20 ) using aseptic techniques. A low midline abdominal incision was made and the uterus exposed. Each horn of the uterus was opened from the cervix to near the uterotubai junction and the majority of visible caruncles (80-95) were removed by cautery. After a minimum period of 8 weeks the ewes were introduced to a ram and the date of mating taken as day 0 of pregnancy. Control fetuses were from unoperated ewes of the same flock. The fetuses were delivered by Caesarian section between 139 and 141 days, perfused with aldehydes and the brains prepared by the rapid Golgi method as described above.
Quantitative analysis Purkinje cells. In each animal, the structure of 10-15 Purkinje cells was analysed. Criteria for selection were that the cell was completely impregnated, not significantly obscured by overlapping cells or Bergmann glial fibres and contained entirely within the section. Dendritic field size. The outline of the dendritic field of each Purkinje cell was drawn at a magnification of × 320 using a drawing tube attached to a microscope. The areas of the fields were measured with a Zeiss MOP-1 Image analyser and the mean calculated for each animal. Branching density. The branching density (cumulative length of dendrites per unit area of dendritic field) was determined by the method described by Weiss and Pysh 34 using the line intersection method. 33 In this method a square lattice graticule was mounted in the microscope eye piece to superimpose an array of crossing lines over the Purkinje cell dendritic tree in a random manner. Each test line of the grid had a spacing and length of 20 p,m at the magnification used ( x 1320; oil immersion). The number of intersections of dendrites per unit length of test line were determined for each sample. Twenty to 24 test line segments per cell were averaged so that about 30% of the dendritic field of each cell was sampled. Branching density (length of dendrites, ram/dendritic field area, mm 2) for each Purkinje cell was calculated according to the formula of Saltykov."33 branching density (LA) ~- ~r/2x PL, where LA is length per unit area and PL is number of intersections per unit length of test line. This method can be used for the comparison of cells in control and experimental groups providing the planar nature of the Purkinje cell dendritic tree does not alter as a result of the experimental manipulation. Therefore the mean thickness of the dendritic tree was determined by focusing up and down at several points across the dendritic field of each cell. Total branch length. The total dendritic branch length for the Purkinje cell was calculated by multiplying the lengths of dendrites per unit area (branching density) by the dendritic field area. Dendritic spines. Dendritic spines were counted under oil immersion ( x 1320) in 10 cells per animal. Ten segments of dendritic shaft, 10 ~m in length, distributed randomly over proximal and distal dendrites were sampled for each cell and averaged. Only spines in the same focal plane as the dendritic shaft were counted; hence the spine density reflects only the number of spines in a given row for each 10 ~,m length of a branch. The total number of spines per row per cell was then
Effects of IUGR on dendritic growth in cerebellum
463
calculated by multiplying the n u m b e r of spines per p,m by the total Purkinje dendritic branch length. All of the above m e a s u r e m e n t s were made for fetuses at 140 days; for fetuses at earlier and later gestational ages, only dendritic field size and spine densities were measured.
Granule cells The n u m b e r and average length of granule cell dendrites were measured in 10-15 granule cells per animal and an average taken.
Statistics The differences between experimental and control groups were treated statistically by using the Student's (unpaired) t-test. RESULTS
Ontogeny study Brain and cerebellar weights were recorded (Table 1) and the results confirmed that growth rates were similar to those already reported in a study on Merino sheep.l~ Table I. Ontogeny study of the fetal sheep cerebellum
Age 80 days (n = 2) 100 days (n = 1) 120- I day (n =3) 140 + - 1 day (n= 7) 3 months postnatal (n = l)
Purkinje cell dendriticfield Purkinje spine density (mm2) (no./10 ~,m dendritic shaft)
Brain wt (g)
Cerebellar wt (g)
8.8 -+0.03 23.7 37.0-+l.l 56.5-+ 1.7
0.37 -+0.01 1.3 2.9-+0.1 5.6-+0.2
0.0039 0.0217-+0.002 0.0361-+0.001
4.0 11.6-+0.3 12.9-+0.6
97.0
9.6
0.0639
13.0
n = number of animals. Results expressed as mean or means of mean -+S.E.M.
Thionin-stained sections. At 80 days (Fig. 1A-C) there is a well-developed external granular layer ( E G L ) of small round cells 5-6 deep, a thin molecular layer and a band of several rows of immature Purkinje cells with lightly stained nuclei. An interesting finding was the presence of a cell-sparse layer between the Purkinje cells and the compact band of inner granule cells. This layer appears to correspond to the lamina dissecans previously illustrated only in man 28 and whales,~6 and shown to contain afferent axon terminals some of which are possibly mossy fibres which have arrived in advance of the granule cells with which they will eventually synapse. 2~ By 100 days (Fig. 1D-F) the E G L has increased to a maximal depth of 7-8 cells, the molecular layer is more distinct and the lamina dissecans is no longer present. Purkinje cells have increased in size, are now round or pear shaped in appearance and are mainly arranged in a monolayer although double layers of cells occur in several folia. Some Purkinje cells can be seen in the inner granular layer ( I G L ) , presumably migrating to the Purkinje cell layer. At 120 days (Fig. 1G, H), the E G L has become thinner (5-6 cells deep), the molecular layer has further increased in width parallel with the growth of the dendritic tree of the Purkinje cell (Fig. 2C) and contains many granule cells migrating to the expanding I G L . Purkinje cells are arranged in a monolayer and have increased in diameter. All of these trends are more pronounced at 140 days (Fig. lI, J). Rapid Golgi sections. At 100 days the Purkinje cell apical dendrites are well developed. There is extensive branching of secondary and tertiary dendrites (Fig. 2A) and dendritic spines are present (Fig. 2A inset). Purkinje cell somas are invested with randomly oriented dendritic processes and somatic spines. By 120 days the somatic spines and processes have disappeared and there has been a marked increase in the branching density and the size of the dendritic tree
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(Fig. 2B). Spines have increased in density and are either long and thin or short and rounded in appearance (Fig. 2B inset). By 140 days the dendritic tree has acquired its mature form (Fig. 2C) and spines are more uniform in shape being predominantly short and rounded (Fig. 2C inset). The terminal spiny branchlets have almost reached the pial surface of the folium. Postnatal growth of the dendritic tree occurs mainly in the vertical direction. Morphometric analysis confirmed that the most rapid growth in dendritic field size occurred between 100 and 120 days when there was a 5.6 fold increase in area (Table 1). Thereafter the rate of growth slowed to a 1.7 fold increase between 120 and 140 days and similarly from 140 days to 3 months postnatal. At this stage the lamb brain has achieved approximately 90% of its adult weight, ~9 so the Purkinje cell dendritic tree has almost attained its adult dimensions. Dendritic spine density also increased most markedly between 100 and 120 days from 4.0 to 11.6 spines/ 10 I~m of dendritic shaft. Thereafter it continued to increase slightly to 13.0/10 ixm of dendritic shaft at 3 months postnatal.
IUGR study Purkinje cells. Fetuses which weighed at least 2 S.D.s below the mean weight for gestational age were defined as (intrauterine) growth retarded (IUGR). The brain and cerebellar weights of these animals have been described in a previous paper 29 and both were 21% lighter than controls. In this study we examined tissue from the cerebella of 7 control and 6 growth-retarded fetuses. The morphology of the Purkinje cell dendritic tree in I U G R is shown in Fig. 2D. Vertical expansion of the dendritic tree was retarded more than lateral growth. The area of the dendritic field was reduced by 20% in I U G R compared with control tissue (P < 0.002; Table 2). The stereological measurement of branching density employed in this study is appropriate for dendritic trees with a monoplanar distribution. It is important to determine whether or not there are any alterations in the planar nature of the dendritic field between the two groups as this would affect the accuracy of any relative difference measured. Results showed that there were no significant differences between the two groups in the depth of the dendritic trees (15.0 --- 0.6 I~m control vs 14.0 - 0.7 I~m I U G R ) . These values fall within the range reported for other species, for example, 7.5 I~m in the postnatal mouse; ~ 10 ~m in the monkey; 1~20 ~m in the rat; 24 25.3 I~m in the kitten 7 and 15-20 Ixm in man. 37 T a b l e 2. T h e effect of i n t r a u t e r i n e g r o w t h r e t a r d a t i o n on the g r o w t h of the P u r k i n j e cell d e n d r i t i c tree
Parameter P u r k i n j e cell d e n d r i t i c field ( m m :) P u r k i n j e cell b r a n c h i n g d e n s i t y ( m m / m m 2) Total dendritic branch length (mm) Spine density no./10 ~ m T o t a l no. of spines/row/cell
Control n= 7 0.0361 - 0.001 323.3 - 6.6 I 1.7 -+ {).5 12.9-+0.6 15,131 -+ 1205
Growth retarded n =6 0.0289-+0.O')1 296.2 -+ 4.3 8.5 -+ 0.2 13.1 -+0.3 1 1 , 1 7 4 - 967
% Difference -20%** -8%* - 2 7 % ** +1% -26%**
n = n u m b e r of animals. R e s u l t s are e x p r e s s e d as m e a n of m e a n s + S . E . M .
*P<0.02; **P
The branching density showed a small but significant reduction of 8% in I U G R ( P < 0 . 0 2 , Table 2). The total dendritic branch length of the Purkinje cell (calculated by multiplying the field area by the branching density) was significantly reduced by 27% ( P < 0 . 0 0 2 , Table 2) in the I U G R fetuses compared with controls. There was no detectable significant difference in the number of spines along a 10 I~m segment of dendritic trunk in the I U G R fetuses compared with controls. Qualitatively the spines looked similar in the two groups although there appeared to be more slender spines in the I U G R group (Fig. 2D inset). When the total number of spines per row per cell was calculated there was a reduction of 26% (P < 0.002, Table 2) in I U G R compared with controls. Granule cells. There was no detectable difference in the number of granule cell dendrites per cell in I U G R compared with controls but their average length was significantly reduced by 14% ( P < 0 . 0 1 , Table 3).
Effects of I U G R on dendritic growth in cerebellum
A.
D
E
G
/
Fig. !. Photomicrographs of thionin-stained mid-sagittal sections (30 p.m)of the fetal sheep cerebellum (Iobule 6) illustrating development of the layers during gestation: (A-C) 80 day fetus; (D-F) 100 day fetus; (G, H) 120 day fetus; (l, J) 140 day fetus. EGL, external granular layer; ML, molecular layer; P, Purkinje cell layer; LD, lamina dissecans; IGL, inner granular layer. (A, D, G, I) Bar = 8 mm; (B, E) bar=0.2 mm; (C, F, H, J) bar= I10 p.m.
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S. Rees a n d R. H a r d i n g
A
8
g.
Fig. 2. Photomicrographs of rapid Golgi-impregnated Purkinje cells from fetal sheep cerebellum (Iobule 6). (A) 100 days: note the presence of perisomatic processes and spines on the cell body. Spines and growth cones are evident on dendrites (inset). (B) 120 days: there has been a marked enlargement of the dendritic tree and of the branching density. Dendritic spines are long and thin or short and stubby (inset). (C) 140 days: further expansion of the height and width of the dendritic tree are evident. Dendritic spines are now predominantly short and stubby (inset). (D) 140 days IUGR: note reduced dendritic field size corapared with control. Dendritic spines are similar to control with a tendency to being thinner (inset). (A-D) Bar= 55 Ixm; (A-D insets) bar = 10 Ixm.
Effects of IUGR on dendritic growth in cerebellum
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Table 3. The effect of intrauterine growth retardation on growth of granule cell dendrites
Parameter No. of dendrites/cell Average length of dendrites
Control n= 7
G row t h re t a rde d n=6
% Difference
3.2 - 0. l 22.7 ± 0.5
3.3 ± 0.2 19.5 ± 0.9
+3% - 14%*
n = number of animals. Results expressed as mean of means -+ S.E.M. *P<0.01.
DISCUSSION The development of the Purkinje cell dendritic tree in the normal sheep fetus, just prior to birth, is more advanced than that in monkey26 and man 37 and markedly more advanced than that of the rat, z'5 mouse, 34 hamster, 23 cat 7 and opossum. ~7 For example, in the kitten (at birth) 7 the total dendritic length and the dendritic field size are 750 ~m and 4500 i.~m2, respectively, while in the sheep they are ll,700 ttm and 36,100 i~m2. Zecevic and Rakic37 state that the prenatal completion of the second stage of Purkinje cell maturation, that is the arrangement of Purkinje cells in a monolayer, the formation of dendritic processes on Purkinje cells and the formation of spines on secondary and tertiary dendrites is exclusive to man and primates. However, we have clearly shown that development of the Purkinje cell in the sheep prenatally is more advanced than that in man and monkey. This state of maturation correlates with the degree of motor coordination required by this species at birth. In this study we have shown that the growth of the Purkinje cell dendritic tree is significantly affected in IUGR. There is a reduction in the total number of spines per Purkinje cell as a result of a reduction in the total dendritic field size and in the branching density. The growth of granule cell dendrites is also retarded in IUGR. Sheep fetuses that are growth retarded as a result of maternal carunclectomy are hypoxic and hypoglycaemic3° and have low levels of circulating thyroid hormone.~5 In the present study it is not possible to distinguish between these factors as being causal to the reduced outgrowth of the Purkinje cell dendritic tree and indeed they might all contribute. In the rodent where development of the Purkinje cell occurs largely postnatally, malnutrition, ~8"25,3m,35hypothyroidism8 and hypoxia~ have all been shown to retard Purkinje cell maturation. In addition to each of the above conditions having a direct influence on Purkinje cell growth, growth could also be affected via a reduction in the presynaptic inductive or trophic influences of climbing fibres6'27 or of parallel fibres, 4"5 the axons of granule cells. In growth-retarded fetuses the lack of persistence of perisomatic processes on Purkinje cells suggests that climbing fibres have not been delayed in translocating from somatic dendrites to the proximal shafts of apical dendrites 5 and that their development is likely to be proceeding normally although there could be differences in biochemical or ionic processes at climbing fibre membranes not detectable in this study. It can be deduced from a reduction in the area of the molecular layer in IUGR 29 that the growth of granule cell axons (parallel fibres) has also been affected as these fibres are a major component of the layer. The smaller Purkinje cell dendritic tree might have partly resulted from reduced induction for the growth of the tertiary and quaternary branches by decreased parallel fibre input. 25 The functional consequences of the reduction in the total number of spines per Purkinje cell are not known. In IUGR in the sheep as opposed to malnutrition, ~2.3~ hypothyroidism2j and hypoxia3~ in rodents, there is no persistence of the EGL, 2° indicating that the formation of granule cells and their migration to the IGL has not been delayed. The molecular layer develops by a stacking process whereby parallel fibres formed by earlier migrating granule cells lie deeper in the layer and synapse on spines of the lower branches of the Purkinje cell dendritic tree. 2 If, as we suggested above, the growth (length) of parallel fibres in general has been retarded then the number of synapses made by each parallel fibre would be reduced proportionately and fewer Purkinje cells would be contacted by each granule cell. Alternatively, if parallel fibre growth is not generally retarded then later migrating granule cells could be restricted in the number of synapses they could make on the reduced dendritic arborization.
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A l t h o u g h a d i r e c t c o r r e l a t i o n w i t h t h e s i t u a t i o n in t h e g r o w t h - r e t a r d e d h u m a n i n f a n t ~s n o t p o s s i b l e , t h i s s t u d y h a s d e m o n s t r a t e d t h e m a n n e r in w h i c h t h e g r o w t h o f t h e f e t a l s h e e p P u r k i n j e cell w i t h its s i m i l a r m a t u r a t i o n a l t i m e t a b l e t o t h a t in m a n , is a d v e r s e l y a f f e c t e d b y c h r o n i c i n t r a u t e r i n e d e p r i v a t i o n . It p r o v i d e s a n i n d i c a t i o n o f p o s s i b l e a b n o r m a l i t i e s in n e u r o n a l s t r u c t u r e w h i c h m i g h t u n d e r l i e t h e c e r e b e l l a r d e f i c i t s s e e n in t h e h u m a n . Acknowledgements--We wish to thank Mr Neville Grant for the preparation of some of the Golgi sections and Mr Peter Angus for assistance with the photography. We are grateful to Dr John Rawson for discussion of the manuscript This work was supported by a grant from the National Health and Medical Research Council of Australia.
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Int. J. Devl. Neuroscience, Vol. 6, No. 5, pp. 461-469, 1988. Printed in Great Britain.
T H E EFFECTS OF I N T R A U T E R I N E G R O W T H R E T A R D A T I O N ON T H E D E V E L O P M E N T OF T H E P U R K I N J E CELL D E N D R I T I C T R E E IN T H E C E R E B E L L A R C O R T E X OF FETAL SHEEP: A NOTE ON T H E O N T O G E N Y OF T H E PURKINJE CELL SANDRA REES* a n d RICHARD HARDING Department of Physiology, Monash University, Clayton, Victoria, 3168, Australia (Received 18 March 1988; in revised form 4 May 1988; accepted 5 May 1988) Al~ln~et--The development of the fetal sheep cerebellum at 80, 100, 120 and 140 days gestation (term = 146 days) and 3 months postnatally was studied using Nissl stained sections and rapid Golgi preparations. The most rapid expansion of the Purkinje cell dendritic tree occurred between 100 and 120 days of gestation (5--6 fold increase in area). By 140 days it had acquired its adult form after which time growth continued mainly in the vertical direction. The effects of intrauterine growth retardation on the growth of granule and Purkinje cell dendrites in the cerebellar cortex of fetal sheep (140 days) were investigated in Golgi preparations. Compared with control cerebella the length (but not the number) of granule cell dendrites was reduced by 14% (P<0.01); the area of the Purkinje cell dendritic field was reduced by 20% (P < 0.01 ); the branching density was reduced by 8% (P < 0.01); the total branch length was reduced by 27% (P < 0.002); the density of dendritic spines per row was not affected. These factors resulted in a decrease of 26% (P<0.002) in the total number of dendritic spines per row per Purkinje cell. These findings show that the growth of granule cell dendrites and the Purkinje cell dendritic tree have been significantly affected by chronic intrauterine deprivation. Such structural abnormalities could affect the pattern of neuronal connectivity and could be associated with functional deficits. Key words: Intrauterine growth retardation, Dendritic growth, Cerebellum, Purkinje cell development.
Infants that are growth-retarded, or small-for-gestationai age, have smaller brains than normal although the brain is spared relative to other organs, m4.2oPersistent neurological deficits are more likely to be present in these infants than in children of normal birthweight. 9""~:3'22 For example, deficits in cerebellar function are suggested by a poorer than normal performance in tests of motor skills, 22 poor motor co-ordination t° and clumsiness.9 At present it is not clear how chronic intrauterine deprivation might compromise the structural development of the brain. For this reason we have been interested in studying aspects of brain growth in a model of chronic placental insufficiency which involves the reduction of placental size in the sheep. ~'-~With this technique, placental size at term must be approximately 50% of normal for there to have been effective growth retardation during fetal development. 3° These placentas have been shown to transport less nutrient than normal to the fetus, t5 The growth-retarded fetus which results is hypoxic and hypoglycaemic~ and the brain weight is reduced by approximately 21% .29 In a previous quantitative study29 of the cerebellum in these animals we reported that the areal density of Purkinje cells in the Purkinje cell layer was higher in intrauterine growth retardation (IUGR); that the area of the molecular layer, of which Purkinje cell dendrites and granule cell axons are the major components, was reduced; and that the granular layer was reduced in area. From these results we deduced that the growth of the neuropil in the cerebellar cortex in IUGR is retarded. In order to study the effects of IUGR on Purkinje and granule cell dendritic growth more directly, we undertook the present quantitative study of rapid Golgi preparations of the fetal sheep cerebellum in growth retardation. Since the normal development of the Purkinje cell in the sheep has not been studied, we made an ontogenetic study before commencing the main project.
Ontogeny study
EXPERIMENTAL PROCEDURES
The ewe and the fetus were anaesthetized with pentobarbitone (30 mg/kg i.v.) and fetuses were delivered by Caesarian section at 80 days (n = 2), 100 days (n = 1) and 120 days (n = 3) (term 146 * Author to whom correspondence should be addressed. Abbreviations: EGL, external granular layer; IGL, internal granular layer; IUGR, intrauterine growth retardation.
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days). Fetuses were perfused through the aorta with 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.3. Two hours after perfusion, the brains were removed, weighed and immersed in fixative for 3-7 days. A 3-month-old lamb brain was fixed by immersion in a similar fixative. A midline incision was made through the cerebellar vermis and blocks of tissue (2-4 mm thick) were taken from Iobule 6 at the midline and placed in a Golgi fixative based on Stensaas '32 modification of the Rio-Hortega technique. 2~ Blocks were impregnated for approximately 60 hr in a solution of aldehydes containing 5% potassium dichromate and then placed in 1.5% AgNO3 for 5-6 days. The tissue was dehydrated in graded alcohols and embedded in low viscosity nitrocellulose. Parasagittal sections (90 p,m) were cut, cleared in Histoclea~ (National Diagnostics) and mounted. From the other half of the cerebellum frozen parasagittal sections (30 ~,m) were cut at the midline, mounted on gelatinized slides and stained with 0.01% thionin in acetate buffer (pH 4.5).
IUGR study Surgery. Endometriai caruncles (potential placental attachment sites) were removed from nonpregnant cross-bred ewes (Border Leicester/Merino). t..~ All surgical procedures were performed under halothane anaesthesia (1.5% in O2/N20 ) using aseptic techniques. A low midline abdominal incision was made and the uterus exposed. Each horn of the uterus was opened from the cervix to near the uterotubai junction and the majority of visible caruncles (80-95) were removed by cautery. After a minimum period of 8 weeks the ewes were introduced to a ram and the date of mating taken as day 0 of pregnancy. Control fetuses were from unoperated ewes of the same flock. The fetuses were delivered by Caesarian section between 139 and 141 days, perfused with aldehydes and the brains prepared by the rapid Golgi method as described above.
Quantitative analysis Purkinje cells. In each animal, the structure of 10-15 Purkinje cells was analysed. Criteria for selection were that the cell was completely impregnated, not significantly obscured by overlapping cells or Bergmann glial fibres and contained entirely within the section. Dendritic field size. The outline of the dendritic field of each Purkinje cell was drawn at a magnification of × 320 using a drawing tube attached to a microscope. The areas of the fields were measured with a Zeiss MOP-1 Image analyser and the mean calculated for each animal. Branching density. The branching density (cumulative length of dendrites per unit area of dendritic field) was determined by the method described by Weiss and Pysh 34 using the line intersection method. 33 In this method a square lattice graticule was mounted in the microscope eye piece to superimpose an array of crossing lines over the Purkinje cell dendritic tree in a random manner. Each test line of the grid had a spacing and length of 20 p,m at the magnification used ( x 1320; oil immersion). The number of intersections of dendrites per unit length of test line were determined for each sample. Twenty to 24 test line segments per cell were averaged so that about 30% of the dendritic field of each cell was sampled. Branching density (length of dendrites, ram/dendritic field area, mm 2) for each Purkinje cell was calculated according to the formula of Saltykov."33 branching density (LA) ~- ~r/2x PL, where LA is length per unit area and PL is number of intersections per unit length of test line. This method can be used for the comparison of cells in control and experimental groups providing the planar nature of the Purkinje cell dendritic tree does not alter as a result of the experimental manipulation. Therefore the mean thickness of the dendritic tree was determined by focusing up and down at several points across the dendritic field of each cell. Total branch length. The total dendritic branch length for the Purkinje cell was calculated by multiplying the lengths of dendrites per unit area (branching density) by the dendritic field area. Dendritic spines. Dendritic spines were counted under oil immersion ( x 1320) in 10 cells per animal. Ten segments of dendritic shaft, 10 ~m in length, distributed randomly over proximal and distal dendrites were sampled for each cell and averaged. Only spines in the same focal plane as the dendritic shaft were counted; hence the spine density reflects only the number of spines in a given row for each 10 ~,m length of a branch. The total number of spines per row per cell was then
Effects of IUGR on dendritic growth in cerebellum
463
calculated by multiplying the n u m b e r of spines per p,m by the total Purkinje dendritic branch length. All of the above m e a s u r e m e n t s were made for fetuses at 140 days; for fetuses at earlier and later gestational ages, only dendritic field size and spine densities were measured.
Granule cells The n u m b e r and average length of granule cell dendrites were measured in 10-15 granule cells per animal and an average taken.
Statistics The differences between experimental and control groups were treated statistically by using the Student's (unpaired) t-test. RESULTS
Ontogeny study Brain and cerebellar weights were recorded (Table 1) and the results confirmed that growth rates were similar to those already reported in a study on Merino sheep.l~ Table I. Ontogeny study of the fetal sheep cerebellum
Age 80 days (n = 2) 100 days (n = 1) 120- I day (n =3) 140 + - 1 day (n= 7) 3 months postnatal (n = l)
Purkinje cell dendriticfield Purkinje spine density (mm2) (no./10 ~,m dendritic shaft)
Brain wt (g)
Cerebellar wt (g)
8.8 -+0.03 23.7 37.0-+l.l 56.5-+ 1.7
0.37 -+0.01 1.3 2.9-+0.1 5.6-+0.2
0.0039 0.0217-+0.002 0.0361-+0.001
4.0 11.6-+0.3 12.9-+0.6
97.0
9.6
0.0639
13.0
n = number of animals. Results expressed as mean or means of mean -+S.E.M.
Thionin-stained sections. At 80 days (Fig. 1A-C) there is a well-developed external granular layer ( E G L ) of small round cells 5-6 deep, a thin molecular layer and a band of several rows of immature Purkinje cells with lightly stained nuclei. An interesting finding was the presence of a cell-sparse layer between the Purkinje cells and the compact band of inner granule cells. This layer appears to correspond to the lamina dissecans previously illustrated only in man 28 and whales,~6 and shown to contain afferent axon terminals some of which are possibly mossy fibres which have arrived in advance of the granule cells with which they will eventually synapse. 2~ By 100 days (Fig. 1D-F) the E G L has increased to a maximal depth of 7-8 cells, the molecular layer is more distinct and the lamina dissecans is no longer present. Purkinje cells have increased in size, are now round or pear shaped in appearance and are mainly arranged in a monolayer although double layers of cells occur in several folia. Some Purkinje cells can be seen in the inner granular layer ( I G L ) , presumably migrating to the Purkinje cell layer. At 120 days (Fig. 1G, H), the E G L has become thinner (5-6 cells deep), the molecular layer has further increased in width parallel with the growth of the dendritic tree of the Purkinje cell (Fig. 2C) and contains many granule cells migrating to the expanding I G L . Purkinje cells are arranged in a monolayer and have increased in diameter. All of these trends are more pronounced at 140 days (Fig. lI, J). Rapid Golgi sections. At 100 days the Purkinje cell apical dendrites are well developed. There is extensive branching of secondary and tertiary dendrites (Fig. 2A) and dendritic spines are present (Fig. 2A inset). Purkinje cell somas are invested with randomly oriented dendritic processes and somatic spines. By 120 days the somatic spines and processes have disappeared and there has been a marked increase in the branching density and the size of the dendritic tree
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(Fig. 2B). Spines have increased in density and are either long and thin or short and rounded in appearance (Fig. 2B inset). By 140 days the dendritic tree has acquired its mature form (Fig. 2C) and spines are more uniform in shape being predominantly short and rounded (Fig. 2C inset). The terminal spiny branchlets have almost reached the pial surface of the folium. Postnatal growth of the dendritic tree occurs mainly in the vertical direction. Morphometric analysis confirmed that the most rapid growth in dendritic field size occurred between 100 and 120 days when there was a 5.6 fold increase in area (Table 1). Thereafter the rate of growth slowed to a 1.7 fold increase between 120 and 140 days and similarly from 140 days to 3 months postnatal. At this stage the lamb brain has achieved approximately 90% of its adult weight, ~9 so the Purkinje cell dendritic tree has almost attained its adult dimensions. Dendritic spine density also increased most markedly between 100 and 120 days from 4.0 to 11.6 spines/ 10 I~m of dendritic shaft. Thereafter it continued to increase slightly to 13.0/10 ixm of dendritic shaft at 3 months postnatal.
IUGR study Purkinje cells. Fetuses which weighed at least 2 S.D.s below the mean weight for gestational age were defined as (intrauterine) growth retarded (IUGR). The brain and cerebellar weights of these animals have been described in a previous paper 29 and both were 21% lighter than controls. In this study we examined tissue from the cerebella of 7 control and 6 growth-retarded fetuses. The morphology of the Purkinje cell dendritic tree in I U G R is shown in Fig. 2D. Vertical expansion of the dendritic tree was retarded more than lateral growth. The area of the dendritic field was reduced by 20% in I U G R compared with control tissue (P < 0.002; Table 2). The stereological measurement of branching density employed in this study is appropriate for dendritic trees with a monoplanar distribution. It is important to determine whether or not there are any alterations in the planar nature of the dendritic field between the two groups as this would affect the accuracy of any relative difference measured. Results showed that there were no significant differences between the two groups in the depth of the dendritic trees (15.0 --- 0.6 I~m control vs 14.0 - 0.7 I~m I U G R ) . These values fall within the range reported for other species, for example, 7.5 I~m in the postnatal mouse; ~ 10 ~m in the monkey; 1~20 ~m in the rat; 24 25.3 I~m in the kitten 7 and 15-20 Ixm in man. 37 T a b l e 2. T h e effect of i n t r a u t e r i n e g r o w t h r e t a r d a t i o n on the g r o w t h of the P u r k i n j e cell d e n d r i t i c tree
Parameter P u r k i n j e cell d e n d r i t i c field ( m m :) P u r k i n j e cell b r a n c h i n g d e n s i t y ( m m / m m 2) Total dendritic branch length (mm) Spine density no./10 ~ m T o t a l no. of spines/row/cell
Control n= 7 0.0361 - 0.001 323.3 - 6.6 I 1.7 -+ {).5 12.9-+0.6 15,131 -+ 1205
Growth retarded n =6 0.0289-+0.O')1 296.2 -+ 4.3 8.5 -+ 0.2 13.1 -+0.3 1 1 , 1 7 4 - 967
% Difference -20%** -8%* - 2 7 % ** +1% -26%**
n = n u m b e r of animals. R e s u l t s are e x p r e s s e d as m e a n of m e a n s + S . E . M .
*P<0.02; **P
The branching density showed a small but significant reduction of 8% in I U G R ( P < 0 . 0 2 , Table 2). The total dendritic branch length of the Purkinje cell (calculated by multiplying the field area by the branching density) was significantly reduced by 27% ( P < 0 . 0 0 2 , Table 2) in the I U G R fetuses compared with controls. There was no detectable significant difference in the number of spines along a 10 I~m segment of dendritic trunk in the I U G R fetuses compared with controls. Qualitatively the spines looked similar in the two groups although there appeared to be more slender spines in the I U G R group (Fig. 2D inset). When the total number of spines per row per cell was calculated there was a reduction of 26% (P < 0.002, Table 2) in I U G R compared with controls. Granule cells. There was no detectable difference in the number of granule cell dendrites per cell in I U G R compared with controls but their average length was significantly reduced by 14% ( P < 0 . 0 1 , Table 3).
Effects of I U G R on dendritic growth in cerebellum
A.
D
E
G
/
Fig. !. Photomicrographs of thionin-stained mid-sagittal sections (30 p.m)of the fetal sheep cerebellum (Iobule 6) illustrating development of the layers during gestation: (A-C) 80 day fetus; (D-F) 100 day fetus; (G, H) 120 day fetus; (l, J) 140 day fetus. EGL, external granular layer; ML, molecular layer; P, Purkinje cell layer; LD, lamina dissecans; IGL, inner granular layer. (A, D, G, I) Bar = 8 mm; (B, E) bar=0.2 mm; (C, F, H, J) bar= I10 p.m.
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A
8
g.
Fig. 2. Photomicrographs of rapid Golgi-impregnated Purkinje cells from fetal sheep cerebellum (Iobule 6). (A) 100 days: note the presence of perisomatic processes and spines on the cell body. Spines and growth cones are evident on dendrites (inset). (B) 120 days: there has been a marked enlargement of the dendritic tree and of the branching density. Dendritic spines are long and thin or short and stubby (inset). (C) 140 days: further expansion of the height and width of the dendritic tree are evident. Dendritic spines are now predominantly short and stubby (inset). (D) 140 days IUGR: note reduced dendritic field size corapared with control. Dendritic spines are similar to control with a tendency to being thinner (inset). (A-D) Bar= 55 Ixm; (A-D insets) bar = 10 Ixm.
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Table 3. The effect of intrauterine growth retardation on growth of granule cell dendrites
Parameter No. of dendrites/cell Average length of dendrites
Control n= 7
G row t h re t a rde d n=6
% Difference
3.2 - 0. l 22.7 ± 0.5
3.3 ± 0.2 19.5 ± 0.9
+3% - 14%*
n = number of animals. Results expressed as mean of means -+ S.E.M. *P<0.01.
DISCUSSION The development of the Purkinje cell dendritic tree in the normal sheep fetus, just prior to birth, is more advanced than that in monkey26 and man 37 and markedly more advanced than that of the rat, z'5 mouse, 34 hamster, 23 cat 7 and opossum. ~7 For example, in the kitten (at birth) 7 the total dendritic length and the dendritic field size are 750 ~m and 4500 i.~m2, respectively, while in the sheep they are ll,700 ttm and 36,100 i~m2. Zecevic and Rakic37 state that the prenatal completion of the second stage of Purkinje cell maturation, that is the arrangement of Purkinje cells in a monolayer, the formation of dendritic processes on Purkinje cells and the formation of spines on secondary and tertiary dendrites is exclusive to man and primates. However, we have clearly shown that development of the Purkinje cell in the sheep prenatally is more advanced than that in man and monkey. This state of maturation correlates with the degree of motor coordination required by this species at birth. In this study we have shown that the growth of the Purkinje cell dendritic tree is significantly affected in IUGR. There is a reduction in the total number of spines per Purkinje cell as a result of a reduction in the total dendritic field size and in the branching density. The growth of granule cell dendrites is also retarded in IUGR. Sheep fetuses that are growth retarded as a result of maternal carunclectomy are hypoxic and hypoglycaemic3° and have low levels of circulating thyroid hormone.~5 In the present study it is not possible to distinguish between these factors as being causal to the reduced outgrowth of the Purkinje cell dendritic tree and indeed they might all contribute. In the rodent where development of the Purkinje cell occurs largely postnatally, malnutrition, ~8"25,3m,35hypothyroidism8 and hypoxia~ have all been shown to retard Purkinje cell maturation. In addition to each of the above conditions having a direct influence on Purkinje cell growth, growth could also be affected via a reduction in the presynaptic inductive or trophic influences of climbing fibres6'27 or of parallel fibres, 4"5 the axons of granule cells. In growth-retarded fetuses the lack of persistence of perisomatic processes on Purkinje cells suggests that climbing fibres have not been delayed in translocating from somatic dendrites to the proximal shafts of apical dendrites 5 and that their development is likely to be proceeding normally although there could be differences in biochemical or ionic processes at climbing fibre membranes not detectable in this study. It can be deduced from a reduction in the area of the molecular layer in IUGR 29 that the growth of granule cell axons (parallel fibres) has also been affected as these fibres are a major component of the layer. The smaller Purkinje cell dendritic tree might have partly resulted from reduced induction for the growth of the tertiary and quaternary branches by decreased parallel fibre input. 25 The functional consequences of the reduction in the total number of spines per Purkinje cell are not known. In IUGR in the sheep as opposed to malnutrition, ~2.3~ hypothyroidism2j and hypoxia3~ in rodents, there is no persistence of the EGL, 2° indicating that the formation of granule cells and their migration to the IGL has not been delayed. The molecular layer develops by a stacking process whereby parallel fibres formed by earlier migrating granule cells lie deeper in the layer and synapse on spines of the lower branches of the Purkinje cell dendritic tree. 2 If, as we suggested above, the growth (length) of parallel fibres in general has been retarded then the number of synapses made by each parallel fibre would be reduced proportionately and fewer Purkinje cells would be contacted by each granule cell. Alternatively, if parallel fibre growth is not generally retarded then later migrating granule cells could be restricted in the number of synapses they could make on the reduced dendritic arborization.
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A l t h o u g h a d i r e c t c o r r e l a t i o n w i t h t h e s i t u a t i o n in t h e g r o w t h - r e t a r d e d h u m a n i n f a n t ~s n o t p o s s i b l e , t h i s s t u d y h a s d e m o n s t r a t e d t h e m a n n e r in w h i c h t h e g r o w t h o f t h e f e t a l s h e e p P u r k i n j e cell w i t h its s i m i l a r m a t u r a t i o n a l t i m e t a b l e t o t h a t in m a n , is a d v e r s e l y a f f e c t e d b y c h r o n i c i n t r a u t e r i n e d e p r i v a t i o n . It p r o v i d e s a n i n d i c a t i o n o f p o s s i b l e a b n o r m a l i t i e s in n e u r o n a l s t r u c t u r e w h i c h m i g h t u n d e r l i e t h e c e r e b e l l a r d e f i c i t s s e e n in t h e h u m a n . Acknowledgements--We wish to thank Mr Neville Grant for the preparation of some of the Golgi sections and Mr Peter Angus for assistance with the photography. We are grateful to Dr John Rawson for discussion of the manuscript This work was supported by a grant from the National Health and Medical Research Council of Australia.
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