Early Human Development, 33 (1993) 145-155 @ 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0378-3782/93/$06.00

145

EHD 01412

Environmental deprivation delays the maturation of motor pyramids during the early postnatal period

Rodrigo Pascual”, Victor Fernbndezb, Samuel Ruiz” and Rodrigo 0. Kuljisd

“Department of Health Sciences, Universidad Catdlica del Maule, Casilla 617, Talca, bDepartment of Physiology and Biophysics, School of Medicine, Universidad de Chile, Casilla 7000.5 Correo 7, Santiago, ‘Laboratory of Neurophysiology and Biophysics, Institute of Nutrition and Food Technology (INTA), Universidad de Chile, P.O. Box 138-11, Santiago (Chile) and dDepartment of Neurology, The University

of Iowa College of Medicine, Veterans Affairs Medical Center, Iowa City, Iowa 52242-1053 (USA)

(Received 23 February 1993; revision received 6 April 1993; accepted 20 April 1993)

Summary

The effects of environmental deterioration upon the development of motor cortex was studied in 30 Sprague-Dawley albino rats during lactation (lst-18th postnatal days). The use of Golgi-Cox-Sholl methodology allowed qualitative and particularly quantitative evaluations since impregnation of neurons take place at random without any selectivity. Morphometric studies were assessed by measuring layers II-III pyramidal neurons, basal dendritic branching, under camera lucida. Early en- vironmental impoverishment results in a highly significant decrease in the number and length of peripherical branches and terminal dendrites. These results extend pre- vious observations made predominantly in non-motor cortices which indicate that during early postnatal life restrictions or enrichments of the environment may be as- sociated with quantitative changes in the differentiation of cerebrocortical neurons. It is of upmost importance to consider that the potential effects of different types of epigenetic cues are highly selective since pyramids of pups subjected to mild nutri- tional manipulation during the same developmental period remained unaffected.

Key words: growth; dendritic branching; development of motor pyramids; en- vironmental deterioration; undernutrition; rat

Correspondence to: Victor FernBndez, Department of Physiology and Biophysics, Medical School, Universidad de Chile, P.O. Box 70005, Correo 7, Santiago, Chile.

146

Introduction

The ontogenesis of the cerebral cortex results from four conceptually distinct but temporally overlapping events: interkinetic nuclear migration in the germinal matrix of the neural tube, cell migration through the corticipetal thalamotelencephalic sys- tem of fibers, histogenesis by deposition of cells in the cortical mantle and cytodif- ferentiation, which continues after birth [ 14,15,23,26]. Postnatal effects of both environmental stimulation and nutrition on the maturation of neurons have been reported in studies focused predominantly on the basilar dendrites of pyramidal neu- rons of the cerebral cortex. Most such studies have been carried out in the visual cor- tex [2,10,21]. Recent studies in the developing primate motor cortex [4,32] demonstrate that basal dendritic branchings, among other aspects, are significantly affected by environmental influences. The work of Salas et al. [29] suggests similar effects on the rodent motor cortex during the suckling period. So far, most such ef- fects have been observed as a consequence of exposure to environmental enrichment. To date, there is considerably less evidence regarding such changes when the habitat is significantly impoverished by sensorial and physical impairment [ 121, The present study was designed to analyze whether the neonatal brain responds to environmental deprivation, in a manner different to mild undernutrition before weaning, while avoiding a severe disruption of mother-offspring interaction.

Methods

One-hundred forty-five male and female Sprague-Dawley albino rats were sub- jected to an experimental paradigm with environmental and nutritional variables (Table I). At birth, litter-mates were randomly assigned to either an impoverished habitat or to a relatively more stimulating environment. Eight litters were raised in an impoverished condition (IC) that was produced by three simultaneous manipula- tions: (1) leaving the pups with their mother in impoverished conditions, with minimal handling, restricted only to that which was required to maintain cleanliness; (2) submitting the litters to air pollution delivered by a local incinerator (a negative condition frequently found in crowded cities of developing countries) and (3) rearing the pups in a monotonous and gray habitat with restricted human presence. The im- poverished pups were housed in standard plastic laboratory cages (30 x 25 x 15 cm). These cages were located in a quiet, sound-proof, dimly-lit chamber, with solid walls, measuring 2.5 m high x 1 m deep x 1.5 m wide. Litters were left undisturbed except for control of body weight twice a week. In contrast, 10 litters were raised in standard conditions (SC). They remained in large 40 x 50 x 25 cm stainless-steel boxes. These boxes were located in a large room (3 m high x 4 m deep x 3 m wide) under gentle handling and relatively normal tactile, auditory and visual influences. The 12 h dark-light schedule was reversed. Both groups were kept in their respective conditions at 21 f 1.2”C with a relative humidity of 50 f 5%. It is not known ex- actly which elements of the former impoverished habitat may induce impairment of neuronal development. Nevertheless differential rearing has been shown to produce significant changes in behavior and brain morphology. This type of effect was also found consistently in all the experiments performed in the developing motor cortex

147

during the present study, as described below. To determine the possible effect of dietary conditions, the nutritional status of all individuals was modified by increas- ing or decreasing the litter size from eight to 13 or from eight to four pups per nurse. Litters of eight animals were used as controls (normally grown animals) (Table I). This manipulation has been shown to affect the nutritional status of pups consistent- ly [6,13]. After 18 days of combined nutritional and environmental treatment, five male pups in each group were randomly selected for quantitative morphological studies (Table I). Litter-mates were sacrificed while under deep ether anesthesia and the brain were processed according to the Golgi-Cox procedure as modified by Sholl [30]. Tissue blocks were dehydrated, embedded in cedukol, and cut serially in the coronal plane at 120 pm intervals as described previously [33]. The motor cortex was identified according to cytoarchitectonic [22] and axonal transport pathway criteria 1341.

In order to establish rigorous morphological comparisons between different experimental groups, a homogeneous population of neurons was examined. At least 10 well-impregnated superficial pyramidal neurons were drawn from each brain with the aid of a camera lucida. A total of 353 neurons positioned at a distance of 300-540 pm from the pia in layers II/III was selected in the frontomedial motor cor- tex for quantitative studies. Neurons were drawn at a magnification of 400 x and selected according to the following criteria: adequate staining of soma and dendrites, pyramidal or pyriform shape, symmetrical basilar dendritic tree, single apical den- drite perpendicular to the pial surface and no extensive overlapping of their process- es with those of neighboring neurons. The drawings included the perikaryon and the complete array of basilar dendrites. The length of the basal dendrites was measured using a map reader [3]. The total number of branches per neuron was counted according to the method of Coleman and Riesen [7]: dendrites leaving the cell body were catalogued as first order, direct branches from first order dendrites were designated as second order, and so on. The number and the longitude of terminal branches were also considered. Neurons were drawn blinded, that is to say, all slides were coded and neither the animal number nor the condition was known by the experimenters.

Results

Figure 1 shows the average and standard error of dendritic span and number of branches observed, in both experimental paradigms, on the 18th postnatal day. Pups raised in standard conditions (SC) exhibited the following values in the length of basilar dendrites (large litters (L) = 1802.5 f 44.8 pm; controls (C) = 1867.4 * 39.9 pm; small litters (S) = 1757.2 f 40.8 pm). These parameters are significantly distinct (P < 0.01 in all cases according to ANOVA test) from those of the group of animals subjected to the same nutritional condition but raised in a impoverished habitat (L = 1420.2 f 28.5 pm; C = 1580.3 f 34.9 pm and S = 1487.3 + 38.8 pm). Moreover the average number of basilar dendritic branches per neuron from pups raised in large, control and small groups, in standard conditions, was also significantly larger (P < 0.01) than those from similar groups raised in the impoverished condition (L = 30.1 f 1.1; C = 33.1 f 0.95 and S = 30.0 f 1.10 vs. L = 25.3 f 1.12; C =

TAB

LE I

Dis

tribu

tion

of e

xper

imen

tal

grou

ps s

ubm

itted

to

env

ironm

enta

l in

fluen

ces:

Sta

ndar

d co

nditi

on

(SC

) an

d Im

pove

rishe

d co

nditi

on

(IC

). C

orpo

ral

deve

lopm

ent

(wei

ght

and

size

), lit

ters

, nu

mbe

r of

bra

ins

and

neur

ons

utili

zed

with

in e

ither

exp

erim

enta

l gr

oup

are

indi

cate

d.

Envi

ronm

enta

l Li

tter

size

N

umbe

r of

st

atus

lit

ters

To

tal

num

ber

Cor

pora

l de

velo

pmen

t of

pup

s Si

ze (

mm

) W

eigh

t (g

)

Num

ber

of

brai

ns

eval

uate

d

Num

ber

of

cells

qua

ntifi

ed

SC

4

3 12

48

.86

f 6.

05

104.

77 ??

5.70

5

60

IC

4 3

12

40.7

2 f

2.33

10

0.00

f 4.

79

5 60

SC

8

4 32

31

.67

f 2.

76

92.0

8 f

5.46

5

60

IC

8 3

24

37.4

6 +

7.10

94

.81

& 5

.33

5 60

SC

13

3

39

27.9

3 &

4.2

4 84

.83

f 6.

71

5 60

IC

13

2

26

23.5

1 f

5.99

86

.38

* 6.

65

5 53

149

2 1800

5 1500

EJ 1200

900

600

300

0 L

L C

42

36 *

30

24

18

12

6

0

S

fast lx

??

L C S

Experimental Groups

Fig. 1. Dendritic development in motor pyramids. Data are presented according to two environmental conditions and three nutritional levels. A: Mean (*S.E.M.) dendritic length per neuron; B: Mean (*S.E.M.) number of dendritic branches per neuron. SC: standard condition; IC: impoverished condi- tion. L: large litters; C: controls; S: small litters. *P < 0.01, ANOVA-test.

26.9 f 0.91 and S = 24.0 f 1.04, respectively; Fig. IB). These observations indicate that superficial pyramidal neurons of the motor cortex of rats raised in impoverished environments prior to weaning develop shorter and decreased dendritic branchings. Greenough and co-workers [19] did not observe such an effect in the frontolateral motor cortex of rats after weaning. Taken together with our findings, their results suggest a lesser degree of neuronal plasticity at that time and/or in that particular

Fig. 2. Photomicrographs of superficial pyramidal neurons found in the motor cortex of 18 day-old rats. A: Highly developed neuron of a subject raised under standard condition. B: Extremely immature neuron in an environmentally deprived pup. Golgi-Cox-Shall. 1250 x Further explanation is provided in the text.

151

region of the motor cortex. In this respect, it is important to consider that the histogenesis and cytodifferentiation of the cortical plate take place according to a latero-medial gradient [ 1,15,16]. Thus, the sectors situated in the medial aspects of the cortex are much more sensitive to epigenetic influences, since they are much more immature at any given stage of development. The data of Fig. 1 show that, the effect of nutritional status upon the maturation of motor pyramids is not statistically signi- ficant when the total dendritic length and number of segments in large, control and small litters is compared. Figure 2A shows a highly differentiated neuron present in the motor cortex of an 18-day-old, normally reared rat. The cell body displays a typical pyramidal shape, from which many first order basilar branches originate. In addition, numerous segments of 2nd, 3rd, 4th and 5th order dendrites are clearly seen. Approximately 50% of the pyramidal neurons in the motor cortex of normally raised pups develop this relatively high degree of differentiation. Figure 2B shows a very immature neuron present in the motor cortex of a deprived pup. The soma exhibits a pyriform shape from which three primary basilar dendrites originate. Varicose structures (arrows) correspond to developing branching of higher orders. This extremely immature form of pyramidal neuron, not detected in standard condi- tions, represents about 40-50% of the total neuronal population observed in depriv- ed animals after impregnation with metallic mercuric salt. The rest of the impregnated neurons (ca. 50-60%) found in such circumstances display improved degrees of dendritic differentiation. Thus, although the statistical analysis in depriv- ed vs. standard subjects is not designed to discriminate these additional qualitative differences among pyramidal neurons, it reveals clearcut quantitative difference be- tween both groups of animals, as shown in Fig. 1. By contrast our results indicate that - at the level of analysis employed in the present study - the superficial

DENDRITIC ORDER DECLINE (%)

100

80

60

40

1st 2nd 3rd 4th 5th 6th

Order of Branch

??sc

Fig. 3. Mean proportion of neurons having segments of each branching order. SC: standard condition; IC: impoverished condition. *P < 0.01, ANOVA-test.

152

TABLE II

Average length and average number of terminal branches per neuron.

SC IC

Average length of terminal 12.30 f 1.33 52.70 zt 2.50 * dendritic segments/neuron (pm)

Average number of tenninal dendritic 19.36 f 0.44 16.58 f 0.48* branches/neuron

*P c 0.01 (ANOVA-test) (x ??S.E.M.)

pyramidal neurons in the motor cortex are apparently not sensitive to mild nutri- tional influences, since no statistical correlation could be detected between nutrition- al status and dendritic branching. However, more severe undernutrition is able to produce detectable effects [29]. As in our previous work [33], the percentage of peripheral branches declines dramatically as shown in Fig. 3. Although proximal branches are present on all neurons of both groups, a greater proportion of neurons from the standard (relatively enriched) habitat have 4th to 6th order dendrites (P < 0.01). Further effects of environmental deprivation upon peripheral dendrites are revealed by the diminishing length of their terminal branches, which also develop a significant decrease in the number of terminal segments (Table II).

Discussion

The present observations support previous findings that peripheral branches of the small pyramidal neurons of layers II/III are highly sensitive to changes in the habitat [20,33]. Studies performed by Stell and Riesen [32] are entirely concordant with our findings. Briefly, they reported that the superficial pyramidal neurons from the monkey motor cortex reared in an enriched environment displayed significantly more numerous basilar branches than controls. Surprisingly, they found this in the motor, but not in the primary somatosensory cortex. In addition, Bryan and Riesen [4], using a similar experimental design, observed that the dendritic development of neurons in monkey cortex is retarded when the subjects were reared in a restricted (or deprived) environment. Further, Carughi et al. [5] have recently shown that the motor cortex of the rat is significantly more sensitive to environmental enrichment than parietal and occipital cortices. Such effects may be related to the increasing ex- ploratory behavior exhibited by the animals subjected to the enriched milieu. Thus, pyramidal neurons in the motor cortex appear to be substantially more responsive to environmental influences during the early postnatal period than similar neurons in other cortical regions. Further, the greater plasticity observed in the present study before weaning contrast with the lack of effect reported by Greenough et al. [ 191 dur- ing the postweaning period, suggesting that the modifiability of pyramidal neurons in response to environmental cues is more marked during early stages of postnatal

153

development. This is perhaps consistent with the concept of ‘critical periods’ during early postnatal life, when various types of experimental manipulations in sensory systems may result in drastic effect in the structure and function of the brain. Similar manipulations in older animals have minor or undetectable effects on cortical organization. We have shown that mild undernutrition in our experimental paradigm does not produce discernible effects in the differentiation of pyramidal neurons in the motor cortex. This relative resistance to a mild reduction of food intake may reflect the compensatory effect of autogenous motor activity or an inherently less vulnerable internal organization in the motor cortex, or perhaps a combination of both factors. The second possibility is supported by the recent finding that some ‘highly specialized modular and laminar organizations of the primate visual cortex’ can develop in the absence of pre and postnatal cues from photoreceptors [24,25]. Taken together, these finding indicate that certain features of the internal organization of the cerebral cortex can be specified intrinsically. Therefore, it is perhaps not surprising that some of these features of cortical develop- ment may also be resistant to mild undernutrition. Certain structures, such as dendrites appear substantially more susceptible to environmental conditions than nutrition, perhaps indicating selective responsiveness or significantly different thresholds to distinct types of environmental cues.

The present study shows the negative effects of environmental deprivation upon cortical differentiation during the early postnatal period. Our findings, together with those from other laboratories, appear to indicate that the ontogenesis of the cerebral cortex depends on a combination of programs, some of which may be influenced by environmental factors. These latter factors may provide a basis to exploit the differ- ential effects of environmental cues on cortical development for therapeutic pur- poses, to alleviate brain and behavioral disturbances. Such environmental ‘therapy’ has proved to be useful in improving the behavioral performance in laboratory sub- jects with deterioration due to early sensory deprivation, undernutrition or hypothy- roidism [8,9,11,21,28,31]. Further, human studies [17,18] taken in conjunction with our own observations in experimental animals, strongly suggest that environmental enrichment during early life may offer similar prospects for humans. Studies to ex- plore morphological, physiological and behavioral effects of enrichment upon cor- tical pyramids in laboratory animals should be continued, since such neurons in man represent a highly evolved stage of the basic pyramidal shape already present in the cortical plate of the rat and other vertebrates [27].

Acknowledgements

This research was supported by Grant FONDECYT 1114-91. The authors thank Dr Ricardo Uauy for careful reading and helpful comments on the manuscript.

References

I Beaulieu, C. and Colonnier, M. (1989): Effects of the richness of the environment on six different cortical areas of the cat cerebral cortex. Brain Res., 495, 382-386.

2 Bhide, P.G. and Bedi, KS. (1984): The effects of environmental diversity on well fed and previously

154

3

4

5

10

11

12

13

14

15

16

17

18

19

20

21

22

23

undernourished rats: neuronal and glial cell measurements in the visual cortex (area 17). J. Anat., 138, 447-461. Boothe, R.G., Greenough, W.T., Lund, J.S. and Wrege, K. (1979): A quantitative investigation of spine and dendrite development of neurons in visual cortex (area 17) of macaca nemestrina monkeys. J. Comp. Neurol., 186, 473-490. Bryan, G.K. and Riesen, A.H. (1989): Deprived somatosensory-motor experience in stumptailed monkey neocortex: dendritic spine density and dendritic branching of layer IIIB pyramidal cells. J. Comp. Neural., 286, 208-217. Cart&i, A., Carpenter, K.J. and Diamond, MC. (1989): Effect of environmental enrichment during nutritional rehabilitation on body growth, blood parameters and cerebral cortical development of rats. J. Nutr., 119, 2005-2016. Chase, H.P., Dorsey, J. and McKhann, G.M. (1967): The effect of malnutrition on the synthesis of myelin lipid. Pediatrics, 40, 551-559. Coleman, P.D. and Riesen, A.H. (1968): Environmental effects on cortical dendritic fields I. Rearing in the dark. J. Anat., 102, 363-374. Davenport, J.W., Gonzalez, L.M., Carey, J.C., Bishop, S.B. and Hagguist, W.W. (1976): En- vironmental stimulation reduces learning deficits in experimental cretinism. Science, 191, 578-579. Davenport, J.W. (1976): Environmental therapy in hypothyroid and other disadvantaged animal populations. In: Environments as therapy for brain dysfunction, pp. 71-114. Editors: R.N. Walsh and W.T. Greenough. Plenum Press, New York. Davies, C.A. and Katz, H.B. (1983): The comparative effects of early-life undernutrition and subse- quent differential environments on the dendritic branching of pyramidal cells in rat visual cortex. J. Comp. Neural., 218, 345-350. Diamond, M.C., Rosenzweig, M.R., Bennett, E.L., Lindner, B. and Lyon, L. (1972): Effects of en- vironmental enrichment and impoverishment on rat cerebral cortex. J. Neurobiol., 3, 47-64. Diamond, M.C., Connor, J.R., Orenberg, E.K., Bissell, M., Yost, M. and Krueger, A. (1980): En- vironmental influences on serotonin and cyclic nucleotides in rat cerebral cortex. Science, 210, 652-654. Dobbing, J. (1963): The influence of early nutrition on the development and myelination of the brain. Proc. Roy. Sot. Med., 159, 503-509. Femandez, V. (1969): An autoradiographic study of the development of the anterior thalamic group and limbic cortex in the rabbit. J. Comp. Neural., 136, 423-452. Fernandaez, V. and Bravo, H. (1974): Autoradiographic study of development of the cerebral cortex in the rabbit. Brain Behav. Evol., 9, 317-332. Fernandez, V. (1990): Cortical ontogenesis: intrinsic and environmental interactions. IBRO- UNESCO WORKSHOP. Perspectives of neuroscience in South America and First Chilean Neuros- cience Meeting. Viria de1 Mar-Chile. Field, T.M., Schanberg, SM., Scafidi, F., Bauer, C.R., Vega-Lahr, N., Garcia, R., Nystrom, J. and Kuhn, CM. (1986): Tactile/Kinesthetic stimulation effects on preterm neonates. Pediatrics, 77, 654-658. Grantham-McGregor, S.M., Powell, C.A., Walker, S.P. and Himes, J.H. (1991): Nutritional sup- plementation, psychosocial stimulation, and mental development of stunted children: the Jamaican Study. Lancet, 338, 1-5. Greenough, W.T., Volkmar, F.R. and Juraska, J.M. (1973): Effects of rearing complexity on den- dritic branching in frontolateral and temporal cortex of the rat. Exp. Neural., 41, 371-378. Juraska, J.M. (1982): The development of pyramidal neurons after eye opening in the visual cortex of hooded rats: A quantitative study. J. Comp. Neurol., 212, 208-213. Katz, H.B., Davies, C.A. and Dobbing, J. (1982): 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, 1362-1368. Krieg, W.J.S. (1946): Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas. J. Comp. Neural., 84, 221-276. Kuljis, R.O. and Fernandez, V. (1981): A model of neocortical histogenesis based on Cajal’s prin- ciples of homotropism and neurocladism. Medical Hypoth., 7, 1431-1439.

24

25

26

21

28

29

30

31

32

33

34

155

Kuljis, R.O. and Rakic, P. (1990): Hypercolumns in primate visual cortex can develop in the absence of cues from photoreceptors. Proc. Natl. Acad. Sci., U.S.A., 87, 5303-5306. Kuljis, R.O. (1991): Development of the visual cortex deprived prenatally of retinal cues. In: The Changing Visual System, pp. 255-267. Editors: P. Bagnoli and W. Hodos. Plenum Press, New York. Petit, T.L., LeBoutillier, J.C., Gregorio, A. and Libstug, H. (1988): The pattern of dendritic develop- ment in the cerebral cortex of the rat. Dev. Brain Res., 41, 209-219. Ramon y Cajal, S. (1909- 1911): Histologie du Systbme Nerveux de 1’Homme et des Vertebres, 2 vols (L. Azoulay, trans). (Reprinted by Instituto Ramon y Cajal de1 Consejo Superior de Investigaciones Cientificas. Madrid, 1955). Rosenzweig, M.R. and Bennett, E.L. (1977): Effects of environmental enrichment or impoverish- ment on learning and on brain values in rodents. In: Genetics, Environment and Intelligence, pp. 163-196. Editor: A. Oliverio. ElsevierNorth-Holland, Biomedical Press, Amsterdam. Salas, M., Diaz, S. and Nieto, A. (1974): Effects of neonatal food deprivation on cortical spines and dendritic development of the rat. Brain Res., 73, 139-144. Sholl, D.A. (1953): Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat., 87, 387-407. Sjiiden, P.O. and Lindovist, M. (1978): Behavioral effects of neonatal thyroid hormones and differ- ential post weaning rearing in rats. Dev. Psychobiol., 11, 371-383. Stell, M. and Riesen, A. (1987): Effects of early environments on monkey cortex. Neuroanatomical changes following somatomotor experience: Effects on layer III pyramidal cells in monkey cortex. Behav. Neurosci., 101, 341-346. Venable, N., Fernandez, V., Diaz, E. and Pinto-Hamuy, T. (1989): Effects of preweaning en- vironmental enrichment on basilar dendrites of pyramidal neurons in occipital cortex:a Golgi study. Dev. Brain Res., 49, 140-144. Wise, S.P., Murray, E.A. and Coulter, J.D. (1979): Somatotopic organization of corticospinal and corticotrigeminal neurons in the rat. Neuroscience, 4, 65-78.