Malnutrition-induced alterations of developing Purkinje cells

EXPERIMENTAL

NEUROLOGY

Malnutrition-Induced W.

The

SUE

T.

University School,

GRIFFIN,

of Texas Department

56,

298-311

(1977)

Alterations DONALD

J.

of Developing WOODWARD,

AND

Purkinje RITA

CHANDA

Health Scicuce Center at Dallas, Southzwestws of Cell Biology, 5323 Harrs Hims Boulevard, Dallas, Texas 75235 Received

Drccrrzber

Cells 1

Medical

16,1976

To test whether or not malnutrition affects postmitotic, but still differentiating neurons, the development of Purkinje cell dendrites was studied in Golgi-Cox-stained preparations at 8, 11, 14, and 17 days postnatally. Significantly more dendritic aberrations involving branching and shape were observed in Purkinje cells from malnourished animals than from well-fed control animals. This work demonstrates that postnatal malnutrition in the rat affects growth of nonmitotic populations of neurons.

INTRODUCTION There have been numerous investigations into the effect of postnatal malnutrition on cerebellar development in the rat (2, 4-7, 9, 15, 17). Such work has concentrated mainly on changes in number, composition, and function of granule cells which, due to the vulnerability of the external granular layer, prove to be the major target of this developmental stress. The major change detected in cerebellar structure has been accounted for by decreases in rates of mitosis, i.e., nutritional stress during development resulting in fewer cerebellar granule cells at maturity. On the other hand, changes detected in the sagittal-area1 extent of the molecular layer have suggested that changes might also occur in the nonmitotic cells, the Purkinje cells ( 10). Here, our aim has been to study the dendritic structure of the Purkinje cells, which complete mitosis before birth and hence whose proliferation is Abbreviations : RNA-ribonucleic acid, MAM-methylazoxymethanol. 1 We thank Ms. Debra Bickett for technical assistance. This work was supported in part by National Institutes of Health Grant No. 5 ROl NS13225 and National Institutes of Health Teacher Investigator Award No. 1 Fll N3 11030 to D. J. Woodward. W. S. T. Griffin was a trainee under U.S. Public Health Service Grant No. 527.555292. 298 Copyright AlI

rights

0 1977 by Academic Press, Inc. of reprtiuction io any form reserved.

ISSN

0014-4886

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not affected by postnatal malnutrition. By focusing our interest on this one cell type, we attempted to separate changes in neuronal differentiation from changes in cell proliferation resulting from a stress such as malnutrition. Golgi-Cox impregnation techniques, as utilized here, clearly demonstrated marked changes in dendritic structure of Purkinje cells induced by nutritional deficiencies in the first three postnatal weeks in the rat.

METHODS Handling of Control and Malnourished Animals. Timed-pregnant rats of the Wistar strain were obtained from Carworth -Farms, New City, New York, on the 17th to 18th days of pregnancy. Chow and water were given ad libitum throughout the experimental period. On the day the dams littered (day 0), the litters were mixed so that some dams had 20 pups, designated as experimental.litters, and some had six pups, designated as control litters. No lactating dams died or were flagrantly cannibalistic during the experiments. A&y.& of Golgi-Cox-Stained Purkinje Cells. A modification of Van der Loos’s (14) Golgi-Cox procedure was used as described below. Six animals each from control and experimental groups were killed on days 8, 11, 14, and 17. The cerebellar vermis was dissected free and placed in Golgi-Cox solution for 3 months, and then was embedded in 10% Parlodion. Sections were cut in the sagittal plane at 100 pm, developed 2 min in a fresh solution of 1: 1 concentrated NHhOH : water, and then placed in F-5 photographic fixer for 5 to 10 min. After dehydration, sections were mounted on slides with Caedex (E. Merck, Darmstadt) and coverslipped. Photomicrographs were made using a Leitz Orthoplan microscope and a Nikon photographic attachment.

RESULTS A single observer examined a total of 3,462 Purkinje cells from 44 animals (21 control; 23 malnourished from birth to day of termination), at ages 8, 11, 14, and 17 days. In order to make systematic observations, a simple system was devised for evaluating dendritic characteristics whose presence or absence could be determined with certainty by a single observer. This method of observation could determine the prevalence of unusual dendritic, somatic, and positional aberrations which are not common in tissue from control animals as well as those common to malnourished animals. The frequency of these occurrences could be determined by scoring cells on an individual basis.

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TABLE Scoring Typical

Only

one main

which

had

A major bifurcation or branching which occurred within 50 pm of the cell soma

1 Criteria cells had

Extrasomal branches

processes

(one or more) with

secondary

Elongated main dendrites which extended more than 50 pm above the cell soma

dendrite

A main dendrite which projected the soma more perpendicular parallel to the pial surface

CHANDA

Atypical

cells had

No extrasomal processes secondary branches

AND

from than

Main dendrites perpendicular

which were not to the pial surface

Elongated, misshapen main dendrites which were first perpendicular, then parallel to the pial surface Dendrites extended from the molecular layer down toward or into the granular layer below the top of the cell soma

Typical Cells After looking at many cells in cerebella from control animals, a morphological characterization of cells that were most often seen at all ages evaluated was described and designated as “typical” (Table 1) . On the other hand, throughout development there were special characteristics, as described below, for the typical cell. Day 8. At this time, typical characteristic features appear to be varied across the cerebellum from folium I to folium X. The more well developed cells (i.e., larger dendritic arborization) appeared in the eighth through tenth folia in midsagittal sections, whereas the less mature cells (i.e., smaller dendritic arborization) appeared in the middle and anterior portion of th? cerebellum. However, the typical 8-day cell could be described as having some somatic processes, a main dendrite, and profuse dendritic branching (Fig. 1A) from the main or secondary dendrites. Day 11. Variation of cellular appearance was much less across the cerebellum by postnatal day 11. At 11 days, somatic processes were absent in typical cells and branching of the main dendrite usually occurred between 10 and 30 ,pm of the soma. Elongated dendrites were declared when major branches did not occur within that range. The main dendrite normally bifurcated and sent many uniformly stained ascending processes to the lower boundary of the external granular layer (Fig. 2A) .

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FIG. 1. Colgi-Cox-impregnated Purkinje cells from animals killed at day 8. The cell in (A) is representative of a typical S-day cell from control animals. Cells in (B) through (F) are examples of cells seen in tissue from malnourished animals. Arrows point to aberrations mentioned in the results. Magnification X230.

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FIG. 2. Golgi-Cox-impregnated Purkinje cells from animals killed at day II. The cell in (A) is representative of a typical 11-day cell from control animals. Cells in (B) through (F) are examples of cells seen in tissue from malnourished animals. Arrows point to aberrations mentioned in the results. Magnification X230.

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Days 14 and 17. By the 14th postnatal day, the typical cell was as described for day 11, but had more fully arborized to take up a rectangular area within the molecular layer, and the cell somata were arranged in an ordered monolayer at the junction of the molecular and internal granular layers (Fig. 3A and 4A). Aty&zl

Cells

Cells from either well-fed or malnourished animals were included in the category considered to possess “dendritic aberrations” if they (a) exhibited dendrites which extended away from the pial surface (Figs. 2C, 3D) toward the internal granular layer at least to the middle of the Purkinje cell soma (Fig. 4D) ; (b) possessed main dendrites which elongated by a distance of one somal diameter before eventually branching (Figs. lC, D, F; 2D; 3E; 4B) or never branched into the characteristic, bifurcated dense arborization (Fig. 2F) ; (c) had main dendrites which were misshapen into an S-form rather than straight, andJor had dendrites with long stretches devoid of branching (see arrows in Figs. 1F; 2D ; 3B, C, D ; and 4C, E, F) ; and (d) possessed enlarged zones of dendrites at the centers of extensively branching dendrites (Fig. 3D, F) . Cells scored in the category called “somal aberrations” included those Purkinje cells possessing: (a) more than one somal process which had secondary and tertiary dendrite-like processes (Figs. 2B, E ; 3F; 4B) ; (b) somas with their major dendrites lying parallel to the pial surface (Figs. lF, 3C, 4E) ; and (c) somas not in the normal monolayered arrangements (Fig. 2D). The results are expressed as the average percentage of total cells from each animal which exhibited the characteristics (Table 1) . Because some cells exhibited more than one characteristic, the percentages do not necessarily total 100%. Day 8. Despite the variation in appearance of cells across the cerebellum from folium I to folium X, variations from the characteristic features of Purkinje cells were seen in all folia from malnourished animals. Dendritic abnormalities included the full range of aberrations described above. The percentage of somal aberrations was less than the percentage of dendritic aberrations in both control and experimental animals. However, the percentage of somal aberrations was significantly greater in experimental than in control animals (Table 2). In addition, the percentage of somal aberrations is greater than at later ages studied. Day 11. At postnatal day 11, dendritic aberrations were much greater than somal aberrations. Both dendritic and somal aberrations are shown in Fig. 2. Dendrites descending below the soma were a common aberration (Fig. 2C). Figure 2C also illustrates an elongated primary dendrite with small tertiary-like perpendicular branches, and, finally, a bifurcation re-

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Purkinje cells from animals killed at day 14. The FIG. 3. Golgi-Cox-impregnated cell in (A) is representative of a typical 14-day cell from control animals. Cells in (B) through (F) are examples of cells seen in tissue from malnourished animals. Arrows point to aberrations mentioned in the results. Magnification X230.

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FIG. 4. Golgi-Cox-impregnated Purkinje cells from animals killed at day 17. The cell in (A) is representative of a typical 17-day cell from control animals. Cells in (B) through (F) are examples of cells seen in tissues from malnourished animals. Arrows point to aberrations mentioned in the results. Magnification x230.

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sulting in two secondary branches high in the dendritic tree. Another type of elongated primary dendrite (Fig. 2D) never bifurcated but ended in a puff-like profusion of branches. Furthermore, Fig. 2F illustrates a third type of elongated primary dendrite where there is no bifurcation into two secondary dendrites but rather a number of perpendicular, tertiary-like dendrites. Both Figs. 2D and 2E illustrate misshapen dendritic arborizations ; Fig. 2D shows several cells close together, each with hanging asymmetrical dendritic trees. Figures 2C and 2F illustrate the “nodules” at branch points rarely seen in normal animals. The large sum of percentages of cells with dendritic aberrations from experimental animals reflects the fact that many cells had more than one dendritic aberration ; for example, elongated main dendrites together with extrasomal processes (Fig. 2E). The particular somal aberrations scored here were not significantly greater in percentage in malnourished animals compared to controls. Nevertheless, interesting and unusual features occurred in experimental which were rarely or never noted in control data. Some of those unusual features included extrasomal dendrite-like processes which were evident in some cells as in Figs. 2B and 2E, and a highly abnormal somal position as noted in the three cells in Fig. 2D. Figure 2F shows that in some instances elongated neuronal somata lie almost parallel to the pia rather than perpendicular to it. Day 14. Dendritic aberrations in malnourished animals were significantly more common than in control animals. Again, somal aberrations were not significantly different between the two groups; yet, highly unusual features were in the experimental group and included extra, dendrite-like, somal processes (Fig. 3F), cell somata below the monolayer (Fig. 3B), and somata parallel to the pial layer (Fig. 3C). The cell in Fig. 3C (f rom an experimental animal) exhibits an S-shape dendritic configuration, and, although not as obvious, a similar type of cell is illustrated in Fig. 3D. Nodules at branch points are exhibited in Figs. 3D and 3F. Dendrites extend below the soma in both Fig. 3C and Fig. 3F. Day 17. The occurrence of both dendritic and somal aberrations was significantly greater among cells from experimental animals (Table 2). Misshapen dendritic arborizations are illustrated in Figs. 4C (long bare dendrites ending in puffs), 4F (dendrites projecting laterally), 4D (elongated S-shape main dendrite), and 4B (elongated main dendrite). Nodules are evident in Fig. 4D. Because few somal aberrations were apparent even in tissue from experimental animals, the trend toward more somal aberrations was clearly present at earlier ages, but the significant difference at day 17 was probably due to the larger sample size. Figures 4B and 4E illustrate somata parallel to the pial surface.

E C

E C

E C

E C

.8

11

14

17

6 5

6 5

6 6

5 5

Number

11 4 1.5 2

28 10 31 11

1.5 5

7 2

S-shaped

208 338

646 686

2

characteristics

24 8

42 14

Elongated

Dendritic

TABLE

13 0

30 8

43 4

7 0

Below soma

on Differentiation

358 584

222 421

Number of cells

of Malnutrition

a E = Experimental, C = control. b Values are mean percentage of total cells examined. c Significantly different from control (P < 0.05).

Groupa

Age (days)

Animals

Effect

0 0

8 0

8 4

10 0

Nodules

(Percentage

of Purkinje

3 0

4 2

6 2

0 0

Extrasomal processes

of cells)

Cells

89.2~ 8.4

55.1C 10.8

113.1c 27.4

92.0~ 16.0

Dendritic aberrations (Percentage of cell@)

15.oc 72.9

15.oc 66.6

12.2= 72.3

13.00 69.0

Dendritic typical (Percentage of cellsb)

ti z m cl P t:

z

5

z 2

z 2 =j

Et $

308

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CHANDA

At each day assessed,the percentage of typical cells was much greater in control than in experimental cerebellum. Conversely, the percentage of atypical cells was greater in experimental than in control cerebella. DISCUSSION Several previous workers investigated changes in growth of the cerebellum during periods of malnutrition (2, 4-7, 9, 15, 17). Those studies showed that postnatal malnutrition administered by a variety of methods resulted in decreased cerebellar weight, cellularity, protein content, and RNA content. The simplest view was that malnutrition during postnatal development of the rat cerebellum resulted in a decreased cell number but otherwise allowed existing cells to achieve a normal state in the adult. However, the current study has demonstrated that the morphological structure of substantial numbers of existing cells is also influenced by malnutrition. Purkinje cell dendritic morphology, as investigated here, might be expected to be altered due to the postnatal reduction of granule cell numbers and the parallel fibers, which constitute space and volume within the molecular layer for growth of dendrites. Supporting our initial hypothesis that changes ought to appear in Purkinje cell dendrites was a finding of Neville and Chase (9)) who reported a decreased ratio of molecular layer to granular cell areas after postnatal malnutrition. This suggested that beyond a reduction in cell numbers, additional changes in Purkinje cells occurred. An issue to be resolved is the extent to which intrinsic, direct effects of malnutrition cause changes in Purkinje cell dendrites as opposed to effects caused by a simple reduction of granule cells and parallel fibers. For purposes of summarizing the alterations observed in Purkinje cells as a consequenceof malnutrition, included is a camera lucida drawing (Fig. 5) of a typical Purkinje cell at 17 postnatal days and one other 17-day cell illustrating the major dendritic and somalaberrations. Our biochemical and histological data available from littermates of these animals (7) showed that the maximum reduction in cell number at the ages studied here was 21% (day 17), and the greatest difference in molecular layer size was 26% (14 days). Because such changes are probably distributed across the cerebellum, one would expect small but detectable changes to occur in Purkinje cells located everywhere in the cerebellum. In fact, the observational methods used here have shown a shift from 20 to 80% in the fraction of Purkinje cells possessingsuch defined “abnormalities.” The present study does not allow us to decide how much of the effect is due to reduced numbers of granule cells and parallel fibers. Some contribution of intrinsic actions can most certainly be anticipated due to fluctuations in cerebellar RNA and protein (7) known to occur in malnourished animals.

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FIG. 5. Camera lzlcida drawings of two Purkinje cells from animals killed at day 17. The layers are denoted as EGL, external granular layer; ML, molecular layer; and IGL, internal granular layer. The cell in (A) is a typical cell from a well-nourished animal. Characteristics of typical 17-day cells as described in Table 1 can be seen. The cell in (B) is an atypical cell from a malnourished animal killed at day 17. Structural aberrations are noted on the drawings.

Numerous studies have shown changes in Purkinje cell dendrites with selective destruction of the external granular layer. Several conditions and agents have led to such granule cell destruction-injections of the antimitotic drug methylazoxymethanol (MAM) (16), doses of X irradiation (1, 3)) perinatal infections with viruses (8)) and actions of gene mutations (12). However, the study of developing Purkinje cells after moderate doses of MAM in the first 4 postnatal days is the only comparable quantitative study of relatively subtle changes similar to those reported here. At low doses of MAM (10 mg/kg), there was a decrease in the number of internal granule cells (3) similar in amount to that seen as a consequence of malnutrition. Dendritic and somal alterations of Purkinje cells after MAM treatment were also similar in both quality and quantity to that reported here in malnourished animals. There were large increases in the percentage of cells with elongated primary dendrites, many were S-shape, and others extended down below the soma and into the internal granular layer. Such effects therefore are probably due to the absence of parallel fibers and granule cells alone. In contrast to malnutrition, with MAM treatment there appears to be more of a tendency toward failure of cell somata to align within the normal monolayer. These differences in effect of MAM and malnutrition could be attributed to the destruction by MAM of the external granular layer in the first 4 postnatal days of that study so that there is insufficient expansion of the folia and reduced room for the Purkinje cells to move up into a normal

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monolayer (3). On the other hand, in malnutrition there is no abrupt loss of the external granular layer and thus no sudden “crowding” of Purkinje cells in the first week. Although there is no clear understanding of the way in which dendritic elongation and branching occurs, there are possibilities for both intrinsic and extrinsic influences. The “nodules” which appear as small lakes of cytoplasm at branch points suggest a preferential production, due to some intrinsic factor, of cytoplasmic elements over some structural elements necessary for dendritic growth. Second, extrinsic factors such as the number of parallel fibers may ultimately regulate the length a dendrite attains before branching and the number of branches emanating from a single dendrite. The decrease in the number of granule cells due to malnutrition (7) and the corresponding decrease in the number of afferents to the Purkinje cells could be responsible for a substantial increase in the distance between the location of a Purkinje cell and a suitable afferent promoting dendritic growth. However, smooth elongated dendrites devoid of branches are seen to traverse zones within portions of the molecular layer which contain branches originating from other dendrites (Fig. 5) suggesting that synaptic sites are available in all zones of the molecular layer. Although the presence of suitable afferents may promote dendritic growth to some extent, our hypothesis is that, as a consequence of malnutrition, there are less nutrients for a selective production of structural elements necessary for dendritic growth. Our finding of less RNA and protein per cell in cerebella from malnourished animals (7)) together with our illustrations of branching in all zones of the molecular layer, are in support of this hypothesis. Our findings included downward-projecting dendrites, some even invading the internal granular layer. A question exists as to whether such low dendrites contact mossy fibers, as has been postulated in totally degranulated cerebellum (lo), due to a lack of appropriate numbers of afferents in the molecular layer. Connectivity between Purkinje cell dendrites and elements of the internal granular layer is currently being investigated by electron microscopy in this laboratory. In summary, Purkinje cells from malnourished animals tend to have elongated, sometimes sparsely branched dendrites which may enter inappropriate synaptic zones. Also, dendritic processes may originate directly from the soma in addition to the usual single main dendrite. Many cells from malnourished animals were reminiscent of earlier ages in well-fed animals and many had few or bare branches scattered throughout the dendritic arborization. The reduction in area of both the molecular layer (7) and sagittal area of dendritic expansion of individual Purkinje cells (11) induced by malnutrition is compatible with the dendritic and somal aberrations reported here.

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4. 5. 6. 7.

8.

CHANDA, R., D. J. WOODWARD, AND S. GRIFFIN. 1973. Cerebellar development in the rat after early postnatal damage by methylazoxymethanol: DNA, RNA and protein during recovery. J. Neurochem. 21: 547-555. CHASE, H. P., W. F. B. LINDSLEY, AND D. O’BRIEN. 1969. Undernutrition and cerebellar development. Nature 221: 554555. CULLEY, W. J., AND R. 0. LINEBFXGER. 1968. Effect of undernutrition on the size and composition of the ,rat brain. J. Nutr. 96 : 375-381. DOBBING, J. 1971. Undernutrition and ,the developing brain: The use of animal models to elucidate the human problem. Adv. Exp. Med. Biol. 13: 399412. GRIFFIN, W. S. T., R. CHANDA, AND D. J. WOODWARD. 1976. Malnutrition and brain development: Cerebellar weight, DNA, RNA, protein and histological correlations. J. Neurochem. In press. HERND~N, R. M., G. MARGOLIS, AND L. KILHAM. 1971. The synaptic organization of the malformed cerebellum induced by prenatal infection with the feline leukopenia virus (PLV). II. The Purkinje cell and its afferents. J. Newopathol.

Exp. Neural. 30 : 557-570. 9. NEVILLE, H. E., AND H. P. CHASE. 1971. Undernutrition ment. Exp. Neurol. 33 : 485-497. 10. PURO, D., AND D. J. WOODWARD. Physiological properties

11. 12. 13.

14. 15. 16. 17.

and cerebellar

develop-

of atferents and ‘synaptic reorganization in the rat cerebellum degranulated by postnatal X-kradiation. J. Neurobiol. In press. PYSH, J. J., AND R. E. PERKINS. 1975. Undernutrition and Purkinje cell development. Neurosci. Abst. 1: 1164. RAKIC, P., AND R. L. SIDMAN. 1973. Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of Weaver mutant mice. J. Camp. Neurol. 152 : 103-132. SHOFER, R. J., G. D. PAPPAS, AND D. P. PURPURA. 1964. Radiation induced changes in morphological and physiological properties of immature cerebellar cortex. Pages 476-508 in T. J. HALEY AND R. S. SNIDER, Eds., Responses of the Nervous System to lo&ring Radiation. Little, Brown and Co., Boston. VAN DER Loos, H. 1956. Une combinaison de deux vieilles methodes histologiques pour le ,systeme nerveux central. Mschr. Psychiat. Neurol. 132 : 330-334. WINICK, M., AND A. NOBLE, 1966. Cellular response in rats during malnutrition at various ages. J. Nutr. 89: 300-306. WOODWAR~, D. J., D. BICKETT, AND R. CHANDA. 1975. Purkinje cell dendritic alterations after transient developmental injury of the external granular layer. Brain Res. 97 : 195-214. ZAMENHOF, S., E. VAN MARTHENS, AND L. GRAVEL. 1971. DNA (cell number) in neonatal brain: Alterations by maternal dietary caloric restriction. Nutr. Rep. Znt. 4 : 26%274.