Morphometric assessment of caudate neurons following fetal X-irradiation

TOXICOLOGY

AND

APPLIED

57, 172- 180 (1981)

PHARMACOLOGY

Morphometric

Assessment of Caudate Fetal X-Irradiation1 B. F. SCHNEIDER

Department

of Pharmacology, Sciences

Received

University and Hospital,

August

AND

S.

Neurons

following

NORTON

of Kansas Medical Center, College Kansas City, Knnsas 66103

5. 1980; accepted

September

of Heulth

30, 1980

Morphometric Assessment of Caudate Neurons following Fetal X-Irradiation. SCHNEIDER, F., AND NORTON, S. (1981). Toxicol. Appl. Phurmacol. 57, 172-180. Four quantitative measures were obtained of spiny interneurons in the caudate nucleus of immature and mature control rats and rats which had been exposed to X-irradiation in utero on gestational Day 15. Dendritic branching patterns were consistently reduced by embryonic X-irradiation, whereas dendritic spine density showed little change. The cell nucleus was significantly smaller at 4 weeks and 4 months postnatally. Soma size was slightly increased in 4-week-old rats but showed no significant change in 4-month-old rats. The long-term changes in the morphology of the irradiated neuron may reflect primary damage to the cell at the time of irradiation or may be a secondary response to an altered environment. such as some decrease in the total neuronal complement in related brain areas. B.

Exposure of fetuses in the late period of gestation, after major organogenesis, to conditions which kill dividing cells can result in severe abnormalities in the structure of the central nervous system. A major factor in the nature of the eventual morphological damage is the unique cephalad progression of development of the central nervous system. The gross structural abnormalities of the developing rat brain which accompany exposure of the fetus to X-irradiation at the time of formation of the forebrain have been well documented (Hicks et al., 1959; Mullenix et al., 1975; Norton, 1979). The degree of damage is dose-related as well as being related to the stage of fetal development. Doses of 30-40 R or greater cause cell death when the cell is irradiated during the synthetic phase of mitosis (Hicks and D’Amato, 1963). The nature of the effect of fetal irradiation on the maturation and dif-

ferentiation of surviving neurons has not been well documented. Two factors are involved in the eventual differentiation of the neuron, the residual damage to the cell chromatin from irradiation and the effects on differentiation consequent to alterations in synaptic contacts available from reduced numbers of surviving neurons. The possible consequences of altered synaptogenesis in the process of differentiation of neurons need to be examined over a relatively long period of postnatal brain development since the time course for the morphological effects of synaptogenesis is not known for many neuronal systems. Morphological changes in neurons which are not killed as a result of X-irradiation may be detected as altered nuclear size, dendritic branching patterns, dendritic spine counts, or soma size. In the present study these parameters were measured to evaluate the kind and duration of effect produced in a selected neuronal type after prenatal irradia-

’ This study was supported in part by USPHS Grants MH 17279, MH 43860, and HD 02528. 0041-008X/81/020172-09$02.00/0 Copyright All rights

8 1981 by Academic Press, Inc. of reproduction in any form reserved.

172

EVALUATION

AGE AND NUMBERS

FOR MORPHOMETRIC

OF NEURONAL

173

DAMAGE

TABLE

1

STUDIES

OF CONTROL RATS AND RATS IRRADIATED DAY 15

(125 R) ON GESTATIONAL

Treatment

Number of mothers

Number of offspring

Histological preparation

Control Control Irradiated Irradiated

5 4 6 5

6 4 6 5

Golgi Toluidine Golgi Toluidine

Control Control Irradiated Irradiated

5 4 5 4

6 4 5 4

Golgi Toluidine Golgi Toluidine

Number of soma

Number of dendrites for branching pattern

Number of dendrites for spine counts

Number of nuclei

79 79 -

67 64 -

160 200

62 62 -

61 60 -

150 150

4 weeks postnatal age blue

79 76 -

4 months postnatal age blue blue

tion. The neuron studied was the mediumsized spiny neuron of the caudate nucleus which constitutes over 95% of the neuronal population of the caudate (Kemp and Powell, 1971; Norton and Culver, 1977). It is well established that the spines on dendrites of these neurons seen with Golgi impregnation represent axo-dendritic contacts, generally one synaptic contact per spine (Fox et al., 1971/1972). METHODS Breeding and irradiation. Female Charles River, Sprague-Dawley derived rats (200-300 g) were mated to males of the same strain. The day on which a spermpositive vaginal washing was found in the morning was called Day 1 of gestation. Pregnant rats were housed singly in standard plastic cages. On the fifteenth gestational day, between 11 AM and 1 PM, the pregnant females were exposed to 125 R whole body irradiation from a GE. Maximar III X-ray machine. This dose produces minimal effects on the mothers and is well below the dose of 200 R which has been reported to produce minor damage to the mother (Sipila, 1960). Irradiation parameters were: half wave rectification, 250 kV and 15 mA, with added filtration of 0.25 mm Cu and 1 mm Al. The source distance to the approximate center of the animal was 50 cm. Rats were placed

59 62 -

in Plexiglas restraining cages on the metal X-ray tumtable and rotated at 3 t-pm during irradiation. Exposure rate was approximately 38 R/min, measured with a Model 570 Condenser Victoreen R-meter. Control rats received the same treatment but without irradiation, Litters were reduced to a maximum of 10 about 24 hr after parturition. The number of pups per litter ranged from 11 to 15 for control and 9 to 15 for irradiated rats. Histological technique. Offspring of control and irradiated mothers were killed by decapitation at 4 weeks or 4 months of age. The brains were removed rapidly and sections containing the caudate nucleus were immersed immediately in buffered formalin for paraffin sections and toluidine blue staining or were fixed in osmic acid-dichromate solution for silver impregnation of neurons following the rapid Golgi technique described by Valverde (1970). Table 1 shows the number of treatment groups and the number of brains studied at each age and the histologic process used. Paraffin sections were cut at a thickness of 6 pm. Golgi-stained brains were sectioned at 100 pm. For examination of neurons in the caudate nucleus, coronal sections approximately 7 mm anterior to the external auditory meatus were used. The sections contained anterior commissure, which was used as a landmark. This is about the anterior-posterior center of the caudate nucleus, equivalent to Fig. 19 in the atlas of Konig and Klippel (1963). Four methods of quantifying neuronal parameters were used: determination of soma size, dendritic spine counts, dendritic branching patterns, and nuclear size.

174

SCHNEIDER

In addition the toluidine blue-stained sections were examined for gross malformations. Nuclear size determination. Sections stained with toluidine blue were used. After an area of the caudate nucleus was selected for study under low power (100x) of the light microscope, the nuclei of cells in the field were measured using oil immersion optics (1250x). The necessary criteria for selection were the presence of a fairly well centered nucleolus and abundant Nissl substance. As further assurance that only Golgi type II neurons were being counted, only the numerous population of medium-sized neurons was used. With a micrometer eyepiece, measurements were made of the long and short axes (at right angles to each other) of the cell nucleus. Differences in nuclear size of irradiated and control neurons were examined statistically with the analysis of variance comparing neurons within and among rats and treated groups. Soma size determination. The size of Golgi type II neuronal cell bodies was measured with the procedure used for nuclear size determination. Caudate neurons in Golgi-stained specimens were randomly selected under low power (100x). The only requirement for further analysis, besides the morphological characteristics of a Golgi type II neuron, was that the neuron have at least one intact dendritic tree. With a micrometer eyepiece and oil immersion optics, the long and short axes of the cell body were measured. Because the soma has an irregular shape, often some uncertainty was present in determining the exact end of the soma and the beginning of the primary dendrite. Dendritic spine counts. Counts of dendritic spines were made on segments of dendrites beginning with their origin at the soma and extending out for 60 km. All counts were made using oil immersion optics (1250x). One dendrite per neuron was selected. The neurons selected for soma size measurements were used. All protrusions were counted as spines if they appeared to be continuous with the shaft of the dendrite. At 4 weeks and 4 months of age the Golgi II caudate neuron typically bears four dendrites originating at the four “comers” of the soma. At a distance of a few microns from the soma the dendritic shafts are generally uniform in size. The size of the dendritic shafts at the first branching point from the soma in both irradiated and control caudate neurons was about 2 pm. Dendritic branching patterns. Branching patterns of dendrites were drawn schematically with 600x magnification of Golgi-stained neurons. These branching patterns were confirmed using oil immersion optics. The most highly branched dendrite from each neuron was studied. Dendritic branches were numbered using the centrifugal ordering method (Uylings et a/. , 1975). The stem of the dendrite coming off the soma was called the first order segment. Beyond each bifurcation of this segment the order was raised by one. The two daugh-

AND NORTON ter segments arising from a mother segment were always given the same order number, one order higher than that of the mother (Fig. 1).

RESULTS X-irradiation (125 R) on gestational Day 15 caused gross structural damage to the brain and a generalized decrease in the size of the brain. At the level of the anterior commissure, this damage included thinning of the cerebral cortex, development of ectopic cortex beneath a deficient corpus callosum, and reduction in the size of caudate nucleus. These changes have been described previously (Hicks et al., 1959: Mullenix et al., 1975; Norton, 1979). All irradiated offspring in this study at both 4 weeks and 4 months of age showed the gross malformations. Rats 4 Weeks Old

The shape of the nuclei of caudate neurons in cross-section was generally oblong although occasionally circular nuclei were observed. In rats receiving embryonic X-irradiation the average length of the long and short axes of the nuclei was significantly decreased (Table 2). The variability in nuclear size in irradiated rats was primarily the result of variability between rats as measured by the analysis of variance. Variability of nuclear size between control animals was no greater than between different nuclei in the same caudate nucleus. The length of the long and short axes of the caudate neuron soma at 4 weeks of age was greater in the irradiated than in the control rats (Table 2). Length of the soma was more variable than width in control and irradiated rats. Dendritic branching of caudate neurons was significantly reduced by embryonic Xirradiation (Table 3). The difference between the complexity of branching in control and irradiated neurons was significant when the number of branches at centrifugal orders from 1 to 7 was compared with a

EVALUATION

OF NEURONAL

175

DAMAGE

FIG. 1. Camera lucida drawing (left) of a caudate interneuron from a Cweek-old control rat and diagram (right) of the centrifugal ordering method of dendritic branching for the same neuron. Circle in diagram representing the soma is 10 pm in diameter.

x2 test. The average number of branches of neurons per rat is another measure of the diminished branching in irradiated rats. Approximately 12 neurons per caudate nu-

cleus were counted. For six control rats the number of branches per dendrite was 16.4, 15.8, 14.1, 13.6, 13.1, and 9.6. For six irradiated rats the values were 12.5, 9.6, 9.2,

TABLE

2

AVERAGE LENGTH (pm ? SE) OF LONG AND SHORT AXES FOR CELL SOMAS AND NUCLEI OF CAUDATE NEURONS IN CONTROL RATS AND RATS IRRADIATED (125 R) ON GESTATIONAL DAY 15 AND EXAMINED AT 4 WEEKS OR 4 MONTHS POSTNATALLY 4 weeks

4 months

Axis

Control

Irradiated

Control

Irradiated

Soma

Long Short

20.4 + 0.4 14.2 t 0.2

21.4 1 0.4a 15.2 2 0.3”

20.2 f 0.4 14.4 2 0.3

19.7 k 0.4 14.4 + 0.3

Nucleus

Long Short

13.5 * 0.1 11.8 * 0.2

12.7 2 O.lb 10.9 2 O.lb

12.5 4 0.1 11.0 % 0.1

11.4 c O.lb 10.0 f 0.1s

a Significantly different from control, p < 0.05, analysis of variance. b Significantly different from control, P < 0.001, analysis of variance.

176

SCHNEIDER

AND

9.0, 8.8, and 8.2. The difference between control and irradiated rats was significant using the Mann-Whitney U test (p = 0.004). There was no significant difference in the density of dendritic spines in irradiated and control caudate neurons (Table 4). A t test was used to compare each 8-pm segment of irradiated and control dendrites. Neurons in both groups of rats showed the typical distribution of spines, increasing from less than 1 in the first 8 pm from the soma to almost 1 spine per pm at 40 pm. The length of some dendrites is considerably greater than the distance shown in Table 4 (62 pm), but it is difficult to follow the course of intact dendrites at greater distance from the soma. Rats 4 Months

Old

Although the general pattern of effects of prenatal irradiation seen in 4 week-old rats was still present at 4 months of age, there were some differences. The range of nuclear sizes in both control and irradiated rats approximated a normal distribution for both axes of the nuclei (Fig. TABLE TOTAL DENDRITIC

DENDRITIC BRANCHING TREE PER CAUDATE

RATS AND RATS TIONAL DAY 15”

I 2 3 4 5 6 7 8 9

” Examined b Significantly mdevendence.

PATTERNS FOR ONE NEURON IN CONTROL

IRRADIATED

Total Branching order number

3

number

(125

R)

of branches

GESTA-

per order

4 weeks

4 months

Control

Irradiated*

79 158 262 281 208 78 28

Control

Irradiatedb

79

62

159 230 168 58 20 0

124 170 224 156 74 24 2 2

62 128

-

at 4 weeks different

ON

and 4 months from control,

postnatally. p < 0.001.

185 176 66 36 6 2 0

x’ test of

NORTON TABLE DENDRITIC

OF CAUDATE IRRADIATED

SPINE

(SPINES

PER 8 pm

IN CONTROL

RATS

R) ON GESTATIONAL

Distance from soma (fim)

DENSITY

NEURONS (125

4

DAY

4 weeks COIltrOl

SE)

i

AND

RATS

IS”

4 months

Irradiated

Cilntr0l

Irradiated --

0-8 8-16 16-24 24-32 32-40 40-48 48-56 56-62

0.2 1.0 2.8 5.0 6.0 7.2 7.4 7.3

2 t f -r k 2 i r

0.1 02 0.3 0.3 0.3 0.3 0.3 0.2

0.1 0.7 2.2 4.9 5.8 6.8 6.9 6.7

2 i_ f t z t + z

0.1 0.1 0.3 0.3 0.3 0.3 0.3 0.3

0.2 0.5 2 I 4.2 5.5 6.X 76 7.2

2 -f i! f t + +

0.1 0. I 0.3 0 3 0.3 0.3 0.3 0.3

0.2 0.6 3.0 50 4.6 7.3 7.4 6.9

r + I * + * * r

0.1 0.1 0.3” 0.4 0.3” 0 3 0.3 0 3

” Examined at 4 weeks or 4 months postnatally. ’ Significantly different from contro1.p < 0.05. I tesf.

2). The increased soma variability in irradiated rats at 4 weeks of age when the neurons were actively differentiating was reduced to control levels. The irradiated nuclei were still significantly smaller than control nuclei (Table 2). The long and short axes of the soma were not significantly different in the control and irradiated rats (Table 2). The complexity of branches of the major dendrites coming from each soma was still significantly reduced (Table 3). Five control rats had average numbers of branches per dendrite of 16.2, 16.1, 13.7, 10.7, and 10.0. The average number of branches for six irradiated rats was 13.3, 11.2, 10.4, 9.8. 8.3, and 7.5. The difference between these two groups is significant (Mann-Whitney CJ test, p < 0.05). No consistent differences were seen in spine density along the dendrites as a result of irradiation. Up to 48 pm from the soma the irradiated rats had a few more spines on the dendrites. The increase was significant at a distance of 16 to 56 pm from the soma (Table 4). DISCUSSION Changes in caudate neuronal morphology were measured after exposing the embryo

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OF NEURONAL

177

DAMAGE

60

50

10

11

12

13

14

15

16

17

18

19

20

SIZE FIG. 2. Frequency distribution plot. Size in units (1 unit = 0.8 pM) of the short and long axes of the nucleus of caudate neurons in control rats and rats irradiated (125 R) on gestational Day 15 plotted against frequency of occurrence at 4 months postnatally. -, Control; - - - -, irradiated.

on gestational Day 15 to 125 R X-irradiation, a treatment known to cause structural damage to the caudate nucleus (Hicks et al., 1959; Mullenix ef al., 1975; Norton, 1979). Morphological change in remaining caudate neurons was therefore expected. Approximately 95% of caudate neurons are the same type, the medium-sized Golgi type II neuron. Therefore, in the caudate nucleus the same type of neuron could be compared in irradiated and untreated rats. By using only those cells which in toluidine bluestained slides displayed a prominent nucleolus and abundant Nissl substance flaring out in the neuronal processes, it was possible to exclude glial cells from the cell population examined. Additionally, glial cell nuclei are smaller and more uniformly stained than neuronal nuclei. Differentiation of the caudate neurons proceeds for several weeks in the postnatal period, achieving maximum development of synaptic spines at 4 to 6 weeks of age, followed by slight regression to a stable,

mature pattern (Norton and Culver, 1977). At 4 months of age the caudate neuron is histologically mature. Spines are slightly decreased in number along the dendritic branches compared with the peak spine count found in immature caudate neurons at 4 to 6 weeks of age. However, at 4 months of age the complexity of dendritic branching has increased slightly. This can be seen in the relative increase in branching orders 6 through 9 in Cmonth-old control rats in Table 3. These two ages were chosen in order to compare the neurons during a period of rapid differentiation (4 weeks of age) with an adult neuron in which synaptogenesis could be considered complete (4 months of age). Of the four methods used to evaluate morphologic changes in caudate neurons, nuclear size and dendritic branching showed consistent prolonged changes after X-irradiation, whereas dendritic spine density was rather stable. Soma size was not markedly affected by age in the control rats.

178

SCHNEIDER

The slightly larger soma in irradiated rats may be the result of lesser dendritic differentiation in the irradiated neuron and retention of more perinuclear cytoplasm. By 4 months of age the irradiated soma was not significantly different from control soma. Size of nuclei decreased from 4 weeks to 4 months in both control and irradiated rats. At both 4 weeks and 4 months, irradiated nuclei were significantly smaller than control nuclei in rats the same age. The nucleus may have been smaller in the irradiated rats because there were reduced demands on the neurons or because the cell was permanently damaged by the irradiation. Either condition would result in smaller nuclei. It is known that nuclear size can change with the functional stage of the cell, at least when demands requiring synthesis are placed on the cell (Bresnick and Schwartz, 1968; Adhami, 1975). Wiesel and Hubel(1965) reported that after bilateral eye closure in kittens there were reductions in nuclear size in neurons of the lateral geniculate, without gross structural damage. Adhami (1975) concluded from his data that determination of nuclear size reflected the functional condition of the cell. Although the reduced size of the nucleus may be a response to nuclear damage from irradiation, Martin (1977) was unable to find changes in RNA/DNA ratios in brains of rats irradiated with 40-240 rads on gestational Day 18. Dendritic branching patterns of caudate neurons seen with Golgi staining were also significantly affected by embryonic X-irradiation when the centrifugal method of ordering dendritic branches was used. In comparison with the Strahler and Horsefield-Cumming methods, Uylings and coworkers (1975) put forth arguments for centrifugal ordering as the preferred method for relating physiologic phenomena to the morphology of the dendritic tree. Other methods have been used to measure dendritic branching. Using Sholl’s

AND

NORTON

method of intersection of concentric rings by the dendrite, Schade and Caveness (1968) showed that dendritic organization in pyramidal cells of the monkey cerebral cortex was altered by X-irradiation. However, Norton (1979), using Shall’s concentric ring technique, found only slight but not significant changes in dendritic branching of caudate neurons from rats irradiated ( 125 R) on gestational Day 15. In the present report, using the centrifugal method of ordering, significant changes were seen in the branching patterns. Unlike changes in nuclear size, which may occur acutely, effects on dendritic branching have all been observed after prolonged damage to neurons, such as deafferentation. When using the Golgi method of staining neurons, there is selective staining of neurons, the process of which is still not well understood. Though there is considerable evidence to indicate that the selection of cells is random, with all cell types staining (Pasternak and Woolsey, 1975: Smit and Colon, 1969), only a small percentage of all the neurons are stained. It is theoretically possible that neuronal damage could affect the staining characteristics of some of the neurons, selecting for cells with smaller branching patterns in this case. However, a more likely situation is that the diminished branching indicates a chronic reduction in function of irradiated neurons. From the data in this experiment it is not possible to distinguish the site of damage from irradiation. The same consequences could follow from either irradiation damage to the DNA of the caudate neuron or from deafferentation of the caudate neurons by damage elsewhere. Since data from the literature indicate that the number of dendritic spines can be increased or decreased by a variety of experimental manipulations (Valverde. 1967: Norton and Culver, 1977), it might be expected that spine density could be more easily affected than dendritic branching patterns. However, in the present experiment

EVALUATION

OF NEURONAL

with irradiated rats significant decreases were seen in dendritic branching but not spine counts. Several factors may be involved. In counting spines it was necessary to decide which protrusions along the dendrite shaft were spines, and it was sometimes difficult to achieve the resolution necessary to decide whether one or more spines were present at a point on the dendrite. Such problems increased variability of the measurements and thus reduced the power of the technique to detect changes. Each dendritic spine seen in the light microscope is a postsynaptic element in contact with one, or occasionally two, presynaptic axonal boutons. Although decreased numbers of both dendritic spines and branches have been reported in neurons after loss of presynaptic terminals, there may be different determinants of these two cellular elements. It is possible that dendritic branching and spine density are independent phenomena, and that one may be selectively affected. This possibility is supported indirectly by data from the literature. After rearing animals in darkness, changes were found in the density of spines (ValVerde, 1967), but no changes were seen in branching of dendrites on pyramidal cells in the visual cortex (Coleman and Riesen, 1968). More direct evidence was obtained by measuring branching patterns of caudate neurons, using the same Golgi slides from which Norton and Culver (1977) demonstrated a significant increase in spine density of these neurons after perinatal carbon monoxide exposure. The results of a small number of measurements (18 neurons from 2 control animals, 32 neurons from 4 carbon monoxide exposed animals) did not reveal significant differences in dendritic branching between the two groups [x2(5) = 3.75, p > 0.051. The effects of prenatal irradiation did not disappear with maturation of the rat except in the cell soma which was different at 4 weeks but was not different from con-

179

DAMAGE

trol soma at 4 months postnatal age. Spine counts tended to increase slightly at the older age. Measurements of both spine density and dendritic branching can be used to estimate the total number of spines on a neuron. The total number of spines on the neuron will be reduced if branches are reduced while spine density for the dendrites is unchanged. Using the Spearman rank correlation (Siegel, 1956) no correlation was found between the measurements of dendritic branching and spine density in the 4-monthold control animals (r, = 0.001). In the rats receiving embryonic irradiation there was an inverse but still not significant correlation ( rs = -0.21, Spearman rank correlation) between measures of branching and spine density at 4 months of age. This further suggests that the measurements of spine density and branching patterns are independent of each other. The reduced dendritic branching seen in the neurons of immature and adult rats after prenatal irradiation may be a reflection of either altered demand on the cell, due to inadequate numbers of presynaptic elements, or of inability of the cell to develop normal dendritic trees. Spine density at 4 months might be increased slightly as partial compensation for the reduction in synaptic connections to the neuron brought about by the reduced branching. The caudate neuron was used in this study for reasons advanced above. The generality of the effects of prenatal X-irradiation on other neurons needs to be demonstrated. REFERENCES H. (1975). Nuclear size independence cell cycle and localization in neuroepithelium.

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