Brain Research, 177 (1979) 347-360 © Elsevier/North-Holland Biomedical Press
347
I M P E D E D C E R E B E L L A R D E V E L O P M E N T A N D R E D U C E D SERUM T H Y R O X I N E LEVELS ASSOCIATED W I T H F E T A L A L C O H O L INTOXICATION
S. E. KORNGUTH, J. J. RUTLEDGE, E. SUNDERLAND, F. SIEGEL, I. CARLSON, J. SMOLLENS, U..IUHL and B. YOUNG University Wisconsin Medical Center Madison, Wisc. 53706 (U.S.A.)
(Accepted March 8th, 1979)
SUMMARY Pregnant albino rats were placed on a complete liquid diet (Ensure) containing either 9 ~ ethanol or an isocaloric amount of sucrose between the third and twentieth day of gestation. The pups born to these rats were sacrificed either day 11 or day 14 postnatum and morphometrical, histological and biochemical analyses were done on their cerebellums and cerebrums. Pups that were exposed to ethanol in utero had significantly smaller body weights, cerebrums and cerebellums than pair-fed controls. The cerebellar mass was reduced by 10 ~o and the cerebral weight by 3 ~ in the pups exposed to alcohol when body weights were normalized to that of pair-fed controls. Cerebellar aspartyl aminotransferase (EC 2.6.1.1) activity was reduced at day 11 and 14 in ethanol treated pups compared with controls. Serum T4 levels were also reduced in the ethanol treated group. Histological analyses revealed that the external granule cell (EGC) layer of ethanol treated pups was significantly thicker at 11 and 14 days postnatum than that of pair-fed control pups. Cerebellar ornithine decarboxylase (ODC, EC 4.1.1.17) activity was higher at day 11 in the ethanol treated pups than in controls. The reduced mass, AAT activity, T4 serum levels and the increased thickness of the EGC layer indicate a delayed or impeded maturation of cerebellum in ethanol treated pups. These data are considered from the viewpoint that ethanol, other drugs such as methadone and prenatal stress (malnutrition) may cause delayed cerebellar maturation by reducing serum T4 levels in the early postnatal period (day 5-14).
INTRODUCTION The normal development of rat cerebellar cortex has been examined from gross anatomical 2a, histological2,S,lS, 5°, ultrastructural30, 31 and biochemicall,a,l~, 50 perspectives. These studies have described the time of maximal appearance of the external
348 granule cell (EGC) layer, the inward migration of EGCs and the superficial migration of presumptive Purkinje cells (PC), the period during which the apical dendrite of the PC develops, the development of synaptic spines and branches on PC dendrites and the increase in oxidative metabolic capacity during PC maturation. Changes in the normal developmental pattern of the cerebellar cortex have been demonstrated to occur in rat pups that were malnourished~,la,2a,28,a2,34,43,48,49,sa, 52, caused to become functionally hypothyroid4,7,10,14,29 or exposed either to ethano16,as,46, 47 or methadone ~3 during the pre- or early postnatal period. It is of particular significance that the characteristics of delayed cerebellar development were similar in rat pups that were malnourished, hypothyroid, ethanol or methadone treated. This suggests that all of these agents may exert their action by impairing the synthesis or release of a common product necessary for normal cerebellar development (i.e. thyroxine). In this report the effect of ethanol on the development of rat cerebellar cortex is examined by gross anatomical, histological and biochemical techniques. It will be shown that the ethanol induced changes may be related to the reduced serum T4 levels in pups exposed to ethanol. The reduced T4 levels in the pup serum are of significance because T4 does not traverse the placenta 11. MATERIALS AND METHODS Selection and diet of rats Albino rats (250-300 g) from an inbred colony, originally obtained from Holtzman Laboratories, Madison, Wisc., were mated in individual cages during a 16 h period. The next morning those female rats with an ejected vaginal plug were selected as pairs on the basis of weight (pairs were within 10 g). During the entirety of pregnancy rats were maintained on a liquid diet. All animals were provided with a solution of Ensure (Ross Laboratories, Columbus, Ohio) containing 7 parts Ensure to 3 parts distilled water ( 7 0 ~ v/v). Ensure contains 1 calorie per ml and is vitamin supplemented. During the first and second day of pregnancy the ethanol treated pregnant rats were maintained on the 70 ~ Ensure diet. From the 3rd through the 20th day they were provided ad libitum with 100 ml of liquid diet containing 9 ~ ethanol (9.4 ml of 95 ~ ethanol, 70 ml Ensure, 20.6 ml water). The amount of liquid taken by each animal was determined between 09.00 and 10.00 h daily and fresh food was provided daily between 10.00 and 11.00 h. On the 21st day the alcohol content was reduced to 5 ~ and on the 22rid day it was reduced to 2.5 0/oo.The day after birth all rats were given solid rat chow (Purina). The pair-fed controls were given an isocaloric amount of sucrose in lieu of ethanol; 100 ml of diet contained 70 ml Ensure, 13.5 ml of 87 ~ sucrose and 16.5 ml water. The pair-fed controls were administered the volume of liquid diet consumed by their pair-mate the previous day. On the second day after birth, all litters were reduced to 8 pups. Four pups from each litter were sacrificed at 32 days and the remainder at 35
349 days after conception, which was usually 11 and 14 days after birth. The animals were anesthetized with ether prior to sacrifice and bled by cardiac puncture. Each pup was weighed and its crown-rump length measured. From each group of 4 pups, the cerebellum and cerebrum of the one animal with a median weight was fixed for histological analysis. The remaining 3 cerebellums were pooled for biochemical analysis as were the three cerebrums. Histology One-half of the brain was placed in formalin and the other half in water/acetic acid/formalin/alcohol (5/3/1/1). After 8 days the AFA fixed material was dehydrated and then serially embedded in celloidin (1 ~ ) and paraffin. Sections were cut at 8/~m, stained with thionin (1 ~ ) and differentiated with ethanol. Histological measurements were made with a graded reticle using a 40 x lens. There are 50 divisions in the reticle and each division is 2.5 # m with the 40 x lens. Only midsagittal sections of the vermis region were used for histological analyses. In each of 6 areas of cerebellum (nodule, uvula, tuber, culmen, central lobe, lingua) measurements were made of EGC layer thickness, depth of molecular layer and the diameter of 8 Purkinje cells. Three sections of cerebellum were evaluated for each animal. A total of 10 ethanol treated pups and 10 pair-fed pups were analyzed histologically at each of the two days (day 11 and 14 postnatum). At least one pup from each dam described in Table I was studied. Biochemical measurements Tissue samples were placed in 5 vols of a 0.32 M sucrose solution containing 0.001 M MgClz and 0.0004 M phosphate buffer, pH 7.2. All samples were kept at 4 °C. The tissues were homogenized in a Dounce tissue grinder with 15 strokes of the B plunger. The aspartyl aminotransferase (AAT, EC 2.6.1.1) activity was determined on the unfractionated homogenate using the Karmen procedure z°. The decrease in absorbance at 340 nm of N A D H was measured in a constant temperature cuvette holder (25 °C). The rate of change was maintained below 0.07 OD units per min. Ornithine decarboxylase (ODC, EC 4.1.1.17) activity was determined by minor modifications of the method described by Snyder and Russell 44. The supernatant prepared by centrifugation of the brain homogenates at 20,000 x g for 30 rain was used as enzyme source. DL-[l-14C]ornithine (purchased from New England Nuclear) was incubated with 0.4 # M pyridoxal phosphate, 1 # M dithiothreitol, 0.2 /~M ornithine, 0.1 ml of tissue sample and 100/~M phosphate buffer, pH 7.2, in the center well of a modified Erlenmeyer Flask. Incubation was for 30 min at 37 °C after which 2.0 M citric acid was added. The 14CO2 was captured in the center well containing ethanolamine and counted in a scintillation counter. Protein determinations were done by the Lowry procedure 24 with bovine serum albumin as the standard. Thyroxine (T4) levels of serum were measured by radioimmunoassay using the
350 commercial kit prepared by Bio-Rad laboratories. The standard curve was prepared using 6 concentrations of T4 (0-20 #g/dl). A coefficient of variation of 4.4 Yohas been obtained from monthly quality control studies; a between-run standard deviation of 0.186 #g/dl was determined at T4 concentrations of 4.27/zg/dl where n=20.
Statistical analyses D a t a were analyzed by least-squares analyses of covariance 42. Linear models for analysis of body, cerebellum a n d cerebrum weight a n d c r o w n - r u m p length included effects of treatment, day of sacrifice, their interaction a n d regressions within treatment on cumulative food intake d u r i n g gestation. F o r cerebellum a n d cerebrum a n additional analysis fitted body weight as a covariate to determine if treatment differences i n these organ weights could be explained solely by body weight. The same statistical procedures were applied to the analysis of E G C layer thickness a n d molecular layer depth. RESULTS The p r e g n a n t rats placed on the liquid Ensure diet d r a n k approximately 100 ml per day prior to the inclusion of ethanol. Those dams placed on the 9 ~o ethanol diet reduced their intake to 40-60 ml of the liquid diet per day. O f the 30 p r e g n a n t rats examined, a total of 14 animals (ethanol treated a n d pair-fed controls) were used in the analyses reported below. These pair-fed a n i m a l s were of similar weight at pregnancy a n d their gestation period was of similar d u r a t i o n (Table I). TABLEI Food and alcohol consumption by pregnant rats Dam
Days gestation
ml EtOH consumed*
ml Ensure consumed**
No. pups in litter at birth
1 2 3 4 5 6 7 8 9 10 11 12 13 14
22 22 23 22 21 22 23 22 22 22 20 21 21 22
83 -87 -76 -72 -57 -71 -62 --
665 693 693 693 763 728 679 644 546 546 707 714 588 609
18 10 7 8 14 15 11 9 10 11 11 11 11 15
EtOH PF1 EtOH PF3 EtOH PF5 EtOH PF7 EtOH PF9 EtOH PFI1 EtOH PF13
* The ml EtOH consumed refers to total amount of 100% EtOH consumed between days 3 and 21 after conception. ** The ml Ensure consumed between days 8 and 21 after conception are reported in this column.
351 TABLE II Least squares means and regression coefficients of the effect of ethanol on body, cerebellar and cerebral weight of pups Characteristic analyzed
Body weight (g) Cerebellar weight (mg) Cerebral weight (mg) Crown-rump length (cm)
Means at day 11"
Means at day 14"
Regression coefficients with food intake* *
Alcohol
Pair-fed
Alcohol
Pair-fed
Alcohol
Pair-fed
25.0 74.9 907 7.1
25.4 83.3 941 7.4
33.3 113.6 1016 8.1
34.6 130.1 1060 8.2
0.020 ~:).054 0.068 0.001
0.073 0.101 0.806 0.005
* Means differed for all characteristics (P < 0.05) as a result of treatment. ** Within treatment regressions differed for all characteristics (P < 0.01). Effects o f ethanol on cerebral, cerebellar and body weight
Ethanol ingestion by pregnant dams had a detrimental effect on the postnatal growth of their pups (Table II). Pups from dams that drank ethanol during pregnancy had smaller body weights (P < 0.01), smaller cerebellar weights (P < 0.05), smaller cerebral weights (P < 0.01) and smaller c r o w n - r u m p lengths (P < 0.05) than pups from pair-fed controls. As expected, pups sacrificed at day 14 postnatum had larger (P < 0.0001) mean values for these characteristics than those sacrificed at day 11. The relative difference between alcohol and pair-fed control pups remained constant from day 11 to day 14. There was no suggestion of an interaction of treatment with day of sacrifice for any of these characteristics. To determine if the relationship between food intake and brain or body weight differed in the ethanol and pair-fed controls, the linear regression on cumulative food intake was calculated. For each characteristic analyzed (Table II) the regressions differed significantly between treatments indicating that the toxic effects of ethanol changed the relationship between a dam's food intake during pregnancy and the subsequent growth of her pup. As an example of this, body weight increased at a rate of 0.020 g/ml food intake in ethanol treated pups, whereas pair-fed control pups increased at a rate of 0.073 g/ml food intake by the pregnant dam. The results for c r o w n - r u m p length were similar. TABLE III Least squares means for cerebellar and cerebral weights adjusted to common body weight by analysis of covariance Character~t~ analyzed
Cerebellar weight Cerebral weight
Means as a function of treatment
Means as a function of age
Alcohol
Pair-fed controls
11 days
14 days
95 962
106 997
86 974
115 985
P < 0.01 P < 0.01
P < 0.001 P < 0.05
352 The regression analyses based on brain weight and maternal food intake differed between ethanol and pair-fed animals (P < 0.01). In the alcohol treated pup group, the regressions were not significantly different from zero. This suggested that the ethanol had a greater effect on brain weight than would be predicted from its effects on body weight. To investigate further whether brain was selectively at a disadvantage when exposed to ethanol in utero, analysis of covariance was used to adjust the brain measures to a common body weight (Table II1). The mean cerebellar weights in the pair-fed controls were 11 O//ogreater (P < 0.01) than in ethanol treated pups on an 'equal body weight basis'. The mean cerebral weights were also greater by 3 ~ in the pair-fed controls than in ethanol treated pups (P < 0.05). The key point of Table III is the demonstration that alcohol ingestion during pregnancy interferes with the allometric relationship between brain and body weight. Many treatments can reduce brain weight at 11 or 14 days but may do so solely by causing a reduction in body weight. That is, the allometric relationship may remain intact; the brains are smaller simply because the pups are smaller. These analyses suggest to us that brain tissue is selectively at a disadvantage after exposure to ethanol in utero. The question remained whether the observed changes in cerebellar and cerebral development were due solely to the intake of alcohol during pregnancy or whether the changes reflected a 'carry-over effect' into the lactation phase of growth. To investigate this point, pups of two sets of alcohol treated and pair-fed dams were examined in the following manner: each dam nursed 4 pups that had been exposed to alcohol in utero and 4 pups that had not. For body, cerebellar and cerebral weights and crown-rump length the same inferences were obtained as in Table II. By these critelia there was no significant difference between the postnatal care of pups by alcohol treated and pair-fed dams and we conclude that the 'carry-over effects' were small. Effect of ethanol on serum thyroxin levels and on cerebellar and cerebral AA T and ODC activities
Table IV describes the serum thyroxine levels and selected enzyme activities in brain samples of the same set of animals reported in Table I. The data reveal that (l) the cerebellar AAT activity of rats exposed in utero to ethanol was significantly lower than that of controls at day 11 and 14 postnatum; (2) the serum T4 levels of ethanol treated pups were significantly lower at 11 days postnatum compared to those of controls; (3) the cerebellar ODC activities of ethanol treated pups were generally higher than that of controls except for those controls which had a low food intake. The reduced AAT activities in the cerebellums of ethanol treated pups suggest an impaired mitochondrial function in this brain region. The normal cerebral and midbrain AAT activities indicate that the cerebellums were selectively affected by ethanol. The AAT may be most affected in cerebellum because this brain region has an unusually high alcohol content in fetuses exposed to ethanol 17 or because neurons in the cerebellum are particularly susceptible to ethanol. The lowered AAT activity however may be directly related to the reduced weight of cerebellum in the ethanol treated rats since mitochondrial accumulations are detected in growing dendrite tips4L
353 TABLE IV Cerebellar and cerebral enzyme activities and serum thyroxine leve
Units: *Karmen units/mg protein; **picomoles ornithine decarboxylated/min/mg protein, ***/~g T4/dl. The cerebellar AAT activities of ethanol treated animals are lower (P < 0.02 at 11 days and P < 0.04 at 14 days) than the controls. The T4 levels are also lower (P < 0.04). Dam
1 EtOH 2 PF1 3 EtOH 4 PF3 5 EtOH 6 PF5 7 EtOH 8 PF7 9 EtOH 10 PF9 11 EtOH 12 P F l l 13 EtOH 14 PFI3
Cerebellar A A T *
Cerebral A A T *
Cerebellar ODC** Cerebral Serum thyroxine*** ODC* *
l l-day
14-day
11-day
14-day
l l-day
229 335 300 417 276 349 347 334 298 353 303 328 264 270
464 538 499 559 405 454 470 488 456 486 408 490 438 391
416 422 426 439 235 399 367 404 352 342 294 246 270 208
. . . . 457 363 434 378 379 343 479 495 433 426
. . . . 2.2 1.4 2.7 1.4 2.5 2.4 1.3 0.7 1.8 1.9
. . . .
14-day
1 l-day
l l-day
14-day
0.39 0.32 0.59 0.41 0.5 0.38 0.32 0.18 0.23 0.28
2.8 4.1 2.3 4.4 2.4 3.5 2.4 3.6 3.2 3.0 3.6 4.2 2.9 2.9
4.2 5.1 4.6 5.6 2.8 3.0 2.6 4.2 3.4 2.6 3.8 4.0 2.6 2.7
. . . .
Ethanol 288 4- 14 447 4- 13 337 4- 27 4364-16 2.1 4-0.2 Pair-fed 340 4- 16 486 4- 20 351 4- 34 401 4- 27 1.6 4- 0.3
0.8 0.4 0.3 0.03 0.19 0.05 0.02 0.01 0.01 0.01
2.84-0.2 3.44-0.3 3.8 4- 0.3 3.8 4- 0.4
These a c c u m u l a t i o n s are p r e s u m e d to p a r t i c i p a t e in the g r o w t h o f the Purkinje cell dendrites. R e d u c e d m i t o c h o n d r i a l activity w o u l d t h e r e b y result in i m p e d e d developm e n t o f the Purkinje cell dendrite. A n a l y s e s o f the m i t o c h o n d r i a l enzyme malic d e h y d r o g e n a s e activity ( M D H ) in the cerebellums o f these rats revealed r e d u c e d M D H activity in e t h a n o l t r e a t e d pups which is consistent with the A A T data. The serum t h y r o x i n e levels o f e t h a n o l treated pups were significantly r e d u c e d at d a y 11 (3.3 # g total T4/dl) c o m p a r e d with controls (3.8/~g/dl). The n o r m a l total T4 level in sera o f 12-day-old rats is r e p o r t e d 11 to be 3.5 #g/dl. By day 14 the p r o g e n y o f e t h a n o l a n d pair-fed d a m s h a d n o r m a l serum T4 levels. Since T4 is r e q u i r e d for n o r m a l cerebellar d e v e l o p m e n t a n d it has been shown t h a t T4 does n o t cross the p l a c e n t a 11, the small size a n d i m p e d e d d e v e l o p m e n t o f the cerebellum in a l c o h o l t r e a t e d pups m a y be due in p a r t to the r e d u c e d T4 levels. Pups b o r n to rats 10 a n d 14 (those pair-fed c o n t r o l s receiving the least food) also h a d r e d u c e d serum T4 levels at days 11 a n d 14. The activity o f O D C in cerebellum rises d u r i n g the first week o f life p o s t n a t u m a n d declines after the eleventh day a. This p e a k is a function o f r a p i d cell division ( p r e s u m a b l y o f granule cells). The cerebellar O D C activity o f the e t h a n o l treated pups was usually b u t n o t u n i f o r m l y increased a b o v e t h a t o f pair-fed controls in o u r study. The p r o g e n y o f c o n t r o l rats l 0 a n d 14 h a d elevated cerebellar O D C activities at d a y 11. By d a y 14 the O D C levels h a d declined to n o r m a l in all a n i m a l sets. The O D C d a t a also reveal t h a t either e t h a n o l ingestion o r r e d u c e d f o o d i n t a k e d u r i n g p r e g n a n c y
354 TABLE V Mean external granule cell layer (EGC) thickness and Purkinje cell soma depth by region Regions: l, nodule; 2, uvula; 3, tuber; 4, culmen; 5, central lobe; 6, lingua. Ten ethanol treated pups and 10 pair-fed pups were analyzed histologically at each of the two days. TreatmentDay
Ethanol Control
11 14 11 14
EGC thickness (Fire)
Purkinje cell depth (t~m)
l
2
3
4
5
6
1
2
3
4
5
6
41.8 22.1 38.6 17.1
42.9 17.5 36.8 14.3
40.4 17.5 37.9 18.3
47.5 24.6 38.2 17.5
40.0 21.4 35.0 15.4
32.1 19.3 33.6 16.3
98 106 95 121
100 126 97 129
109 126 109 126
108 134 118 141
115 145 120 143
97 126 110 130
may result in delayed cerebellar d e v e l o p m e n t o f progeny. Th e observation o f Schanberg 46 that withdrawal o f e th a n o l f r o m the diet o f n e o n a t a l rats results in elevation o f cerebellar O D C activity is consistent with the data presented in o u r study. T h e cerebral O D C levels in pups f r o m e th an o l treated and co n t r o l dams were low even at day 11 p o s t n a t u m , consistent with previous reports a that cerebral O D C levels decline earlier than cerebellar O D C .
}~i~%~Lii~;.~i}~i ~i}i~d~
~ L~LVk~IjI~
~L~L;
!x~viW~/~xu ¸ z ~
~i ~; ~ } ~
Fig. 1. Sagittal sections through the cerebellar vermis of ethanol treated and pair-fed control pups. Section a is from an ethanol treated pup sacrificed 11 days postnatum and section b is from a control at 11 days. Section c is from an ethanol treated pup sacrificed 14 days postnatum and section d is from the control. Arrows indicate apparent fusion of adjacent folia in the 14-day-old cerebellums. Sections were thionin stained, x 10.7.
355
Effect of ethanol on cerebellar histogenesis For each group of 4 pups sacrificed either at day 11 or 14, the cerebellum of the one animal with median body weight was examined histologically to determine the thickness of the external granule cell layer, the depth of the molecular layer and the diameter of the Purkinje cells. Six regions (nodule, uvula, tuber, culmen, central lobe and lingua) were examined and the datum for an individual rat at a region was the mean of three measurements. Table V indicates the thinning of the EGC layer associated with the maturation of the cerebellar cortex (P < 0.001). Ethanol effectively retarded the thinning of the EGC layer (P < 0.05) in the culmen. When the data from all 6 regions were treated as a common pool, the EGC layer of ethanol treated pups was significantly thicker at day 11 (P < 0.02) and 14 (P < 0.01) than that of pair-fed controls. For Purkinje cell depth and diameter (16 # m at day 11 and 18 # m at day 14) there was no significant effect of ethanol treatment in any region. Figure 1 shows sagittal sections through the vermis of ethanol treated and pairfed rat pups at days 11 and 14 postnatum. The cerebellar areas of ethanol treated rats were smaller at both days and the EGC layer appears thicker at day 14 consistent with the other data presented above. Sites of apparent fusion between adjacent folia were observed both in the ethanol treated and pair-fed control cerebellums obtained from the 14-day-old pups. The significance of this is unclear. DISCUSSION In this report it has been shown that the administration of ethanol to pregnant rat dams between the 3rd and 20th day of gestation resulted in an impeded development of the cerebellum and to a lesser extent of cerebrum in their progeny. The abnormal cerebellar development was characterized by a reduction in cerebellar mass, a significant increase in the thickness of the EGC layer at days 11 and 14 postnatum and a reduced level of AAT and M D H activity, reflecting reduced mitochondrial activity. The normal occurrence of AAT and M D H activities in the cytosol and mitochondria precludes a conclusion that the entire reduction in activity was from the mitochondrial compartment. The pups exposed to ethanol in utero also had a reduced serum level of T4 at day 11. A delayed maturation of the cerebellar cortex has been observed in several conditions: (1) in rat pups born to dams that were placed on a very restricted caloric intake during pregnancy or shortly after deliveryS,13,26,28,3z,34,43,4s,49;(2) in rats that were made hypothyroid in the perinatal period4,7,1°,14,29; (3) in rat pups exposed in utero to alcoho16,a8,46, 47 or methadoneSa; (4) in children born with cystic kidneys z2 or Menkes kinky hair disease 18. In certain of these cases the delay in cerebellar development occurred at the stage in ontogenesis when Purkinje cells have begun to elaborate their apical dendrite but still retained their perisomatic protuberances16, 22. In the malnutrition, hypothyroid, methadone and fetal alcohol syndromes the primary affect appears to be on the proliferation of granule cellsSA a,27 and elaboration of the Purkinje cell dendrite and dendritic spines. The increased thickness of the EGC layer in the alcohol treated pups at days 11
356 and 14 postnatum also suggests that the inward migration of their granule cells may have been delayed. Delayed migration of cerebellar granule cells has been demonstrated to occur in kittens subsequent to cortical ablation 15 and in other species following chemical, infectious and physical insults 36. It has been postulated that exposure of fetuses to alcohol does result in aberrant neuronal migration patterns ~6. Our observations suggest that the proliferation and migration of granule cells may be selectively affected by the in utero exposure of the rat fetuses to alcohol. The cerebellar changes have been readily detected by measurements of the cerebellar mass, the EGC layer thickness, and the synaptic bouton density on Purkinje cell dendrites. Cerebral changes have been described in hypothyroid rats 1l, fetal alcohol syndrome6,19,41; changes in the density of synaptic spines on pyramidal cells in hippocampus were described in rats exposed to alcohol 40. From these reports it is concluded that the development of several CNS regions are affected by ethanol and malnutrition, but that the cerebellar changes are most pronounced. The questions arising from these observations are threefold: (1) is the delayed cerebellar development due to the exposure of rat fetuses to ethanol or to reduced food intake by the pregnant dams; (2) if ethanol does cause the delay in cerebellar development is this chemical acting directly on the cerebellar anlage either during its administration or as a result of withdrawal; (3) does the ethanol cause a delay in the synthesis or release of a hormone (i.e. thyroxine) necessary for cerebellar development ? Although both malnutrition and the exposure of rat pups in utero to ethanol have very similar effects on cerebellar development, the results presented here on the ethanol treated pups are not due primarily to malnutrition. The reasons for this conclusion are: (1) the body weights of pups exposed to ethanol were similar to that of controls in this experiment whereas the weights of malnourished rats may be 50-60 of controls13; (2) the caloric and nutritional diet of ethanol treated and control rats was similar during their gestation period; (3) the young rats with the most marked reduction in cerebellar/body mass ratio were born to the dams consuming the most food and the most alcohol. Although the results of ethanol treatment in the present experiment cannot be explained as due to a malnutrition syndrome, the reduced food intake in ethanol treated and control rats did affect cerebellar development. The weights of cerebellums from ethanol treated and pair-fed controls were reduced from approximately 100 mg (normal cerebellar weight of I 1-day-old pups born to dams fed solid laboratory chow) to 74-83 mg. The similarity between the effects of the alcohol, malnutrition and methadone on the developing cerebellums suggests that they may act via a common pathway. The ethanol may act on the cerebellar anlage either in the prenatal period and thereby affect cerebellar development or in the postnatal period as a consequence of its withdrawal. In both situations the ethanol may be considered to effect changes in cerebellar development by acting directly on the anlage as opposed to acting via a hormone. For example ethanol causes a dose dependent reduction of cerebellar c G M P levels z5,39. The ethanol effect on cGMP however may be reversed by TRH, implying that the adult cerebellum responds directly to ethanol and TRH.
357 The studies of Schanberg and colleagues reveal that the developing cerebellum is responsive to ethanol withdrawal46. They have shown that if ethanol is withdrawn at birth, cerebellar ODC activity in ethanol treated neonatal tissue is similar to that of controls. If ethanol is withdrawn from the maternal diet during the third day postnatum the neonatal cerebellar ODC activity doubles by the fourth day and if withdrawn on the fifth day postnatum, the ODC activity increases 50 ~ by day eight. The differences in cerebellar ODC activity are dependent upon the time of ethanol withdrawal. Although it does not appear possible to conclude unambiguously whether withdrawal from alcohol significantly affected our results, the 11-day delay between withdrawal and tissue analysis reduces this likelihood. It is relevant in this regard that cerebellar development is most rapid between the 5th and 14th day postnatumS, 9,50 which is longer than 4 days after ethanol withdrawal. During the 5th to 14th day the following changes occur: basket cells appear at day 5; succinic dehydrogenase and lactic dehydrogenase activity appears as in adult at day 7 and by day 11-14 the Purkinje cell apical dendrites develop and the Bergmann astrocytes cover Purkinje soma. We have reported here that AAT and MDH activity were reduced markedly in the cerebellums of ethanol treated pups. Malnutrition also caused a delay in the appearance of mitochondrial oxidative activity in the developing apical dendrite of Purkinje cells84 with a prolonged elevation of glycolytic activity. If mitochondrial aggregates are involved in dendritic growth as proposed by Sotelo and Palay45 the reduced oxidative activity in ethanol treated or malnourished rats may be causally related to the reduced cerebellar mass seen in these animals. We do not consider however that ethanol is acting primarily on the cerebellar anlage because methadone withdrawal, hypothyroidism and malnutrition all effect similar changes. It would be more parsimonious to postulate that each of these conditions causes malfunction of a signal (i.e. hormone) necessary for normal development of the cerebellum. The hormones most likely to be involved are thyroxine and growth hormone. Previous studies have demonstrated that cerebellar development in ratsS, 11,50, macaques 21,a5 and humans 11,a7,54 occurs coordinately with the appearance of T4 in their sera. More directly, it has been demonstrated that thyroid deficient neonatal rats have a reduced cerebellar mass, but that there is no reduction in cerebellar cell density or Purkinje cell body diameter4,14,29. Ethanol significantly reduced sera T4 levels in the neonatal rat (the data presented this paper) and in adult rats as. These data are consistent with the postulate that the primary action of ethanol may be to delay the elevation of T4 concentrations in the sera during the neonatal period. This delayed T4 elevation may then cause the delayed cerebellar maturation. The determination that functional hypothyroidism in neonatal rats may be a primary result of the administration of alcohol to pregnant dams is important because administration of T4 to the neonates may then reverse the delay in cerebellar development. Since T4 also regulates the plasma growth hormone level in rat 27 it is possible that cerebellar development is also dependent upon growth hormone levels.
358 ACKNOWLEDGEMENTS
The authors express their appreciation to E. Langer, D. Voss and N. Millan for their assistance in this project. This project was supported by grants from the Medical and Graduate Schools of the University of Wisconsin.
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359 23 Larsell, O., The Comparative Anatomy and Histology of the Cerebellumfrom Monotremes through Apes, J. Jansen (Ed.), University of Minnesota Press, Minneapolis, 1970. 24 Lowry, O., Rosebrough, N., Farr, A. and Randall, R., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 25 Mailman, R., Frye, G., Muller, R. and Breese, G., Thyrotropin-releasing hormone reversal of ethanol induced decreases in cerebellar cGMP, Nature (Lond.), 272 (1978) 832-833. 26 McConnell, P. and Berry, M., The effects of undernutrition on Purkinje cell dendritic growth in the rat, J. comp. Neurol., 177 (1978) 159-172. 27 Montes, A., Herras, F. and Jolin, T., Effects of thyroidectomy and thyroxine on plasma growth hormone and insulin levels in rats, Hormone Res., 8 (1977) 148-158. 28 Neville, H. and Chase, H., Undernutrition and cerebellar development, Exp. Neurol., 33 (1971) 458-497. 29 Nicholson, J. and Altman, J., Effects of early hypothyroidism and hyperthyroidism on development of rat cerebellar cortex I, Brain Research, 44 (1972) 13-23. 30 O'Leary, J., Inukai, J. and Smith, J., Histogenesis of the cerebellar climbing fiber in the rat, J. comp. NeuroL, 142 (1971) 377-391. 31 Palay, S. and Chan-Palay, V., Cerebellar cortex; Cytology and Organization, Springer, New York, 1974. 32 Patel, A., Balazs, R. and Johnson, A., Effect of undernutrition on cell formation in the rat brain, J. Neurochem., 20 (1973) 1151-1165. 33 Patel, D., Singh, S., Kabir, M. and Premachandra, B., Effect of chronic ethanol on thyroid function in rats, Fed. Proc., 37 (1978) 519. 34 Persson, L. and Sima, A., The effect of pre- and postnatal undernutrition on the development of the cerebellar cortex in the rat, Neurobiology, 5 (1975) 151-166. 35 Pickering, D. and Kontaxis, N., Thyroid function in the foetus of the macaque monkey (Macaca mulatta), II, J. EndocrinoL, 23 (1961) 267-275. 36 Rakic, P., Cell migration and neuronal ectopias in the brain, Birth Defects: Original Article Series, 11 (1975) 95-130. 37 Rakic, P. and Sidman, R., Histogenesis of cortical layers in human cerebellum, particularly the lamina dissecans, J. comp. Neurol., 139 (1970) 473-500. 38 Rawat, A., Ribosomal protein synthesis in the fetal and neonatal rat brain as influenced by maternal alcohol consumption, Res. Commun. chem. path. Pharmac., 12 (1975) 723-732. 39 Redos, J., Hunt, W. and Catravas, G., Ethanol induced depletion of cerebellar guanosine 3',5'cyclic monophosphate, Science, 193 (1976) 58-59. 40 Riley, J. and Walker, D., Morphological alterations in hippocampus after long-term alcohol consumption in mice, Science, 201 (1978) 646-648. 41 Rosman, N. and Malone, M., Reversal of delayed myelinogenesis in the fetal alcohol syndrome, Neurology (Minneap.), 27 (1977) 369. 42 Searle, S. R., Linear Models, Wiley, New York, 1971, 340 pp. 43 Sima, A. and Persson, L., The effect of pre and postnatal undernutrition on the development of rat cerebellar cortex I, Neurobiology, 5 (1975) 23-34. 44 Snyder, S. and Russell, D., Polyamine synthesis in rapidly growing tissues, Fed. Proc., 29 (1970) 1575-1582. 45 Sotelo, C. and Palay, S., Fine structure of lateral vestibular nucleus in rat. I. Neurons and neuroglial cells, J. Cell Biol., 36 (1968) 151-179. 46 Thadani, P., Lau, C., Slotkin, T. and Schanberg, S., Effect of maternal ethanol ingestion on neonatal rat brain and heart ornithine decarboxylase, Biochem. Pharmacol., 26 (1977) 523-527. 47 Volk, B., Delayed cerebellar histogenesis in embryo-fetal alcohol syndrome: light microscopic study of the rat cerebellum, Acta neuropath. (BerL), 39 (1977) 157-163. 48 Winick, M., Effects of malnutrition on the maturing CNS. In W. J. Friedlander (Ed.), Advances in Neurology 13, Current Reviews, Raven Press, New York, 1975, pp. 193-246. 49 Winick, M. and Nobel, A., Cellular response in rats during malnutrition at various ages, J. Nutrit., 89 (1966) 300-306. 50 Woodward, D., Hoffer, B. and Lapham, L., Postnatal development of electrical and enzyme histochemical activity in Purkinje cells, Exp. NeuroL, 23 (1969) 120-139. 51 Yu, M., Lee, J. and Bakay, L., The ultrastructure of the rat central nervous system in chronic undernutrition, Acta neuropath. (BerL), 30 (1974) 197-210.
360 52 Yu, M., Yu, W.-H., Ultrastructural changes in the developing rat cerebellum in chronic undernutrition, Neuropath. appl. Neurol., 3 (1977) 391-402. 53 Zagon, I. and McLaughlin, D., Perinatal methadone exposure and brain development: A biochemical study, J. Neurochem., 31 (1978) 49-54. 54 Zecevic, N. and Rakic, P., Differentiation of Purkinje cells and their relationship to other components of developing cerebellar cortex in man, J. comp. Neurol., 167 (1976) 27-48.
347
I M P E D E D C E R E B E L L A R D E V E L O P M E N T A N D R E D U C E D SERUM T H Y R O X I N E LEVELS ASSOCIATED W I T H F E T A L A L C O H O L INTOXICATION
S. E. KORNGUTH, J. J. RUTLEDGE, E. SUNDERLAND, F. SIEGEL, I. CARLSON, J. SMOLLENS, U..IUHL and B. YOUNG University Wisconsin Medical Center Madison, Wisc. 53706 (U.S.A.)
(Accepted March 8th, 1979)
SUMMARY Pregnant albino rats were placed on a complete liquid diet (Ensure) containing either 9 ~ ethanol or an isocaloric amount of sucrose between the third and twentieth day of gestation. The pups born to these rats were sacrificed either day 11 or day 14 postnatum and morphometrical, histological and biochemical analyses were done on their cerebellums and cerebrums. Pups that were exposed to ethanol in utero had significantly smaller body weights, cerebrums and cerebellums than pair-fed controls. The cerebellar mass was reduced by 10 ~o and the cerebral weight by 3 ~ in the pups exposed to alcohol when body weights were normalized to that of pair-fed controls. Cerebellar aspartyl aminotransferase (EC 2.6.1.1) activity was reduced at day 11 and 14 in ethanol treated pups compared with controls. Serum T4 levels were also reduced in the ethanol treated group. Histological analyses revealed that the external granule cell (EGC) layer of ethanol treated pups was significantly thicker at 11 and 14 days postnatum than that of pair-fed control pups. Cerebellar ornithine decarboxylase (ODC, EC 4.1.1.17) activity was higher at day 11 in the ethanol treated pups than in controls. The reduced mass, AAT activity, T4 serum levels and the increased thickness of the EGC layer indicate a delayed or impeded maturation of cerebellum in ethanol treated pups. These data are considered from the viewpoint that ethanol, other drugs such as methadone and prenatal stress (malnutrition) may cause delayed cerebellar maturation by reducing serum T4 levels in the early postnatal period (day 5-14).
INTRODUCTION The normal development of rat cerebellar cortex has been examined from gross anatomical 2a, histological2,S,lS, 5°, ultrastructural30, 31 and biochemicall,a,l~, 50 perspectives. These studies have described the time of maximal appearance of the external
348 granule cell (EGC) layer, the inward migration of EGCs and the superficial migration of presumptive Purkinje cells (PC), the period during which the apical dendrite of the PC develops, the development of synaptic spines and branches on PC dendrites and the increase in oxidative metabolic capacity during PC maturation. Changes in the normal developmental pattern of the cerebellar cortex have been demonstrated to occur in rat pups that were malnourished~,la,2a,28,a2,34,43,48,49,sa, 52, caused to become functionally hypothyroid4,7,10,14,29 or exposed either to ethano16,as,46, 47 or methadone ~3 during the pre- or early postnatal period. It is of particular significance that the characteristics of delayed cerebellar development were similar in rat pups that were malnourished, hypothyroid, ethanol or methadone treated. This suggests that all of these agents may exert their action by impairing the synthesis or release of a common product necessary for normal cerebellar development (i.e. thyroxine). In this report the effect of ethanol on the development of rat cerebellar cortex is examined by gross anatomical, histological and biochemical techniques. It will be shown that the ethanol induced changes may be related to the reduced serum T4 levels in pups exposed to ethanol. The reduced T4 levels in the pup serum are of significance because T4 does not traverse the placenta 11. MATERIALS AND METHODS Selection and diet of rats Albino rats (250-300 g) from an inbred colony, originally obtained from Holtzman Laboratories, Madison, Wisc., were mated in individual cages during a 16 h period. The next morning those female rats with an ejected vaginal plug were selected as pairs on the basis of weight (pairs were within 10 g). During the entirety of pregnancy rats were maintained on a liquid diet. All animals were provided with a solution of Ensure (Ross Laboratories, Columbus, Ohio) containing 7 parts Ensure to 3 parts distilled water ( 7 0 ~ v/v). Ensure contains 1 calorie per ml and is vitamin supplemented. During the first and second day of pregnancy the ethanol treated pregnant rats were maintained on the 70 ~ Ensure diet. From the 3rd through the 20th day they were provided ad libitum with 100 ml of liquid diet containing 9 ~ ethanol (9.4 ml of 95 ~ ethanol, 70 ml Ensure, 20.6 ml water). The amount of liquid taken by each animal was determined between 09.00 and 10.00 h daily and fresh food was provided daily between 10.00 and 11.00 h. On the 21st day the alcohol content was reduced to 5 ~ and on the 22rid day it was reduced to 2.5 0/oo.The day after birth all rats were given solid rat chow (Purina). The pair-fed controls were given an isocaloric amount of sucrose in lieu of ethanol; 100 ml of diet contained 70 ml Ensure, 13.5 ml of 87 ~ sucrose and 16.5 ml water. The pair-fed controls were administered the volume of liquid diet consumed by their pair-mate the previous day. On the second day after birth, all litters were reduced to 8 pups. Four pups from each litter were sacrificed at 32 days and the remainder at 35
349 days after conception, which was usually 11 and 14 days after birth. The animals were anesthetized with ether prior to sacrifice and bled by cardiac puncture. Each pup was weighed and its crown-rump length measured. From each group of 4 pups, the cerebellum and cerebrum of the one animal with a median weight was fixed for histological analysis. The remaining 3 cerebellums were pooled for biochemical analysis as were the three cerebrums. Histology One-half of the brain was placed in formalin and the other half in water/acetic acid/formalin/alcohol (5/3/1/1). After 8 days the AFA fixed material was dehydrated and then serially embedded in celloidin (1 ~ ) and paraffin. Sections were cut at 8/~m, stained with thionin (1 ~ ) and differentiated with ethanol. Histological measurements were made with a graded reticle using a 40 x lens. There are 50 divisions in the reticle and each division is 2.5 # m with the 40 x lens. Only midsagittal sections of the vermis region were used for histological analyses. In each of 6 areas of cerebellum (nodule, uvula, tuber, culmen, central lobe, lingua) measurements were made of EGC layer thickness, depth of molecular layer and the diameter of 8 Purkinje cells. Three sections of cerebellum were evaluated for each animal. A total of 10 ethanol treated pups and 10 pair-fed pups were analyzed histologically at each of the two days (day 11 and 14 postnatum). At least one pup from each dam described in Table I was studied. Biochemical measurements Tissue samples were placed in 5 vols of a 0.32 M sucrose solution containing 0.001 M MgClz and 0.0004 M phosphate buffer, pH 7.2. All samples were kept at 4 °C. The tissues were homogenized in a Dounce tissue grinder with 15 strokes of the B plunger. The aspartyl aminotransferase (AAT, EC 2.6.1.1) activity was determined on the unfractionated homogenate using the Karmen procedure z°. The decrease in absorbance at 340 nm of N A D H was measured in a constant temperature cuvette holder (25 °C). The rate of change was maintained below 0.07 OD units per min. Ornithine decarboxylase (ODC, EC 4.1.1.17) activity was determined by minor modifications of the method described by Snyder and Russell 44. The supernatant prepared by centrifugation of the brain homogenates at 20,000 x g for 30 rain was used as enzyme source. DL-[l-14C]ornithine (purchased from New England Nuclear) was incubated with 0.4 # M pyridoxal phosphate, 1 # M dithiothreitol, 0.2 /~M ornithine, 0.1 ml of tissue sample and 100/~M phosphate buffer, pH 7.2, in the center well of a modified Erlenmeyer Flask. Incubation was for 30 min at 37 °C after which 2.0 M citric acid was added. The 14CO2 was captured in the center well containing ethanolamine and counted in a scintillation counter. Protein determinations were done by the Lowry procedure 24 with bovine serum albumin as the standard. Thyroxine (T4) levels of serum were measured by radioimmunoassay using the
350 commercial kit prepared by Bio-Rad laboratories. The standard curve was prepared using 6 concentrations of T4 (0-20 #g/dl). A coefficient of variation of 4.4 Yohas been obtained from monthly quality control studies; a between-run standard deviation of 0.186 #g/dl was determined at T4 concentrations of 4.27/zg/dl where n=20.
Statistical analyses D a t a were analyzed by least-squares analyses of covariance 42. Linear models for analysis of body, cerebellum a n d cerebrum weight a n d c r o w n - r u m p length included effects of treatment, day of sacrifice, their interaction a n d regressions within treatment on cumulative food intake d u r i n g gestation. F o r cerebellum a n d cerebrum a n additional analysis fitted body weight as a covariate to determine if treatment differences i n these organ weights could be explained solely by body weight. The same statistical procedures were applied to the analysis of E G C layer thickness a n d molecular layer depth. RESULTS The p r e g n a n t rats placed on the liquid Ensure diet d r a n k approximately 100 ml per day prior to the inclusion of ethanol. Those dams placed on the 9 ~o ethanol diet reduced their intake to 40-60 ml of the liquid diet per day. O f the 30 p r e g n a n t rats examined, a total of 14 animals (ethanol treated a n d pair-fed controls) were used in the analyses reported below. These pair-fed a n i m a l s were of similar weight at pregnancy a n d their gestation period was of similar d u r a t i o n (Table I). TABLEI Food and alcohol consumption by pregnant rats Dam
Days gestation
ml EtOH consumed*
ml Ensure consumed**
No. pups in litter at birth
1 2 3 4 5 6 7 8 9 10 11 12 13 14
22 22 23 22 21 22 23 22 22 22 20 21 21 22
83 -87 -76 -72 -57 -71 -62 --
665 693 693 693 763 728 679 644 546 546 707 714 588 609
18 10 7 8 14 15 11 9 10 11 11 11 11 15
EtOH PF1 EtOH PF3 EtOH PF5 EtOH PF7 EtOH PF9 EtOH PFI1 EtOH PF13
* The ml EtOH consumed refers to total amount of 100% EtOH consumed between days 3 and 21 after conception. ** The ml Ensure consumed between days 8 and 21 after conception are reported in this column.
351 TABLE II Least squares means and regression coefficients of the effect of ethanol on body, cerebellar and cerebral weight of pups Characteristic analyzed
Body weight (g) Cerebellar weight (mg) Cerebral weight (mg) Crown-rump length (cm)
Means at day 11"
Means at day 14"
Regression coefficients with food intake* *
Alcohol
Pair-fed
Alcohol
Pair-fed
Alcohol
Pair-fed
25.0 74.9 907 7.1
25.4 83.3 941 7.4
33.3 113.6 1016 8.1
34.6 130.1 1060 8.2
0.020 ~:).054 0.068 0.001
0.073 0.101 0.806 0.005
* Means differed for all characteristics (P < 0.05) as a result of treatment. ** Within treatment regressions differed for all characteristics (P < 0.01). Effects o f ethanol on cerebral, cerebellar and body weight
Ethanol ingestion by pregnant dams had a detrimental effect on the postnatal growth of their pups (Table II). Pups from dams that drank ethanol during pregnancy had smaller body weights (P < 0.01), smaller cerebellar weights (P < 0.05), smaller cerebral weights (P < 0.01) and smaller c r o w n - r u m p lengths (P < 0.05) than pups from pair-fed controls. As expected, pups sacrificed at day 14 postnatum had larger (P < 0.0001) mean values for these characteristics than those sacrificed at day 11. The relative difference between alcohol and pair-fed control pups remained constant from day 11 to day 14. There was no suggestion of an interaction of treatment with day of sacrifice for any of these characteristics. To determine if the relationship between food intake and brain or body weight differed in the ethanol and pair-fed controls, the linear regression on cumulative food intake was calculated. For each characteristic analyzed (Table II) the regressions differed significantly between treatments indicating that the toxic effects of ethanol changed the relationship between a dam's food intake during pregnancy and the subsequent growth of her pup. As an example of this, body weight increased at a rate of 0.020 g/ml food intake in ethanol treated pups, whereas pair-fed control pups increased at a rate of 0.073 g/ml food intake by the pregnant dam. The results for c r o w n - r u m p length were similar. TABLE III Least squares means for cerebellar and cerebral weights adjusted to common body weight by analysis of covariance Character~t~ analyzed
Cerebellar weight Cerebral weight
Means as a function of treatment
Means as a function of age
Alcohol
Pair-fed controls
11 days
14 days
95 962
106 997
86 974
115 985
P < 0.01 P < 0.01
P < 0.001 P < 0.05
352 The regression analyses based on brain weight and maternal food intake differed between ethanol and pair-fed animals (P < 0.01). In the alcohol treated pup group, the regressions were not significantly different from zero. This suggested that the ethanol had a greater effect on brain weight than would be predicted from its effects on body weight. To investigate further whether brain was selectively at a disadvantage when exposed to ethanol in utero, analysis of covariance was used to adjust the brain measures to a common body weight (Table II1). The mean cerebellar weights in the pair-fed controls were 11 O//ogreater (P < 0.01) than in ethanol treated pups on an 'equal body weight basis'. The mean cerebral weights were also greater by 3 ~ in the pair-fed controls than in ethanol treated pups (P < 0.05). The key point of Table III is the demonstration that alcohol ingestion during pregnancy interferes with the allometric relationship between brain and body weight. Many treatments can reduce brain weight at 11 or 14 days but may do so solely by causing a reduction in body weight. That is, the allometric relationship may remain intact; the brains are smaller simply because the pups are smaller. These analyses suggest to us that brain tissue is selectively at a disadvantage after exposure to ethanol in utero. The question remained whether the observed changes in cerebellar and cerebral development were due solely to the intake of alcohol during pregnancy or whether the changes reflected a 'carry-over effect' into the lactation phase of growth. To investigate this point, pups of two sets of alcohol treated and pair-fed dams were examined in the following manner: each dam nursed 4 pups that had been exposed to alcohol in utero and 4 pups that had not. For body, cerebellar and cerebral weights and crown-rump length the same inferences were obtained as in Table II. By these critelia there was no significant difference between the postnatal care of pups by alcohol treated and pair-fed dams and we conclude that the 'carry-over effects' were small. Effect of ethanol on serum thyroxin levels and on cerebellar and cerebral AA T and ODC activities
Table IV describes the serum thyroxine levels and selected enzyme activities in brain samples of the same set of animals reported in Table I. The data reveal that (l) the cerebellar AAT activity of rats exposed in utero to ethanol was significantly lower than that of controls at day 11 and 14 postnatum; (2) the serum T4 levels of ethanol treated pups were significantly lower at 11 days postnatum compared to those of controls; (3) the cerebellar ODC activities of ethanol treated pups were generally higher than that of controls except for those controls which had a low food intake. The reduced AAT activities in the cerebellums of ethanol treated pups suggest an impaired mitochondrial function in this brain region. The normal cerebral and midbrain AAT activities indicate that the cerebellums were selectively affected by ethanol. The AAT may be most affected in cerebellum because this brain region has an unusually high alcohol content in fetuses exposed to ethanol 17 or because neurons in the cerebellum are particularly susceptible to ethanol. The lowered AAT activity however may be directly related to the reduced weight of cerebellum in the ethanol treated rats since mitochondrial accumulations are detected in growing dendrite tips4L
353 TABLE IV Cerebellar and cerebral enzyme activities and serum thyroxine leve
Units: *Karmen units/mg protein; **picomoles ornithine decarboxylated/min/mg protein, ***/~g T4/dl. The cerebellar AAT activities of ethanol treated animals are lower (P < 0.02 at 11 days and P < 0.04 at 14 days) than the controls. The T4 levels are also lower (P < 0.04). Dam
1 EtOH 2 PF1 3 EtOH 4 PF3 5 EtOH 6 PF5 7 EtOH 8 PF7 9 EtOH 10 PF9 11 EtOH 12 P F l l 13 EtOH 14 PFI3
Cerebellar A A T *
Cerebral A A T *
Cerebellar ODC** Cerebral Serum thyroxine*** ODC* *
l l-day
14-day
11-day
14-day
l l-day
229 335 300 417 276 349 347 334 298 353 303 328 264 270
464 538 499 559 405 454 470 488 456 486 408 490 438 391
416 422 426 439 235 399 367 404 352 342 294 246 270 208
. . . . 457 363 434 378 379 343 479 495 433 426
. . . . 2.2 1.4 2.7 1.4 2.5 2.4 1.3 0.7 1.8 1.9
. . . .
14-day
1 l-day
l l-day
14-day
0.39 0.32 0.59 0.41 0.5 0.38 0.32 0.18 0.23 0.28
2.8 4.1 2.3 4.4 2.4 3.5 2.4 3.6 3.2 3.0 3.6 4.2 2.9 2.9
4.2 5.1 4.6 5.6 2.8 3.0 2.6 4.2 3.4 2.6 3.8 4.0 2.6 2.7
. . . .
Ethanol 288 4- 14 447 4- 13 337 4- 27 4364-16 2.1 4-0.2 Pair-fed 340 4- 16 486 4- 20 351 4- 34 401 4- 27 1.6 4- 0.3
0.8 0.4 0.3 0.03 0.19 0.05 0.02 0.01 0.01 0.01
2.84-0.2 3.44-0.3 3.8 4- 0.3 3.8 4- 0.4
These a c c u m u l a t i o n s are p r e s u m e d to p a r t i c i p a t e in the g r o w t h o f the Purkinje cell dendrites. R e d u c e d m i t o c h o n d r i a l activity w o u l d t h e r e b y result in i m p e d e d developm e n t o f the Purkinje cell dendrite. A n a l y s e s o f the m i t o c h o n d r i a l enzyme malic d e h y d r o g e n a s e activity ( M D H ) in the cerebellums o f these rats revealed r e d u c e d M D H activity in e t h a n o l t r e a t e d pups which is consistent with the A A T data. The serum t h y r o x i n e levels o f e t h a n o l treated pups were significantly r e d u c e d at d a y 11 (3.3 # g total T4/dl) c o m p a r e d with controls (3.8/~g/dl). The n o r m a l total T4 level in sera o f 12-day-old rats is r e p o r t e d 11 to be 3.5 #g/dl. By day 14 the p r o g e n y o f e t h a n o l a n d pair-fed d a m s h a d n o r m a l serum T4 levels. Since T4 is r e q u i r e d for n o r m a l cerebellar d e v e l o p m e n t a n d it has been shown t h a t T4 does n o t cross the p l a c e n t a 11, the small size a n d i m p e d e d d e v e l o p m e n t o f the cerebellum in a l c o h o l t r e a t e d pups m a y be due in p a r t to the r e d u c e d T4 levels. Pups b o r n to rats 10 a n d 14 (those pair-fed c o n t r o l s receiving the least food) also h a d r e d u c e d serum T4 levels at days 11 a n d 14. The activity o f O D C in cerebellum rises d u r i n g the first week o f life p o s t n a t u m a n d declines after the eleventh day a. This p e a k is a function o f r a p i d cell division ( p r e s u m a b l y o f granule cells). The cerebellar O D C activity o f the e t h a n o l treated pups was usually b u t n o t u n i f o r m l y increased a b o v e t h a t o f pair-fed controls in o u r study. The p r o g e n y o f c o n t r o l rats l 0 a n d 14 h a d elevated cerebellar O D C activities at d a y 11. By d a y 14 the O D C levels h a d declined to n o r m a l in all a n i m a l sets. The O D C d a t a also reveal t h a t either e t h a n o l ingestion o r r e d u c e d f o o d i n t a k e d u r i n g p r e g n a n c y
354 TABLE V Mean external granule cell layer (EGC) thickness and Purkinje cell soma depth by region Regions: l, nodule; 2, uvula; 3, tuber; 4, culmen; 5, central lobe; 6, lingua. Ten ethanol treated pups and 10 pair-fed pups were analyzed histologically at each of the two days. TreatmentDay
Ethanol Control
11 14 11 14
EGC thickness (Fire)
Purkinje cell depth (t~m)
l
2
3
4
5
6
1
2
3
4
5
6
41.8 22.1 38.6 17.1
42.9 17.5 36.8 14.3
40.4 17.5 37.9 18.3
47.5 24.6 38.2 17.5
40.0 21.4 35.0 15.4
32.1 19.3 33.6 16.3
98 106 95 121
100 126 97 129
109 126 109 126
108 134 118 141
115 145 120 143
97 126 110 130
may result in delayed cerebellar d e v e l o p m e n t o f progeny. Th e observation o f Schanberg 46 that withdrawal o f e th a n o l f r o m the diet o f n e o n a t a l rats results in elevation o f cerebellar O D C activity is consistent with the data presented in o u r study. T h e cerebral O D C levels in pups f r o m e th an o l treated and co n t r o l dams were low even at day 11 p o s t n a t u m , consistent with previous reports a that cerebral O D C levels decline earlier than cerebellar O D C .
}~i~%~Lii~;.~i}~i ~i}i~d~
~ L~LVk~IjI~
~L~L;
!x~viW~/~xu ¸ z ~
~i ~; ~ } ~
Fig. 1. Sagittal sections through the cerebellar vermis of ethanol treated and pair-fed control pups. Section a is from an ethanol treated pup sacrificed 11 days postnatum and section b is from a control at 11 days. Section c is from an ethanol treated pup sacrificed 14 days postnatum and section d is from the control. Arrows indicate apparent fusion of adjacent folia in the 14-day-old cerebellums. Sections were thionin stained, x 10.7.
355
Effect of ethanol on cerebellar histogenesis For each group of 4 pups sacrificed either at day 11 or 14, the cerebellum of the one animal with median body weight was examined histologically to determine the thickness of the external granule cell layer, the depth of the molecular layer and the diameter of the Purkinje cells. Six regions (nodule, uvula, tuber, culmen, central lobe and lingua) were examined and the datum for an individual rat at a region was the mean of three measurements. Table V indicates the thinning of the EGC layer associated with the maturation of the cerebellar cortex (P < 0.001). Ethanol effectively retarded the thinning of the EGC layer (P < 0.05) in the culmen. When the data from all 6 regions were treated as a common pool, the EGC layer of ethanol treated pups was significantly thicker at day 11 (P < 0.02) and 14 (P < 0.01) than that of pair-fed controls. For Purkinje cell depth and diameter (16 # m at day 11 and 18 # m at day 14) there was no significant effect of ethanol treatment in any region. Figure 1 shows sagittal sections through the vermis of ethanol treated and pairfed rat pups at days 11 and 14 postnatum. The cerebellar areas of ethanol treated rats were smaller at both days and the EGC layer appears thicker at day 14 consistent with the other data presented above. Sites of apparent fusion between adjacent folia were observed both in the ethanol treated and pair-fed control cerebellums obtained from the 14-day-old pups. The significance of this is unclear. DISCUSSION In this report it has been shown that the administration of ethanol to pregnant rat dams between the 3rd and 20th day of gestation resulted in an impeded development of the cerebellum and to a lesser extent of cerebrum in their progeny. The abnormal cerebellar development was characterized by a reduction in cerebellar mass, a significant increase in the thickness of the EGC layer at days 11 and 14 postnatum and a reduced level of AAT and M D H activity, reflecting reduced mitochondrial activity. The normal occurrence of AAT and M D H activities in the cytosol and mitochondria precludes a conclusion that the entire reduction in activity was from the mitochondrial compartment. The pups exposed to ethanol in utero also had a reduced serum level of T4 at day 11. A delayed maturation of the cerebellar cortex has been observed in several conditions: (1) in rat pups born to dams that were placed on a very restricted caloric intake during pregnancy or shortly after deliveryS,13,26,28,3z,34,43,4s,49;(2) in rats that were made hypothyroid in the perinatal period4,7,1°,14,29; (3) in rat pups exposed in utero to alcoho16,a8,46, 47 or methadoneSa; (4) in children born with cystic kidneys z2 or Menkes kinky hair disease 18. In certain of these cases the delay in cerebellar development occurred at the stage in ontogenesis when Purkinje cells have begun to elaborate their apical dendrite but still retained their perisomatic protuberances16, 22. In the malnutrition, hypothyroid, methadone and fetal alcohol syndromes the primary affect appears to be on the proliferation of granule cellsSA a,27 and elaboration of the Purkinje cell dendrite and dendritic spines. The increased thickness of the EGC layer in the alcohol treated pups at days 11
356 and 14 postnatum also suggests that the inward migration of their granule cells may have been delayed. Delayed migration of cerebellar granule cells has been demonstrated to occur in kittens subsequent to cortical ablation 15 and in other species following chemical, infectious and physical insults 36. It has been postulated that exposure of fetuses to alcohol does result in aberrant neuronal migration patterns ~6. Our observations suggest that the proliferation and migration of granule cells may be selectively affected by the in utero exposure of the rat fetuses to alcohol. The cerebellar changes have been readily detected by measurements of the cerebellar mass, the EGC layer thickness, and the synaptic bouton density on Purkinje cell dendrites. Cerebral changes have been described in hypothyroid rats 1l, fetal alcohol syndrome6,19,41; changes in the density of synaptic spines on pyramidal cells in hippocampus were described in rats exposed to alcohol 40. From these reports it is concluded that the development of several CNS regions are affected by ethanol and malnutrition, but that the cerebellar changes are most pronounced. The questions arising from these observations are threefold: (1) is the delayed cerebellar development due to the exposure of rat fetuses to ethanol or to reduced food intake by the pregnant dams; (2) if ethanol does cause the delay in cerebellar development is this chemical acting directly on the cerebellar anlage either during its administration or as a result of withdrawal; (3) does the ethanol cause a delay in the synthesis or release of a hormone (i.e. thyroxine) necessary for cerebellar development ? Although both malnutrition and the exposure of rat pups in utero to ethanol have very similar effects on cerebellar development, the results presented here on the ethanol treated pups are not due primarily to malnutrition. The reasons for this conclusion are: (1) the body weights of pups exposed to ethanol were similar to that of controls in this experiment whereas the weights of malnourished rats may be 50-60 of controls13; (2) the caloric and nutritional diet of ethanol treated and control rats was similar during their gestation period; (3) the young rats with the most marked reduction in cerebellar/body mass ratio were born to the dams consuming the most food and the most alcohol. Although the results of ethanol treatment in the present experiment cannot be explained as due to a malnutrition syndrome, the reduced food intake in ethanol treated and control rats did affect cerebellar development. The weights of cerebellums from ethanol treated and pair-fed controls were reduced from approximately 100 mg (normal cerebellar weight of I 1-day-old pups born to dams fed solid laboratory chow) to 74-83 mg. The similarity between the effects of the alcohol, malnutrition and methadone on the developing cerebellums suggests that they may act via a common pathway. The ethanol may act on the cerebellar anlage either in the prenatal period and thereby affect cerebellar development or in the postnatal period as a consequence of its withdrawal. In both situations the ethanol may be considered to effect changes in cerebellar development by acting directly on the anlage as opposed to acting via a hormone. For example ethanol causes a dose dependent reduction of cerebellar c G M P levels z5,39. The ethanol effect on cGMP however may be reversed by TRH, implying that the adult cerebellum responds directly to ethanol and TRH.
357 The studies of Schanberg and colleagues reveal that the developing cerebellum is responsive to ethanol withdrawal46. They have shown that if ethanol is withdrawn at birth, cerebellar ODC activity in ethanol treated neonatal tissue is similar to that of controls. If ethanol is withdrawn from the maternal diet during the third day postnatum the neonatal cerebellar ODC activity doubles by the fourth day and if withdrawn on the fifth day postnatum, the ODC activity increases 50 ~ by day eight. The differences in cerebellar ODC activity are dependent upon the time of ethanol withdrawal. Although it does not appear possible to conclude unambiguously whether withdrawal from alcohol significantly affected our results, the 11-day delay between withdrawal and tissue analysis reduces this likelihood. It is relevant in this regard that cerebellar development is most rapid between the 5th and 14th day postnatumS, 9,50 which is longer than 4 days after ethanol withdrawal. During the 5th to 14th day the following changes occur: basket cells appear at day 5; succinic dehydrogenase and lactic dehydrogenase activity appears as in adult at day 7 and by day 11-14 the Purkinje cell apical dendrites develop and the Bergmann astrocytes cover Purkinje soma. We have reported here that AAT and MDH activity were reduced markedly in the cerebellums of ethanol treated pups. Malnutrition also caused a delay in the appearance of mitochondrial oxidative activity in the developing apical dendrite of Purkinje cells84 with a prolonged elevation of glycolytic activity. If mitochondrial aggregates are involved in dendritic growth as proposed by Sotelo and Palay45 the reduced oxidative activity in ethanol treated or malnourished rats may be causally related to the reduced cerebellar mass seen in these animals. We do not consider however that ethanol is acting primarily on the cerebellar anlage because methadone withdrawal, hypothyroidism and malnutrition all effect similar changes. It would be more parsimonious to postulate that each of these conditions causes malfunction of a signal (i.e. hormone) necessary for normal development of the cerebellum. The hormones most likely to be involved are thyroxine and growth hormone. Previous studies have demonstrated that cerebellar development in ratsS, 11,50, macaques 21,a5 and humans 11,a7,54 occurs coordinately with the appearance of T4 in their sera. More directly, it has been demonstrated that thyroid deficient neonatal rats have a reduced cerebellar mass, but that there is no reduction in cerebellar cell density or Purkinje cell body diameter4,14,29. Ethanol significantly reduced sera T4 levels in the neonatal rat (the data presented this paper) and in adult rats as. These data are consistent with the postulate that the primary action of ethanol may be to delay the elevation of T4 concentrations in the sera during the neonatal period. This delayed T4 elevation may then cause the delayed cerebellar maturation. The determination that functional hypothyroidism in neonatal rats may be a primary result of the administration of alcohol to pregnant dams is important because administration of T4 to the neonates may then reverse the delay in cerebellar development. Since T4 also regulates the plasma growth hormone level in rat 27 it is possible that cerebellar development is also dependent upon growth hormone levels.
358 ACKNOWLEDGEMENTS
The authors express their appreciation to E. Langer, D. Voss and N. Millan for their assistance in this project. This project was supported by grants from the Medical and Graduate Schools of the University of Wisconsin.
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