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Postnatal growth and development of the gerbil brain
EXPERIMENTAL
Postnatal
NEUROLOGY
Growth
69,290-298
and Development
K. S. WATANABE Departments
(1980)
of Pediatrics, Neurology, University of California, Received
of the Gerbil
Brain
AND R. J. SCHAIN’ and Psychiatry, Neuropsychiatric Los Angeles, California 90024 February
Institute,
6, 1980
The Mongolian gerbil brain undergoes most of its postnatal growth and development during the first 2 weeks of life. At birth the gerbil weighs only 4% of its adult body weight, and the cerebrum and cerebellum are only 24 and 9.9%, respectively, oftheir adult weights. The cerebrum is more mature at birth than is the cerebellum. At 7 days of age, the cerebrum contains 55.7% of its adult DNA content, whereas the cerebellum has amassed only 17.2% ofits adult content. By 21 days of age, cerebral growth is essentially complete, as measured by DNA, RNA, and protein accumulation. Cholesterol content approaches adult values in the cerebrum by 60 days of age. The rate of growth of the cerebrum and rate of RNA accumulation peak at 7 to 8 days of age. The cerebral protein velocity curve reaches its maximum at 10 to 11 days, that of DNA at 12 days, and cholesterol at 17 days of age. Conversely, the cerebellum at 21 days ofage has only 65.6% ofits adult protein content and 77.4% of its adult weight, although DNA multiplication is nearly complete. Myelination,‘as measured by accumulation of cholesterol, is not complete until sometime between 90 and 180 days of age. The velocity maxima of the other cerebellar constituents occur at about 12 to 13 days of age.
INTRODUCTION The Mongolian gerbil (Meriones unguiculatus) is becoming increasingly popular as a laboratory animal because of its convenient size, fecundity, and tractibility. In recent years a number of reports have appeared on many aspects of its physiology and behavior. Increasing interest in the gerbil’s unusual ability to produce spontaneous seizures (4,7, 12) and attempts to manipulate that susceptibility with drug interventions (3,8, 13,14) create a 1 The authors would like to thank Gay Bailey for her excellent technical assistance and Jeffrey Zagun for his help with the graphs and tables. These studies were supported by U.S. Public Health Service grants HD 04612 and HD 05615. Requests for reprints should be addressed to Dr. Schain, Department of Pediatrics. 290 00144886/80/080290-09$02.00/O Copyright All tights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
GERBIL
BRAIN
MATURATION
291
need for some knowledge of the timing and duration of its brain growth. This study examines the rate of accumulation of DNA, RNA, protein, and cholesterol in the gerbil brain during the first 6 months of life, when adult body weight has been reached. MATERIALS
AND METHODS
Mongolian gerbils (Meriones unguiculatus), bred in our colony, were weighed and killed by decapitation at various postnatal ages. The cerebellum was removed and the cerebrum separated from the brain stem by a transcollicular cut. The parts were first weighed together (for total brain weight) and then separately. After weighing, cerebrum and cerebellum were immediately frozen on dry ice and stored at -70°C for biochemical analysis. The brains were homogenized in a Potter-Elvejem glass-Teflon homogenizer in 20 vol 2: 1 chloroform-methanol. After the addition of 4 vol methanol, the brei was centrigued at 2°C. The resulting pellet was rinsed with 2: 1 chloroform-methanol and methanol. The pooled supernatants were saved for cholesterol determinations. Successive washes with 95% ethanol, 5% trichloroacetic acid, and absolute ethanol yielded a pellet that was hydrolized overnight in 1 N sodium hydroxide. It was then extracted with cold 70% perchloric acid to yield RNA and hot 1 N perchloric (90°C for 15 min) to yield the DNA fraction. Cholesterol was measured calorimetrically by the method of Leffler (5) after an aqueous extraction of the choloroform-methanol supernatants (2). Protein was determined by the Lowry method (9) and DNA and RNA by the spectrophotometric method of Santen and Agranoff (11). Velocity curves were obtained by plotting the mean percentage of adult value (180) days) of the parameter at various ages and then computing the incremental change at 5-day intervals from the curve (6). Mean cell size was derived by dividing the total protein content of the tissue in milligrams by the total DNA in micrograms. The sample size from which each mean value was derived depended on the type and age of tissue used: cerebella were so small that they were pooled for extraction. The 48 3-day cerebella were pooled into three batches, each of which weighed between 0.150 and 0.245 g. The 30 7-day tissues were assayed in three groups of 10, each weighing between 0.245 and 0.260 g. The 25 lo-day tissues were extracted in fourgroups weighing between 0.190 and 0.3 10 g. The 24 15-day tissues were extracted in six groups, each of which weighed between 0.220 and 0.280 g. The 15 21-day cerebella were assayed in five batches, each of which weighed between 0.285 and 0.330 g. The 30- to 180-day tissues were extracted in (five or six) pairs, each of which weighed between 0.215 and 0.265 g. Newborn brains
292
WATANABE
AND
SCHAIN
were too undifferentiated to be dissected into cerebrum and cerebellum and were extracted whole. The appropriate sample sizes are indicated in the tables. RESULTS Body weights and brain wet weights are presented as a function of age in Table 1. Weights of brain parts and total content of DNA, RNA, protein, and cholesterol are shown in Tables 2 and 3. The gerbil acquires 96% of its body weight after birth. By 90 days of age the period of rapid body growth is completed and the animal has essentially achieved its adult size. Some animals, particularly males, will continue to gain at a much reduced rate thereafter, and a few adults may weigh more than 100 g. By 20 days of age, 93% of the cerebral weight is achieved, as opposed to only 76% of the cerebellar weight. Not until 45 days of age does the cerebellum attain 96% of its adult weight. The dichotomy between cerebrum and cerebellum is more easily seen in Fig. 1, which depicts the velocity of weight changes in body, cerebrum, and cerebellum. The velocity curve of body growth shows a slow increase in weight from birth to 90 days of age, with no discernible peak. The rate of growth of the cerebrum peaks at 7 to 8 days of age and declines rapidly thereafter; that of the cerebellum peaks at 12 to 13 days of age and declines somewhat more gradually. Simultaneous with the cerebral growth rate peak at 7 to 8 days, there is a peak in the rate of accumulation of RNA (Fig. 2). Shortly thereafter, at 10 to 11 days, the protein curve attains its maximum velocity (Fig. 3) as does TABLE Changes
in Body
Weight
Age
and Brain Body
in the Developing Brain
2.8 3.6 5.8 7.9 10.8 16.1 22.5 34.7 45.2 57.8 66.5 as mean
t f f k t 2 2 + + ? r
0.2 0.5 0.6 1.2 1.8 3.4 2.9 5.6 5.8 7.0 8.9
k SD. Figures
Gerbil”
wet weight
(g)
0 3 7 10 15 21 30 45 60 90 180 are given
Wet Weight
weight
(days)
a Values
1
(9) (11) (48) (34) (35) (25) (30) (20) (20) (25) (20) (19) in parentheses
0.154 0.224 0.367 0.512 0.654 0.798 0.834 0.887 0.893 0.938 0.937 refer
k 2 k + + + + 2 iz r ”
0.006 (11) 0.025 (48) 0.020 (34) 0.041 (35) 0.036 (25) 0.045 (30) 0.028 (20) 0.035 (20) 0.031 (23) 0.050 (20) 0.038 (20)
to number
of samples.
GERBIL
293
BRAIN MATURATION TABLE
2
Changes in the Cerebrum Wet Weight and Total Content of Various Constituents of the Cerebrum in the Developing Gerbil”
Age
Wet weight (is9
DNA (/a)
RNA (Pi39
3
0.183 2 0.019 (53)
433.9 + 35.2 (10)
550.8 2 44.6 (10)
8.0 k 0.8 (10)
0.9 k 0.1 (10)
7
0.294 2 0.018 (38)
401.9 + 39.6 (11)
909.0 rfr 62.3 (11)
13.0 + 1.6 (10)
1.9 r 0.2 (11)
10
0.404 2 0.030 (47)
462.3 -+ 50.5 (9)
1208.2 f 34.2 (9)
18.7 r 1.8 (9)
2.8 2 0.1 (9)
15
0.509 5 0.037 (36)
577.8 + 20.6 (11)
1539.1 + 52.9 (11)
27.3 + 1.8 (10)
4.6 c 0.2 (10)
21
0.603 k 0.033 (41)
676.2 2 25.7 (10)
1831.6 2 63.1 (10)
39.7 2 3.2 (10)
7.6 2 0.4 (10)
30
0.620 rt 0.026 (24)
662.8 + 40.2 (10)
1697.8 + 64.4 (10)
40.8 t 3.5 (10)
9.2 + 0.4 (10)
45
0.640 2 0.024
690.8 k 21.4 (10)
1573.1 k 63.0 (10)
44.1 2 4.0 (10)
10.6 2 0.5 (10)
694.7 k 26.0 (10)
1562.0 + 41.8 (10)
43.6 iz 1.9 (10)
11.7 + 1.1 (10)
697.4 k 43.5 (10)
1496.6 f 101.2 (10)
42.3 zt 2.9 (10)
13.2 4 1.2 (10)
700.5 f 39.8 (10)
1437.9 + 75.8 (10)
44.4 k 3.6 (10)
12.3 -c 0.8 (10)
(days)
(20) 60
0.652 2 0.030
(28) 90
0.677 t 0.027
(23 180
0.669 2 0.027
(24) (LValues are means t
SD.
Protein (mg)
Cholesterol bid
Figures in parentheses refer to number of samples.
DNA (Fig. 2) at 12 days. Cholesterol, the parameter used to determine the state of myelination, achieves its maximum at 17 days of age, well after the other constituents studied (Fig. 3). A somewhat different picture is seen in the cerebellum (Figs. 2, 3). Here all parameters, with the exception of cholesterol, appear to achieve their maximum velocities at 12 to 13 days of age. Apparently, both cell proliferation and cellular growth are occurring most rapidly at this time. Myelination, as seen in the accumulation of cholesterol, lags behind those processes, reaching its peak velocity at 20 days of age. The curve of DNA accumulation in the cerebrum shows a rather interesting anomaly. From 3 to 7 days of age, the DNA content decreases
294
WATANABE
AND SCHAIN
by 10% (see Table 2), from 434 to 402 pg, respectively. Newborn cerebral DNA values are not available, but the whole brain value of 450 pg reflects primarily the DNA content of the cerebrum. This loss appears to be real: the difference in content between newborn and 7 days is significant at P < 0.001 and between 3 and 7 days at P < 0.05. One-fifth (18%) of the cerebral protein is present at 3 days, 42% at 11 days (when maximal velocity is attained), and 92% by 30 days of age. By comparison, the cerebellum has only 5% (one-twentieth) of its adult protein at 3 days of age. At 12 days, when it reaches its peak velocity, one-third of the adult content has been attained, but not until 90 days of age does it begin to approach adult values (87%). TABLE
3
Changes in Cerebellum Wet Weight and Total Content of Various Constituents of the Cerebellum in the Developing Gerbil”
Age (days) 3
Wet Weight (Ii!)
DNA h-4
0.012 2 0.002
(61)
RNA h-G3
Protein b-w)
Cholesterol (mg)
49.0 zt 5.6 (3)
47.9 + 4.2 (3)
0.5 + 0.1 (3)
0.1 2 0.0 (3)
7
0.025 * 0.002 (38)
104.7 + 3.9 (3)
94.3 ? 4.8 (3)
1.2 k 0.1 (3)
0.1 2 0.0 (3)
10
0.040 f 0.005 (41)
205.4 k 48.7 (5)
171.1 + 25.0 (5)
1.9 ” 0.2 (5)
0.2 t 0.0 (5)
1.5
0.066 2 0.006 (30)
406.2 k 31.2
303.9 t 19.6
4.4 ” 0.5
0.5 2 0.1
0.098 f 0.009
557.8 2 46.0 (10)
381.8 f 34.0 (10)
6.3 c 0.9 (10)
I.1 2 0.1 (10)
538.7 + 14.1 (10)
331.6 2 12.3 (10)
7.3 k 0.9 (10)
1.5 + 0.1 (10)
579.8 -c 20.9 (10)
328.7 + 13.2 (10)
8.5 2 0.8 (10)
2.0 2 0.1 (10)
554.7 f 31.0 (10)
316.0 2 17.1 (10)
8.3 t 1.0 (10)
2.0 k 0.1 (10)
(23)
575.8 2 29.5 (10)
310.6 2 18.2 (10)
8.4 ” 1.1 (10)
2.3 2 0.2 (10)
0.123 ? 0.007 (24)
561.4 2 26.7 (10)
293.3 k 13.3 (10)
9.6 + 0.5 (10)
2.6 -t 0.2 (10)
21
(6)
(36) 30
0.105 k 0.006
(24 45
0.117 + 0.005
(21) 60
0.119 k 0.006
(28) 90 180
0.126 * 0.008
a Values are means _’
SD.
(6)
(6)
Figures in parentheses refer to number of samples.
(6)
GERBIL
BRAIN MATURATION
A----A 0-o 0-0
10
20
30
40 POSTNATAL
50 AGE
295
CEREBRUM BODY CEREBELLUM
60
70
80
90
(days1
FIG. 1. Velocity curves of changes in body, cerebrum, and cerebellum weights. The mean percentage of adult (180 days) value was plotted. From this plot the incremental change at 5-day intervals was computed and plotted to yield the velocity curve.
’
u) 0 > ki -; 21 :
6-
A. DNA
----
CEREBELLUM CEREBRUM
5-
POSTNATAL
AGE
(days)
2. Velocity curves ofpostnatal changes in DNA content (A) and RNA content(B) in the gerbil cerebrum and cerebellum. The percentage figures indicate amount of adult values accumulated by that age. Rate changes were calculated as stated for Fig. 1. Sample size was 9 to 10 for the cerebrum and 20 to 48 for the cerebellum. Cerebella were pooled for extraction. See Materials and Methods for details. FIG.
296
WATANABE A.
AND SCHAIN B. CHOLESTEROL
PROTEIN
POSTNATAL
AGE (days)
FIG. 3. Velocity curves ofpostnatal changes in protein (A) and cholesterol(B) content in the cerebrum and cerebellum. The percentage figures indicate amount of adult values present at that age. Rate changes were calculated as stated for Fig. 1. Sample size was 9 to 10 for the cerebrum and 20 to 48 for the cerebellum. Cerebella were pooled for extraction. See Materials and Methods for details.
The RNA velocity curves are of interest because they reflect regional synthetic activity. The cerebrum at 3 days of age has 38% of its adult content of RNA present and 62.4% at 7 to 8 days, the time of peak velocity. At the period of maximum RNA content (20 days) the cerebrum has 27% more RNA present than exists in the adult tissue. The cerebellum has 16% of its RNA at 3 days, 81% at 12 to 13 days, and, as in the cerebrum, 130% of adult values at 20 days of age. After the 20th day, the values decrease rapidly in the cerebellum to 113% of the adult value at 30 days, and then more gradually to adult values at 180 days. In the cerebrum there is a fairly linear decrease from 127% at 20 days to 109% of adult values at 45 days, followed by a gradual diminution to adult values by 180 days. Both the accumulation and the loss of RNA occur more rapidly in the cerebellum than in the cerebrum. Although the adult weight of the gerbil cerebrum is 5.3 times that of the. cerebellum, its total DNA content is only 1.2 times greater. Per unit of weight, the cerebellum contains almost 4.5 times as much DNA as does the cerebrum and it acquires more than 90% of it during the postnatal period. From 20 days to adulthood, a slight increase in the concentration of cholesterol can be seen in the cerebellum, again indicative of the process of myelination, which begins later in the cerebellum than in the cerebrum.
GERBIL
297
BRAIN MATURATION
DISCUSSION The timing of the most rapid growth of the brain or “brain growth spurt” has been related to the event of mammalian birth in such a manner that species can be divided into “prenatal,” “perinatal,” or “postnatal” brain developers (1). Like the rat, a rodent relative, the gerbil is a postnatal brain developer; that is, the brain growth spurt occurs after the event of birth. The tempo of whole brain growth and accumulation of protein, nucleic acid, and cholesterol components is quite similar to that found in the rat (1, 15). Regional patterns also resemble those of the rat in that the cerebellum exhibits its growth spurt somewhat later than the cerebrum. The overall immaturity of the gerbil brain at birth is evident in the fact that it has amassed only 4% of its adult body weight, 24% of its cerebral weight, and 9.9% of its cerebellar weight. Cerebral growth is rapid and essentially complete at 21 days for all parameters studied, with the exception of cholesterol, which approaches asymptote at 60 days. For DNA, 64.2% of the adult cerebral content is present at birth, compared with only 8.7% of the cerebellar DNA. The cerebellum clearly lags behind the cerebrum in the timing of its growth spurt and, for that reason, is far more vulnerable to early postnatal events. The peculiar disposition of the gerbil to manifest spontaneous seizure activity is not reflected in any special alteration in the early growth of the brain or accumulation of major brain constituents, at least on a quantitative level. The present study provides a background for further investigations into structural or biochemical properties of the gerbil brain that may account for its epileptogenic properties. Certain dendritic ultrastructural correlates of spontaneous seizures in the gerbil were recently recognized (10). These may provide a clue to the biological characterization of the basis of spontaneous seizure activity in the Mongolian gerbil. REFERENCES 1. DAVISON, A. N., AND J. DOBBING. 1968. The developing brain. Page 258 in A. N. DAVISON AND J. DOBBING, Eds., Applied Neurochemistry. Davis, Philadelphia. 2. FOLCH, J., M. LEES, AND G. H. SLOANE-STANLEY. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Bid/. Chem. 226: 497-509.
GOLDBLATT, D., A. KONOW, I. SHOULSON, AND T. MACMATH. 1971. Effect of anticonvulsants on seizures in gerbils. Neurology (Minneapolis) 21: 433. 4. KAPLAN, H., AND C. MIEZEJESKI. 1972. Development of seizures in the Mongolian gerbil
3.
(Meriones
unguiculatus).
.I. Comp.
Physiol.
Psychol.
81: 267-273.
5. LEFFLER, H. H., 1959. Estimation of cholesterol in serum. Am. J. Clin. Pathol. 310-313.
31:
298
WATANABE
AND
SCHAIN
6. LINDEN, F. P. G. M., VAN DER. 1970. The interpretation of incremental data and velocity growth curves. Growth 34: 221-224. 7. LOSKOTA, W. J., P. LOMAX, AND S. T. RICH. 1974. The gerbil as a model for the study of epilepsies: seizure patterns and ontogenesis. Epilepsia 15: 109- 119. 8. LOSKOTA, W. J., AND P. LOMAX. 1974. The Mongolian gerbil as an animal model for the study of the epilepsies: anticonvulsant screening. Proc. West. Pharmocol. Sot. 17: 40-45. 9. LOWRY, 0. H., N. J. ROSEBROUGH, A. 0. FARR, AND R. J. RANDALL. 19.51. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-274. 10. PAUL, L., I. FRIED, K. WATANABE, J. DIAZ, AND A. B. SCHEIBEL. 1978. Possible structural correlates of spontaneous seizures in Mongolian gerbils. Sot. Neurosci. Abstr. 4: 145. 11. SANTEN, R. J., AND B. W. AGRANOFF. 1963. Studies on the estimation of deoxyribonucleic acid and ribonucleic acid in rat brain. Biochim. Biophys. Acta 72: 251-262. 12. THIESSEN, D. D., G. LINDZEY, AND H. C. FRIEND. 1968. Spontaneous seizures in the Mongolian gerbil. Psychon. Sci. 11: 227-228. 13. WATANABE, K. S., R. J. SCHAIN, AND B. G. BAILEY. 1978. Effects of phenobarbital on seizure activity in the gerbil. Pediat. Res. 12: 918-922. 14. WATANABE, K. S., B. G. BAILEY, AND R. J. SCHAIN. 1978. Convulsant effects of phenobarbital on the seizure-susceptible Mongolian gerbil. Neurology (Minneapolis) 28: 410. 15. WINICK, M., AND A. NOBLE. 1965. Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. Deve/op. Biol. 12: 451-466.
Postnatal
NEUROLOGY
Growth
69,290-298
and Development
K. S. WATANABE Departments
(1980)
of Pediatrics, Neurology, University of California, Received
of the Gerbil
Brain
AND R. J. SCHAIN’ and Psychiatry, Neuropsychiatric Los Angeles, California 90024 February
Institute,
6, 1980
The Mongolian gerbil brain undergoes most of its postnatal growth and development during the first 2 weeks of life. At birth the gerbil weighs only 4% of its adult body weight, and the cerebrum and cerebellum are only 24 and 9.9%, respectively, oftheir adult weights. The cerebrum is more mature at birth than is the cerebellum. At 7 days of age, the cerebrum contains 55.7% of its adult DNA content, whereas the cerebellum has amassed only 17.2% ofits adult content. By 21 days of age, cerebral growth is essentially complete, as measured by DNA, RNA, and protein accumulation. Cholesterol content approaches adult values in the cerebrum by 60 days of age. The rate of growth of the cerebrum and rate of RNA accumulation peak at 7 to 8 days of age. The cerebral protein velocity curve reaches its maximum at 10 to 11 days, that of DNA at 12 days, and cholesterol at 17 days of age. Conversely, the cerebellum at 21 days ofage has only 65.6% ofits adult protein content and 77.4% of its adult weight, although DNA multiplication is nearly complete. Myelination,‘as measured by accumulation of cholesterol, is not complete until sometime between 90 and 180 days of age. The velocity maxima of the other cerebellar constituents occur at about 12 to 13 days of age.
INTRODUCTION The Mongolian gerbil (Meriones unguiculatus) is becoming increasingly popular as a laboratory animal because of its convenient size, fecundity, and tractibility. In recent years a number of reports have appeared on many aspects of its physiology and behavior. Increasing interest in the gerbil’s unusual ability to produce spontaneous seizures (4,7, 12) and attempts to manipulate that susceptibility with drug interventions (3,8, 13,14) create a 1 The authors would like to thank Gay Bailey for her excellent technical assistance and Jeffrey Zagun for his help with the graphs and tables. These studies were supported by U.S. Public Health Service grants HD 04612 and HD 05615. Requests for reprints should be addressed to Dr. Schain, Department of Pediatrics. 290 00144886/80/080290-09$02.00/O Copyright All tights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
GERBIL
BRAIN
MATURATION
291
need for some knowledge of the timing and duration of its brain growth. This study examines the rate of accumulation of DNA, RNA, protein, and cholesterol in the gerbil brain during the first 6 months of life, when adult body weight has been reached. MATERIALS
AND METHODS
Mongolian gerbils (Meriones unguiculatus), bred in our colony, were weighed and killed by decapitation at various postnatal ages. The cerebellum was removed and the cerebrum separated from the brain stem by a transcollicular cut. The parts were first weighed together (for total brain weight) and then separately. After weighing, cerebrum and cerebellum were immediately frozen on dry ice and stored at -70°C for biochemical analysis. The brains were homogenized in a Potter-Elvejem glass-Teflon homogenizer in 20 vol 2: 1 chloroform-methanol. After the addition of 4 vol methanol, the brei was centrigued at 2°C. The resulting pellet was rinsed with 2: 1 chloroform-methanol and methanol. The pooled supernatants were saved for cholesterol determinations. Successive washes with 95% ethanol, 5% trichloroacetic acid, and absolute ethanol yielded a pellet that was hydrolized overnight in 1 N sodium hydroxide. It was then extracted with cold 70% perchloric acid to yield RNA and hot 1 N perchloric (90°C for 15 min) to yield the DNA fraction. Cholesterol was measured calorimetrically by the method of Leffler (5) after an aqueous extraction of the choloroform-methanol supernatants (2). Protein was determined by the Lowry method (9) and DNA and RNA by the spectrophotometric method of Santen and Agranoff (11). Velocity curves were obtained by plotting the mean percentage of adult value (180) days) of the parameter at various ages and then computing the incremental change at 5-day intervals from the curve (6). Mean cell size was derived by dividing the total protein content of the tissue in milligrams by the total DNA in micrograms. The sample size from which each mean value was derived depended on the type and age of tissue used: cerebella were so small that they were pooled for extraction. The 48 3-day cerebella were pooled into three batches, each of which weighed between 0.150 and 0.245 g. The 30 7-day tissues were assayed in three groups of 10, each weighing between 0.245 and 0.260 g. The 25 lo-day tissues were extracted in fourgroups weighing between 0.190 and 0.3 10 g. The 24 15-day tissues were extracted in six groups, each of which weighed between 0.220 and 0.280 g. The 15 21-day cerebella were assayed in five batches, each of which weighed between 0.285 and 0.330 g. The 30- to 180-day tissues were extracted in (five or six) pairs, each of which weighed between 0.215 and 0.265 g. Newborn brains
292
WATANABE
AND
SCHAIN
were too undifferentiated to be dissected into cerebrum and cerebellum and were extracted whole. The appropriate sample sizes are indicated in the tables. RESULTS Body weights and brain wet weights are presented as a function of age in Table 1. Weights of brain parts and total content of DNA, RNA, protein, and cholesterol are shown in Tables 2 and 3. The gerbil acquires 96% of its body weight after birth. By 90 days of age the period of rapid body growth is completed and the animal has essentially achieved its adult size. Some animals, particularly males, will continue to gain at a much reduced rate thereafter, and a few adults may weigh more than 100 g. By 20 days of age, 93% of the cerebral weight is achieved, as opposed to only 76% of the cerebellar weight. Not until 45 days of age does the cerebellum attain 96% of its adult weight. The dichotomy between cerebrum and cerebellum is more easily seen in Fig. 1, which depicts the velocity of weight changes in body, cerebrum, and cerebellum. The velocity curve of body growth shows a slow increase in weight from birth to 90 days of age, with no discernible peak. The rate of growth of the cerebrum peaks at 7 to 8 days of age and declines rapidly thereafter; that of the cerebellum peaks at 12 to 13 days of age and declines somewhat more gradually. Simultaneous with the cerebral growth rate peak at 7 to 8 days, there is a peak in the rate of accumulation of RNA (Fig. 2). Shortly thereafter, at 10 to 11 days, the protein curve attains its maximum velocity (Fig. 3) as does TABLE Changes
in Body
Weight
Age
and Brain Body
in the Developing Brain
2.8 3.6 5.8 7.9 10.8 16.1 22.5 34.7 45.2 57.8 66.5 as mean
t f f k t 2 2 + + ? r
0.2 0.5 0.6 1.2 1.8 3.4 2.9 5.6 5.8 7.0 8.9
k SD. Figures
Gerbil”
wet weight
(g)
0 3 7 10 15 21 30 45 60 90 180 are given
Wet Weight
weight
(days)
a Values
1
(9) (11) (48) (34) (35) (25) (30) (20) (20) (25) (20) (19) in parentheses
0.154 0.224 0.367 0.512 0.654 0.798 0.834 0.887 0.893 0.938 0.937 refer
k 2 k + + + + 2 iz r ”
0.006 (11) 0.025 (48) 0.020 (34) 0.041 (35) 0.036 (25) 0.045 (30) 0.028 (20) 0.035 (20) 0.031 (23) 0.050 (20) 0.038 (20)
to number
of samples.
GERBIL
293
BRAIN MATURATION TABLE
2
Changes in the Cerebrum Wet Weight and Total Content of Various Constituents of the Cerebrum in the Developing Gerbil”
Age
Wet weight (is9
DNA (/a)
RNA (Pi39
3
0.183 2 0.019 (53)
433.9 + 35.2 (10)
550.8 2 44.6 (10)
8.0 k 0.8 (10)
0.9 k 0.1 (10)
7
0.294 2 0.018 (38)
401.9 + 39.6 (11)
909.0 rfr 62.3 (11)
13.0 + 1.6 (10)
1.9 r 0.2 (11)
10
0.404 2 0.030 (47)
462.3 -+ 50.5 (9)
1208.2 f 34.2 (9)
18.7 r 1.8 (9)
2.8 2 0.1 (9)
15
0.509 5 0.037 (36)
577.8 + 20.6 (11)
1539.1 + 52.9 (11)
27.3 + 1.8 (10)
4.6 c 0.2 (10)
21
0.603 k 0.033 (41)
676.2 2 25.7 (10)
1831.6 2 63.1 (10)
39.7 2 3.2 (10)
7.6 2 0.4 (10)
30
0.620 rt 0.026 (24)
662.8 + 40.2 (10)
1697.8 + 64.4 (10)
40.8 t 3.5 (10)
9.2 + 0.4 (10)
45
0.640 2 0.024
690.8 k 21.4 (10)
1573.1 k 63.0 (10)
44.1 2 4.0 (10)
10.6 2 0.5 (10)
694.7 k 26.0 (10)
1562.0 + 41.8 (10)
43.6 iz 1.9 (10)
11.7 + 1.1 (10)
697.4 k 43.5 (10)
1496.6 f 101.2 (10)
42.3 zt 2.9 (10)
13.2 4 1.2 (10)
700.5 f 39.8 (10)
1437.9 + 75.8 (10)
44.4 k 3.6 (10)
12.3 -c 0.8 (10)
(days)
(20) 60
0.652 2 0.030
(28) 90
0.677 t 0.027
(23 180
0.669 2 0.027
(24) (LValues are means t
SD.
Protein (mg)
Cholesterol bid
Figures in parentheses refer to number of samples.
DNA (Fig. 2) at 12 days. Cholesterol, the parameter used to determine the state of myelination, achieves its maximum at 17 days of age, well after the other constituents studied (Fig. 3). A somewhat different picture is seen in the cerebellum (Figs. 2, 3). Here all parameters, with the exception of cholesterol, appear to achieve their maximum velocities at 12 to 13 days of age. Apparently, both cell proliferation and cellular growth are occurring most rapidly at this time. Myelination, as seen in the accumulation of cholesterol, lags behind those processes, reaching its peak velocity at 20 days of age. The curve of DNA accumulation in the cerebrum shows a rather interesting anomaly. From 3 to 7 days of age, the DNA content decreases
294
WATANABE
AND SCHAIN
by 10% (see Table 2), from 434 to 402 pg, respectively. Newborn cerebral DNA values are not available, but the whole brain value of 450 pg reflects primarily the DNA content of the cerebrum. This loss appears to be real: the difference in content between newborn and 7 days is significant at P < 0.001 and between 3 and 7 days at P < 0.05. One-fifth (18%) of the cerebral protein is present at 3 days, 42% at 11 days (when maximal velocity is attained), and 92% by 30 days of age. By comparison, the cerebellum has only 5% (one-twentieth) of its adult protein at 3 days of age. At 12 days, when it reaches its peak velocity, one-third of the adult content has been attained, but not until 90 days of age does it begin to approach adult values (87%). TABLE
3
Changes in Cerebellum Wet Weight and Total Content of Various Constituents of the Cerebellum in the Developing Gerbil”
Age (days) 3
Wet Weight (Ii!)
DNA h-4
0.012 2 0.002
(61)
RNA h-G3
Protein b-w)
Cholesterol (mg)
49.0 zt 5.6 (3)
47.9 + 4.2 (3)
0.5 + 0.1 (3)
0.1 2 0.0 (3)
7
0.025 * 0.002 (38)
104.7 + 3.9 (3)
94.3 ? 4.8 (3)
1.2 k 0.1 (3)
0.1 2 0.0 (3)
10
0.040 f 0.005 (41)
205.4 k 48.7 (5)
171.1 + 25.0 (5)
1.9 ” 0.2 (5)
0.2 t 0.0 (5)
1.5
0.066 2 0.006 (30)
406.2 k 31.2
303.9 t 19.6
4.4 ” 0.5
0.5 2 0.1
0.098 f 0.009
557.8 2 46.0 (10)
381.8 f 34.0 (10)
6.3 c 0.9 (10)
I.1 2 0.1 (10)
538.7 + 14.1 (10)
331.6 2 12.3 (10)
7.3 k 0.9 (10)
1.5 + 0.1 (10)
579.8 -c 20.9 (10)
328.7 + 13.2 (10)
8.5 2 0.8 (10)
2.0 2 0.1 (10)
554.7 f 31.0 (10)
316.0 2 17.1 (10)
8.3 t 1.0 (10)
2.0 k 0.1 (10)
(23)
575.8 2 29.5 (10)
310.6 2 18.2 (10)
8.4 ” 1.1 (10)
2.3 2 0.2 (10)
0.123 ? 0.007 (24)
561.4 2 26.7 (10)
293.3 k 13.3 (10)
9.6 + 0.5 (10)
2.6 -t 0.2 (10)
21
(6)
(36) 30
0.105 k 0.006
(24 45
0.117 + 0.005
(21) 60
0.119 k 0.006
(28) 90 180
0.126 * 0.008
a Values are means _’
SD.
(6)
(6)
Figures in parentheses refer to number of samples.
(6)
GERBIL
BRAIN MATURATION
A----A 0-o 0-0
10
20
30
40 POSTNATAL
50 AGE
295
CEREBRUM BODY CEREBELLUM
60
70
80
90
(days1
FIG. 1. Velocity curves of changes in body, cerebrum, and cerebellum weights. The mean percentage of adult (180 days) value was plotted. From this plot the incremental change at 5-day intervals was computed and plotted to yield the velocity curve.
’
u) 0 > ki -; 21 :
6-
A. DNA
----
CEREBELLUM CEREBRUM
5-
POSTNATAL
AGE
(days)
2. Velocity curves ofpostnatal changes in DNA content (A) and RNA content(B) in the gerbil cerebrum and cerebellum. The percentage figures indicate amount of adult values accumulated by that age. Rate changes were calculated as stated for Fig. 1. Sample size was 9 to 10 for the cerebrum and 20 to 48 for the cerebellum. Cerebella were pooled for extraction. See Materials and Methods for details. FIG.
296
WATANABE A.
AND SCHAIN B. CHOLESTEROL
PROTEIN
POSTNATAL
AGE (days)
FIG. 3. Velocity curves ofpostnatal changes in protein (A) and cholesterol(B) content in the cerebrum and cerebellum. The percentage figures indicate amount of adult values present at that age. Rate changes were calculated as stated for Fig. 1. Sample size was 9 to 10 for the cerebrum and 20 to 48 for the cerebellum. Cerebella were pooled for extraction. See Materials and Methods for details.
The RNA velocity curves are of interest because they reflect regional synthetic activity. The cerebrum at 3 days of age has 38% of its adult content of RNA present and 62.4% at 7 to 8 days, the time of peak velocity. At the period of maximum RNA content (20 days) the cerebrum has 27% more RNA present than exists in the adult tissue. The cerebellum has 16% of its RNA at 3 days, 81% at 12 to 13 days, and, as in the cerebrum, 130% of adult values at 20 days of age. After the 20th day, the values decrease rapidly in the cerebellum to 113% of the adult value at 30 days, and then more gradually to adult values at 180 days. In the cerebrum there is a fairly linear decrease from 127% at 20 days to 109% of adult values at 45 days, followed by a gradual diminution to adult values by 180 days. Both the accumulation and the loss of RNA occur more rapidly in the cerebellum than in the cerebrum. Although the adult weight of the gerbil cerebrum is 5.3 times that of the. cerebellum, its total DNA content is only 1.2 times greater. Per unit of weight, the cerebellum contains almost 4.5 times as much DNA as does the cerebrum and it acquires more than 90% of it during the postnatal period. From 20 days to adulthood, a slight increase in the concentration of cholesterol can be seen in the cerebellum, again indicative of the process of myelination, which begins later in the cerebellum than in the cerebrum.
GERBIL
297
BRAIN MATURATION
DISCUSSION The timing of the most rapid growth of the brain or “brain growth spurt” has been related to the event of mammalian birth in such a manner that species can be divided into “prenatal,” “perinatal,” or “postnatal” brain developers (1). Like the rat, a rodent relative, the gerbil is a postnatal brain developer; that is, the brain growth spurt occurs after the event of birth. The tempo of whole brain growth and accumulation of protein, nucleic acid, and cholesterol components is quite similar to that found in the rat (1, 15). Regional patterns also resemble those of the rat in that the cerebellum exhibits its growth spurt somewhat later than the cerebrum. The overall immaturity of the gerbil brain at birth is evident in the fact that it has amassed only 4% of its adult body weight, 24% of its cerebral weight, and 9.9% of its cerebellar weight. Cerebral growth is rapid and essentially complete at 21 days for all parameters studied, with the exception of cholesterol, which approaches asymptote at 60 days. For DNA, 64.2% of the adult cerebral content is present at birth, compared with only 8.7% of the cerebellar DNA. The cerebellum clearly lags behind the cerebrum in the timing of its growth spurt and, for that reason, is far more vulnerable to early postnatal events. The peculiar disposition of the gerbil to manifest spontaneous seizure activity is not reflected in any special alteration in the early growth of the brain or accumulation of major brain constituents, at least on a quantitative level. The present study provides a background for further investigations into structural or biochemical properties of the gerbil brain that may account for its epileptogenic properties. Certain dendritic ultrastructural correlates of spontaneous seizures in the gerbil were recently recognized (10). These may provide a clue to the biological characterization of the basis of spontaneous seizure activity in the Mongolian gerbil. REFERENCES 1. DAVISON, A. N., AND J. DOBBING. 1968. The developing brain. Page 258 in A. N. DAVISON AND J. DOBBING, Eds., Applied Neurochemistry. Davis, Philadelphia. 2. FOLCH, J., M. LEES, AND G. H. SLOANE-STANLEY. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Bid/. Chem. 226: 497-509.
GOLDBLATT, D., A. KONOW, I. SHOULSON, AND T. MACMATH. 1971. Effect of anticonvulsants on seizures in gerbils. Neurology (Minneapolis) 21: 433. 4. KAPLAN, H., AND C. MIEZEJESKI. 1972. Development of seizures in the Mongolian gerbil
3.
(Meriones
unguiculatus).
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81: 267-273.
5. LEFFLER, H. H., 1959. Estimation of cholesterol in serum. Am. J. Clin. Pathol. 310-313.
31:
298
WATANABE
AND
SCHAIN
6. LINDEN, F. P. G. M., VAN DER. 1970. The interpretation of incremental data and velocity growth curves. Growth 34: 221-224. 7. LOSKOTA, W. J., P. LOMAX, AND S. T. RICH. 1974. The gerbil as a model for the study of epilepsies: seizure patterns and ontogenesis. Epilepsia 15: 109- 119. 8. LOSKOTA, W. J., AND P. LOMAX. 1974. The Mongolian gerbil as an animal model for the study of the epilepsies: anticonvulsant screening. Proc. West. Pharmocol. Sot. 17: 40-45. 9. LOWRY, 0. H., N. J. ROSEBROUGH, A. 0. FARR, AND R. J. RANDALL. 19.51. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-274. 10. PAUL, L., I. FRIED, K. WATANABE, J. DIAZ, AND A. B. SCHEIBEL. 1978. Possible structural correlates of spontaneous seizures in Mongolian gerbils. Sot. Neurosci. Abstr. 4: 145. 11. SANTEN, R. J., AND B. W. AGRANOFF. 1963. Studies on the estimation of deoxyribonucleic acid and ribonucleic acid in rat brain. Biochim. Biophys. Acta 72: 251-262. 12. THIESSEN, D. D., G. LINDZEY, AND H. C. FRIEND. 1968. Spontaneous seizures in the Mongolian gerbil. Psychon. Sci. 11: 227-228. 13. WATANABE, K. S., R. J. SCHAIN, AND B. G. BAILEY. 1978. Effects of phenobarbital on seizure activity in the gerbil. Pediat. Res. 12: 918-922. 14. WATANABE, K. S., B. G. BAILEY, AND R. J. SCHAIN. 1978. Convulsant effects of phenobarbital on the seizure-susceptible Mongolian gerbil. Neurology (Minneapolis) 28: 410. 15. WINICK, M., AND A. NOBLE. 1965. Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. Deve/op. Biol. 12: 451-466.