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The effects of neonatal androgenization on the in vivo transport of alpha-aminoisobutyric acid into specific regions of the rat brain
Brain Research, 132 (1977) 287-299 i!) Elsevier/North-Holland Biomedical Press
287
T H E EFFECTS OF N E O N A T A L A N D R O G E N I Z A T I O N ON T H E IN VlVO T R A N S P O R T OF A L P H A - A M I N O I S O B U T Y R | C A C I D I N T O SPECIFIC R E G I O N S OF T H E R A T BRAIN
M. LITTERIA Nenroh)gy Research, Veterans Admhtistration Hospital, North Chicago, Ill. 60064 (U.S.A.)
(Accepted December 10th, 1976)
SUMMARY The purpose of the present study was to determine if the administration of testosterone propionate (TP) to neonatal rats is followed in vivo by alterations in the transport of the non-metabolizable amino acid, a-aminoisobutyric acid (AIB), into specific regions of the brain. Forty-eight hours after birth, male and female rats were injected s.c. with either 1.25 mg TP or an equivalent volume of vehicle. Five, 10 and 17 days after birth, control and TP-treated rats were decapitated at intervals of 2, 5, 60 and 300 rain after the i.p. injection of 0.25 #Ci [1-14C]a-aminoisobutyric acid/g body weight. Twelve brain regions, i.e., amygdala, cerebellum, corpora quadrigemina, frontal cortex, hypothalamus, medulla, occipital cortex, olfactory bulbs, olfactory tubercles, parietal cortex, pons, pyriform cortex and samples of serum were analyzed in terms of disint./min/mg tissue and as tissue/serum (T/S) ratios. At the end of 300 min there was a significant increase in the active transport of AIB in all brain regions of the 5-day-old TP-treated rats. Similarly, by 300 rain, the active transport of AIB was significantly increased in all brain regions except cerebellum and pons of the 10-dayold TP-treated rats. The administration of TP to neonatal rats did not alter the accumulation and/or active transport of AI B in brain regions of the 17-day-old rat at any of the tested intervals. These data indicate that (I) neonatally administered TP enhanced (either directly or indirectly) the transport and/or accumulation of AIB in specific brain regions of 5- and 10-day-old rats and (2) the effectiveness of the steroid decreased with the age of the rat.
INTRODUCTION The administration of testosterone propionate (TP) to neonatal female rats during the critical period of brain differentiation is followed postpuberally by a syn-
288 drome characterized by anovulation, polycystic ovaries, persistent vaginal cornification and masculinization of sexual behavior a. This syndrome is probably related to TP-induced alterations in hypothatamic 2° and timbic 1~ structures associated with the synthesis and/or release of the pituitary gonadotropins. Although the biochemical nature of these alterations Is unknown, they may be directly or indirectly related to changes in brain nucleic acid z6,29 and or protein synthesis 10 following neonatatly administered TP. Quantitative autoradiographic studies from this laboratory have localized some of the neuroanatomic sites metabolically responsive to neonatallv administered TP u~.ls. The incorporation of [aH]lysine into proteins of specific hypothalamic nuclei (arcuate. periventricular, paraventricular and supraoptict was sign> ficantly inhibited in adult female rats treated neonatally with TP 16. Similarily, the Purkinje cells, but not the granule or stellate cells of the cerebellar cortex incorporated less [aH]lysine into proteins ~s Autoradiography is an invaluable technique for localizing newly synthesized protein 7 However. differences in silver grains between the TP-treated rats and their controls does not imply that the steroid inhibited protein synthesis per se. For example, the steroid might have affected the transport of [aH]lysine into the brain, or produced alterations in the endogenous free lysine paol and or nucleic acid metabolism. The purpose of the present study was to determine if the administration of TP to neonatal rats during the critical period of brain differentiation is followed by alterations in the transport of an amino acid across the blood-brain barrier into specific regions of the brain. Alpha-aminoisobutync acid {AIB} is a small neutral amino acid that enters the brain via career mediated processes z7, but is neither incorporated into proteins nor metabolized by either the brain or other organs ~. AIB was selected ['or this study in order to avoid the problems associated with amino acids that are metabolized by the brain. MATERIALS AND METHODS Three month old virgin Sprague-Dawley rats (Madison. Wisc.) were exposed to a 14:10 h light/dark schedule and fed Purina Rat Chow and water ad libitum in our animal facilities which are maintained at approximately 72 °F. These animals served as our breeding stock. Litter size was reduced to 8 pups (4 males and 4 females whenever possible) within 24 h after birth. The day of birth is called day one. Forty-eight hours after birth, male and female rats were mjected s.c. with either 1.25 mg testosterone propionate (TP) dissolved in sesame oil or with an equivalent volume of vehicle. At 5, 10 and 17 days after birth, a minimum of 8 control and 8 TP-treated rats were each injected i.p. with 0.25 uCi [I-14C]a-aminoisobutyric acid/g body weight (sp. act., 9.0 mCi/mmole. New England Nuclear). Prior to sacrifice, the pups were placed in a small beaker (warmed by a lampl containing bedding from the home cage. The rats were decapitated at intervals of 2.5. 60 and 300 rain following the injection of the [I-t4C]a-aminoisobutyric acid (AIB). The brain was rapidly removed, placed on a Petri dish over ice and freed of adhering meninges and blood. Twelve brain regions, i.e., amygdala (with overlying cortexl (AMY), cerebellum (CB), corpora
289 quadrigemina (CQ), frontal cortex (FCX), hypothalamus (HYPO), medulla (MED), occipital cortex (OCX), olfactory bulbs (OB), olfactory tubercles (OT), parietal cortex (PCX), pons and pyriform cortex (PYCX) were placed in a Petri dish resting on a block of dry ice. It has been shown that only 5 ~o of whole brain radioactivity is due to plasma AIB la. Therefore, it was not necessary to make corrections for tile amount of radioactivity contributed by the blood present in the individual brain regions la. Each frozen tissue was weighed and transferred while still frozen into scintillation vials containing 1.0 ml of solubilizer (Soluene-350, Packard Instrument Co., Inc., Downers Grove, lll.). Blood collected at the time of sacrifice was allowed to clot and serum was separated following centrifugation at 2 C for 30 rain at 600 × g. Ten to 50 #1 aliquots of serum (S) were added to 1.0 ml Soluene-350. Fifteen ml of liquid scintillation cocktail (Dimilume TM-30, Packard Instrument Co., Inc.) was added to the vials following the overnight solubilization of tissues at room temperature. Counts/min (CPM) were determined in a Packard Tri-Carb Liquid Scintillation Spectrometer, Model 3385, following temperature and dark adaptation. Quench corrections were made using the external standardization and least squares methods. Data were expressed in terms of disint./min (DPM)/mg tissue, DPM/I,I serum and as tissue/serum (T/S) ratios. In a separate experiment, the total water content of each of the 12 brain regions was determined for the 5-, 10- and 17-day-old control and TP-treated rats. The removal and handling of tissues is described above. The tissues were dried to constant weight and the amount of water was calculated from the difference between the weights of the frozen and dried tissues. The significance of differences between the control and TP-treated groups was determined by Student's t-test. P --< 0.05 was considered significant. RESULTS A IB dose response
The in vivo uptake and accumulation of AIB by the 12 brain regions and serum were determined at three different concentrations of AIB in order to insure that the dose selected for this study would be below saturation kinetics. Five- and 10-day-old control rats were each injected i.p. with C1 0.125 #Ci; C2 -- 0.25 ~Ci; Ca = 0.40 /~Ci of AIB/g body weight. The rats were sacrificed 5 rain after the injection of the isotope. DPM/mg tissue and DPM/#I serum were twice as great for Cz than Ci, and approximately 1.6 times higher for Ca than 02. There were no significant differences between T/S ratios for any given tissue for either the 5 or 10 day old rats at C1, C2 and Ca. These concentrations of AIB were, therefore, below saturation kinetics and 0.25 /zCi/g body weight was chosen for this study. T/S ratios rather than tissue concentration ( D P M / m g tissue) are an accurate measure of AIB transport. Ratios greater than unity' indicate the occurrence of active transport. Tissue water content
In agreement with others a0, normal neonatal rats showed no sex differences in
290 the water c o n t e n t o f specific b r a i n regions. Similarly, there were n o sex :differences in the water content o f the 12 b r a i n regions o f the 5-, 10- a n d 17-day-old r a t f o l l o w i n g the a d m i n i s t r a t i o n o f TP. D a t a f r o m b o t h sexes were, therefore; combined: for the c o n t r o l or T P - t r e a t e d groups. Differences in water content were f o u n d between control a n d T P - t r e a t e d rats for some tissues at 5, 10 a n d 17 days o f age. However, these changes were so small that corrections m a d e for these differences did n o t alter the significance o f the T/S ratios between c o n t r o l a n d T P - t r e a t e d groups, E f f e c t s o f T P on the transport o f A I B S e x differences. There were no sex differences in the c o n c e n t r a t i o n o f A I B in serum or tissues o f control or T P - t r e a t e d 5-, 10- a n d 17-day-old rats at any o f the tested time intervals. D a t a from both sexes were, therefore, c o m b i n e d in the control o r TPtreated groups. Serum. In all age groups the c o n c e n t r a t i o n o f A I B was highest in the serum o f b o t h c o n t r o l a n d T P - t r e a t e d rats 5 minutes after a d m i n i s t r a t i o n o f t h e motope (Table 1). In pilot studies, A I B levels were lower t h a n t h e 5 min values in samples o f serum from rats sacrificed 15 and 30 min after t h e injection o f the isotope. This indicates t h a t the actual p e a k in serum A I B was between 2 a n d 15 min. Serum A I B levels for 5-day-old c o n t r o l or T P - t r e a t e d rats were m a r k e d l y lower t h a n those o f the 10- and 17-day-old rats 2 a n d 5 min after a d m i n i s t r a t i o n o f the i s o t o p e (Table 1). O t h e r workers have r e p o r t e d that the low c o n c e n t r a t i o n o f A t B p r e s e n t in the p l a s m a o f n e w b o r n rats could not be a t t r i b u t e d to a greater a c c u m u l a t i o n o f the i s o t o p e in the neonatal liver a n d kidney aa. Differences in serum A I B levels between c o n t r o l a n d T P - t r e a t e d groups occurred only in the 5-day-old rats 2 and 60 min after a d m i n i s t r a t i o n o f the isotope (Table I). S e r u m A I B levels in the TP-
TABLE 1 Concentration of AIB in rat serum 2. 5, 60 and 300 min after admin&tration
Number of rats used is listed in parentheses. C, control: TP, treated with testosterone prop~onate. Age
DPM/ttI* ( Min after injection o1 AIB ) 2
5-day C TP 10-day C TP 17-day C TP
5
604.9±17.3(8) 766.9 -L 36.3 (81"*
60
797.3 ~ 64.6(10) 818.4 m 36.1 (10)
30o
255.1r~ 10.8(10) 174.7~ 12.6(101 331.0-t 21.7 (10~*** 161.1 11.8 (10/
1128.0±25.3(101 1091.7 + 29.9 ( 1 0 )
1177.8~: 38.2 (10) 1163.0:, 32.5 (10~
228.6 j : l l . 4 ( 1 0 ) 230.7• 1t.9 (101
153.7 i 15.4(10) 141.5 : 4.0 (10~
1115.1 ~39.2(10) 1096.4±69.2(101
1232.1 ~ 51.2(8) 1175.23 29.8(81
277.3-~ 9.4(10) 250.9 ~_ 10.5(10)
163.2-~ 3.8(10~ 156.3 ~. 5.6(i0)
* Mean ± S,E.M. ** P <~ 0.005. *** P -< 0.01.
291 TABLE 1l
Effects of neonatal androgenization on T/S ratios o[' A IB in brain regions o]' the 5-day-oM rat T/S ratios are expressed as mean ± S.E.M. Number of rats used is listed in parentheses. C, control; TP, treated with testosterone propionate. Abbreviations for tissues are listed in the Materials and Methods section. P values are indicated as * < 0.05; ** < 0.025; *** < 0.02; § < 0.01 ; §§ < 0.005 ; ~§~ -< 0.001.
Braitt
T/S (Mitt aJ?er injection o / A I B )
regions
2
5
60
300
0.0400±0.0015~§§ (8) 0.0250~0.0025 (8)
0.0685-20.0038 (10) 0.0750±0.0062 (10)
0.6994±0.0303§§§ (10) 0.4495±0.0332 (10)
1.3044 ±0.0661.~§¢ (10) 2.3523i0.0450 (10)
0.1074±0.0087"** 0.0751 ±0.0070
0.1246±0.0045 0.1235±0.0034
1.3262 ±0.0460§~ 0.7930~0.0381
2.3920i 0.0561§§§ 4.0913 ±0.0578
0.0543±0.0040*** 0.0400±0.0037
0.0545 ~¢O.0064 0.0568±0.0054
0.5490 ±0.0290§,~ 0.3515:£0.0513
1.1051 ~0.0492,~ 1.8904±0.0786
0.0540£=0.0037*** 0.0390±0.0036
0.0831 ±0.0068 0.0986i0.0137
0.9444 £0.0502§~,~ 0.5410±0.0194
1.6293±0.1060~§ 3.3321 ±0.0507
0.0342 ±0.0008~ 0.0273±0.0015
0.0459±0.0040 0.0555±0.0047
0.5522 ~:0.0209§,~ 0.4042i0.0200
1.2711 ±0.0586,~ 1.9880i0.0405
0.1118 :~ 0.0074"** 0.0806-~ 0.0090
0.0965±0.0066 0.0990 ~ 0.0095
0.7851 i 0 . 0 4 7 7 ~ 0.4578 :~0.0244
1.7015 ±0.0754§§~ 2.5506 ~0.0792
0.0548 ~0.0040"** 0.0420±0.0027
0.0751 -E0.0045 0.0739±0.0066
0.8503±0.03080§§ 0.4751 i0.0098
1.7351 ±0.0814§~ 2.9699±0.0762
0.1098 ±0.0081 ~ 0,0733+0.0090
0.1069 i: 0.0111 0.1044 ~0.0070
1.3127±0.0537§§~ 0.8942:~ 0.0319
2.6491 ±0.0877§§~ 3.2842i0.0322
0.0331 ±0.0008* 0.0281 ±0.0020
0.0520~0.0074 0.0553 tO.0053
0.5301 i0.0308§§~ 0.2917 f 0.0140
1.1577 t 0.0601 §§,~ 1.8408 ±0.0213
0.0529±0.0047* 0.0400~0.0037
0.0752 i0.0062 0.0811 50.0036
0.8602 ±0.0356,~§§ 0.4772 t:0.0328
1.7883 i 0 . 1 0 0 4 - ~ 3.0000 ~:0.0609
0.0706+0.0065*** 0.0506£0.0038
0.0742 ~0.0077 0.0819 %0.0082
0.7826±0.0291 ~§~ 0.4722 90.0156
1.9387±0.1101 §§~ 2.7196 ~0.0731
0.0560±0.0022~§ 0.0410i0.0037
0.0788 ~0.0050 0.0884~0.0077
0.8818±0.0232§§8 0.55101 0.0135
1.5932 ±0.1079§§,~ 2.5635 i0.0553
AMY C TP CB C TP CQ C TP FCX C TP HYPO C TP MED C TP OCX C TP OB C TP OT C TP PCX C TP PONS C TP PYCX C TP
treated rats were significantly greater than the control values at both intervals. This f i n d i n g m a y p o s s i b l y be a t t r i b u t e d t o t h e a b i l i t y o f T P t o d e c r e a s e t h e r e n a l c l e a r a n c e o f A I B in r a t s 2'5.
Brain regions. T h e t r a n s p o r t o f A I B i n c r e a s e d w i t h t i m e in all b r a i n r e g i o n s o f t h e 5-, I0- a n d 1 7 - d a y - o l d c o n t r o l a n d T P - t r e a t e d r a t s ( T a b l e s II, I I | a n d IV). T h e s e
292
results are in agreement with the m vivo study by Kuttner et al.~% in which the uptake of AIB by the whole brain of adult rats increased slowly with time and reached a maximum at approximately 20 h after administration. There is considerable regional variation in the uptake of AIB by the brain of both control and TP-treated rats. However, the accumulation of AIB was consistently higher in the CB. OB and P O N S T A B L E I11
Effects o[ neonatal androgenization on T/S rath~s o f AIB in brain regions oJ the lO-day-old rat T/S ratios are expressed as mean _~ S.E.M. N u m b e r of rats used is listed in parentheses, C. control: TP, treated with testosterone propionate. Abbreviations for tissues are listed in the Materials and Methods section. P values are indicated as * - 0.05 : ** --~ 0.025 : *** - 0.02 : ~ . 0.01 ; ~ - " 0.005 : §§,~ .: 0.001.
Brain
T/S ( Min after injection o f AIB)
regkms
2
5
60
300
AMY C
0.0283±0.0012 (10)
0.0513 -~ 0.0011 I101
0.0289 ~O.0016 (10)
0.0567 :~: 0.0037 ~10)
0.6059 ~ 0 . 0 3 9 0 . ~ (10} 0.9347 3:0,0348 ( 1 0 1
2.1764 0,0757~§~ (10p 2,9205 }0,1118~10]
0.0573 ~0.0029 0.0558-2.0.0030
0,0916 i0,0041 0.1011 ~0.0031
1.3774. 0.0473 1.4455 5 0.0646
4.2221 0.0082 4.4810-k O, 1879
0.0295_+0.001 I 0.0328 ~ 0.0031
0.0488-;0.0008 0.0488 -r 0.0009
0.6599 T 0.0480" 0.7817 -v0.0307
1.9721 0,0563§§6 2.6060 ~ 0.1248
0.0362 :E0.0020 0.0370+0.0018
0.0737d 0.0021 0.0806 3 0.0033
0,9300s 0.0419~ 1.2115-c 0.0714
2.712t-: 0 1069~ 3.8881 0.3608
0.0235 ~:0.0009 0.0231 ~ 0.0013
0.0384 ~ 0.0014 0.0380-1 0.0023
0.5884 _: 0.0364 0.5981- 0.0222
1.7892 i 0.0440.~ 2.3518 : 0.0832
0.0437 3 0.0045 0.0089 ~0.0031
0.0777 ~0.0040 0,0745 ~ 0.0023
0.8012~ 0.0339§§~ 1.0253 ~ 0.0332
2.8329 0.0514,~.~,~ 3.5890 ~O.1367
0.0351 t 0 . 0 0 1 4 0.0353.3 0.0030
0.0692 -c 0.0022 0,0699,0.0025
0.8077-L0.0329 §~,~ 1.0615 -i 0.0472
2.6827 -_0.0311 ,~§ 3.4031 40.1163
0.0476 ~0.0016 0.0460 ~ 0.0031
0.0823 --0.0031 0.0945-50.0076
1.1019:a 0.0045"** I 3246 m0.0640
3.0127 r_0.0585~,~ 3.9640~ 0.1161
0.0245 _k0.0013 0.0260%O.0019
0.0444T0.0014 0.0498::-0.0041
0.5405 _L0.0232 ~'~'~ 0.71013 :~ 0.0314
1.8723 ~. 0.648 ~i§~ 2 4154 £0,1040
0.0364 3.0.0023 0.0389~ 0.0024
0.0682 - 0.0020 0.0641 ~ 0.0025
0 8554~0.0469"** 1.0664 ~ 0.0619
2.6287 ~ t),0600§§-~ 3.4468" 0.1236
0.0452~_0.0032 0.0068+0.0040
0.0718 T0.003 I 0.0714~ 0.0036
0 8260=. 0.0054 0.8677 ~ 0.0000
3 0573 :O.1200 3.2065 -0.2231
0.0335-t0.0018 0,0345 j-0.0023
0.0655 ~_0.0020 0.0726-c0.0035
0.9378 ~0.0041 1.0384 7 0 0854
2,5827 ~ 0 . 0 6 6 9 ~ 3,3765 <0.1377
TP CB C TP
CQ C TP FCX C TP HYPO C TP MED C TP OCX C TP OB C TP OT C TP PCX C TP PONS C TP PYCX C TP
293
of the 5- and 10-day-old control rats 300rain after administration of the isotope (Tables ii and lII). The uptake of AlB was also highest in the CB and PONS of the 17-day-old control rats at the 300 min interval (Table IV). By 60 rain, AIB had been actively transported into the CB and OB of the 5- and 10-day-old control rats and into the CB, FCX, MED, OCX, OB, PCX and PYCX of the 10-day-old TP-treated rats (Tables 1I and l If). AI B was actively transported into T A B L E IV
Ej]~wts qf neonatal androgenization
on
T/S ratios of A IB in brain regions o]' the l 7-day-old rat
T/S ratios are expressed as m e a n ± S.E.M. N u m b e r o f rats used is listed in parentheses. C, control; TP, treated with testosterone propionate. Abbreviations for tissues are listed in the Materials and Methods section.
Brahl
T/S (minutes t~?er injection of AIB)
re~ions
2
5
60
300
0.0227 ± 0.0014 (10) 0.0231 ~ 0.0008 (10)
0.0362 i 0.0013(8) 0.0363 i 0.0019 (8)
0.5401 ± 0.0196 (10) 0.5464 ~ 0.0352 (10)
1.7814 L 0.0469 (10) 1.7765 ± 0.0521 (10)
0.0444 :~z 0.0028 0.0421 ~ 0.0036
0.0560 i: 0.0032 0.0602 ± 0.0033
0.7402 ~_ 0.0457 0.7624 ~ 0.0355
2.5268 ~ 0.0762 2.5559 i: 0.0597
0.0381 _L 0.0016 0.0368 ~ 0.0014
0.0454 ± 0.0014 0.0485 +_ 0.0028
0.4708 k 0.0150 0.5115 i 0.0187
1.7221 i 0.0436 1.7170 ± 0.0430
0.0410 i 0.0029 0.0425 ± 0.0024
0.0543 i 0.0021 0.0554 ± 0.0046
0.6400 ~ 0.0140 0.6542 ~ 0.0217
2.1969 £ 0.0472 2.2035 ± 0.0559
0.0212 ± 0.0013 0.0216 ± 0.0008
0.0300 ± 0.0009 0.0310 i 0.0005
0.4547 -- 0.0162 0.4639 5z 0.0129
1.6123 ± 0.0400 1.6722 -- 0.0340
0.0499 ~ 0.0056 0.0534 ± 0.0047
0.0904 ± 0.0082 0.0922 ± 0.0106
0.6124 Az 0.0207 0.6758 ~ 0.0325
2.4321 ± 0.0625 2.4886 ± 0.0479
0.0327 ± 0.0014 0.0344 { 0.0013
0.0493 ± 0.0013 0.0501 ~ 0.0035
0.5810 & 0.0215 0.5914 ~ 0.0248
2.0136 i 0.0576 2.0665 ± 0.0896
0.0367 ± 0.0027 0.0383 i 0.0031
0.0653 t: 0.0027 0.0670 -- 0.0015
0.7228 ~ 0.0483 0.7547 ~ 0.0175
2.0787 ± 0.0656 2.1318 ± 0.0559
0.0224 ~ 0.0015 0.0228 ± 0.0010
0.0391 ± 0.0014 0.0412 i 0.0029
0.4606 ± 0.0241 0.4882 -- 0.0224
1.4372 t~ 0.0342 1.4670 i 0.0255
0.0298 ± 0.0015 0.0310 ± 0.0034
0.0512 ± 0.0035 0.0502 i 0.0032
0.5718 £ 0.0278 0.5852 ± 0.0212
2.1071 ± 0.0605 2.1660 ± 0.0560
0.0378 ~ 0.0018 0.0379 ± 0.0019
0.0600 ± 0.0029 0.0634 £ 0.0023
0.7600 ± 0.0327 0.7693 t 0.0494
2.7681 ± 0.0890 2.8150 ± 0.0664
0.0297 ± 0.0019 0.0299 i 0.0021
0.0489 % 0.0018 0.0524 ~ 0.0023
0.5702 k 0.0114 0.5743 ~ 0.0243
1.9128 t 0.0455 1.8856 k 0.0428
AMY C TP CB C TP
co C TP FCX C TP HYPO C TP MED C TP OCX C TP OB C TP OT C TP PCX C TP PONS C TP PYCX C TP
294 all brain regions of both control and TP-treated 5-. 10- and 17-day-old rats 300 min following administration of the isotope (Tables I1.11t and IV). Administration of TP to neonatal rats did not affect the absolute concentration of AIB (DPM/mg tissue) in brain regions of the 5-day-old rat 2 min after injecnon of the isotope. Therefore, the lower T S ratios observed at this interval are due to the higher concentration of AIB in the serum of the TP-treated rats (Tables i and i[). There were no differences in either the absolute concentrations or transport of AIB into brain regions of the control or TP-treated 5-day-old rats at the 5 min interval (Table I!). With the exception of the CQ and HYPO. the absolute concentrations of AIB in the remaining 10 brain areas of the 5-day-old TP-treated rats were significantly lower than their controls at the 60 min interval. The lower T S ratios observed for the CQ and HYPO of the 5-day-old TP-treated rats are due to the higher level of AIB in the serum of these animals at 60 min. Two factors contributed to the lower T/S ratios observed in the remaining 10 brain regions of the 5-day-old TP-treated rats. i.e., significantly lower absolute concentrations (DPM/mg tissue)of A1B present in these brain areas and the higher level of isotope in the serum of the steroid treated rats (Tables 1 and IlL By 300 min. T/S ratios tbr all brain regions of the 5-day-old TP-treated rats were significantly increased above those of the controls (Table I11. Serum levels of AIB were the same for both control and TP-treated rats at 300 min (Table I). Therefore, the higher T'S ratios are entirely attributable to the greater concentration of isotope in the brain regions of the steroid treated rats (Table I1). The administration of TP to neonatal rats did not affect either the absolute concentrations or the transport of AIB into brain regions of the 10-day-old rats 2 and 5 min after the injection of the isotope (Table 111). With the exceptions of CB. HYPO, PONS and PYCX. the T S ratios for brain regions of the 10-day-old TPtreated rats were significantly higher than the controls at the 60 min interval. By 300 min, T S ratios were significantly greater in all brain regions of the steroid treated rats with the exception of the CB and PONS (Table !11). There were no differences in serum AIB levels between the 10-day-old control and TP-treated rats (Table t). Therefore. the higher T/S ratios for certain brain regions of the TP-treated rats are entirely due to the higher absolute concentrations of isotope in these regions at 60 and 300 rain. The administration of TP to neonatal rats did not alter the transport of AIB into specific brain regions of the 17-day-old rat at any of the tested intervals ITables 1 and IVI. In general, the ability of anaino acids to enter the brain in vivo ~s greater in younger animals than in the adult 28. Therefore. it was of interest to determine if the transport of AIB into individual brain regions followed this pattern. A comparison of the data for the control groups in Tables 11, I11 and IV indicate that there are numerous exceptions to the general rule. For example, the transport of AIB into CQ, FCX and M E D at the 2 min interval is greater for the 17-day-old rats in comparison to the 10-day age group, and at 5 rain, transport of AIB into the MED of the 17-dayold age group exceeded that of the 10-day-old rats. In view of these variations, an
295 TABLE V
Calculated mean values (T/S ratio ± S.E.M.) for control and TP-treated brain regions at each age groltp 2, 5, 60 and 300 rain after injection o I A I B Number of rats used is listed in parentheses.
Min
Z/S (age in days) 5
Significam'e levels (P) lO
17
5vs. lO
IOvs. 17 5vs. 17
0.0367±0.0020 (10) 0.0378±0.0026 (10)
0.0339±0.0022 (10) 0.0345 r:0.0022 (10)
< 0.001 NS* NS NS
0.0779~ 0.0062 (10) 0.0828±0.0068 (10)
0.0652±0.0023( 10) 0.0685±0.0033 (10)
0.0522±0.0026 (8) 0 . 0 5 4 0 i 0 . 0 0 3 3 (8)
NS NS
0.8395 ~ 0.0356 (10) 0.5132 ~ 0.0253 (10)
0.8360 c0.0403 (10) 1.0055-c0.0489(10)
0.5936-~ 0.0248 (10) 0.6148±0.0263(10)
NS 0.001 ": 0.001 0.001 , 0.001 , 0.02
1.6888~0.0799 (10) 2.7152:50.0559 (10)
2.6284±0.0653 (10) 3.3041 ± 0 . 1 5 2 2 ( 1 0 )
2.0491 ±0.0557 (10) 2.0788:L0.0524(10)
0.001 ~: 0.001 0.005 ~-- 0.005 0.001 • 0.001
2 C 0.0649J 0,0044 (8) TP 0.0468±0,0044 (8)
~ 0,001 0.025
5 C TP 6O C TP 3O0 C TP
0.005 - 0.005 < 0.01 ~: 0.005
* P is not significant.
average T/S ratio +: S.E.M. was calculated for the 12 brain regions of control and TP-treated groups for each age group at 2, 5, 60 and 300 rain (Table V). It can be seen that 2, 5 and 60 rain after injection, the average transport of AIB in all brain regions of the controls followed the expected pattern and progressively decreased with maturation (Table V). However, by 300 rain, the lowest average T/S ratio for AIB occurred in the brain regions of the 5-day-old rats and the highest ratio was observed in the 10-day-old age group. Two and 5 rain after injection, the average transport of AIB into all brain regions of the TP-treated rats followed the expected pattern and progressively decreased with age. However, variations from this pattern were observed at the 60 and 300 rain intervals (Table V). During the interval between 2 and 300 rain, the average uptake of AIB by all brain regions of the 5-day-old TP-treated rats was approximately 2.2 times greater than that of the controls, whereas the average transport into brain regions of the 10day-old TP-treated rats was only 1.2 times greater than their controls (Table V). There were no differences in transport between brain regions of control and TPtreated 17-day-old rats. DISCUSSION
Recent in vivo studies indicate that amino acids cross the blood-brain barrier (BBB) by specific carrier mediated transport systems presumed to be localized in plasma membranes of the endothelium of the cerebral capillaries'),~,2z,23,27, 2s. Stereospecific and saturable carrier mediated transport systems have been described for the neutral, basic and acidic amino acids~,2z, zT. The in vivo transport of amino acids out
296 of the brain against a concentration gradient also suggests the presence of carrier mediated efltux systems 15. In general, the ability of amino acids to cross the BBB is greater in the younger animal than in the adult ~8. This has been attributed to incomplete maturation of bidirectional carrier mediated transport systems z3,~s. Although important discrepancies exist between the transport of amino acids into brain slices and their transport across the BBB 2,6. the in vitro active transport system described for AIB~, a4 may have a distinct in vivo counterpart 6.27. in vitro. the uptake of neutral amino acids by brain slices has been divided into somewhat arbitrary and overlapping transport classes ~. These include one ['or large neutral amino acids (e.g., leucine, valine and phenylalanine), one for small neutral amino acids (e.g., AIB. alanine, serine and glycine), and a separate transport system for G A B A 6. In vivo. AIB and cycloteucine (a non-metabolizable neutral amino acid) were found to enter the brain of the infant guinea pig by separate transport mechanisms z7. The regional heterogeneity observed in the transport of AIB by the brain is probably due (among others) to differences in the concentration or availability of carrier molecules in the cell membranes of neurons and]or subceltular particles 4,21. Nuclei and mitochondria prepared from neonatal or adult rat brains actively transported AIB against a concentration gradient in vitro ~1. However. the failure to demonstrate radioactivity in brain nuclei and mitochondria 3 h after the rejection of AIB in rats argues against the subcellular compartmentation of the isotope m vivo ~:~. Separate carrier mediated influx and efflux systems have been proposed for AIB both in vivo 9,t:~ and in vitro'5, ~.5. The differential responses of the influx and efftux systems to drugs 9,15 are of some value in attempting to explain the effects o f TP on the accumulation of AIB in specific brain regions. At any gtven moment the accumulation of AIB is dependent upon the activities of the influx and efflux systems: therefore. it is reasonable to propose that TP preferentially or predominantly influenced a structural, enzymic or metabolic component of one of the two systems. Thus. the significantly higher T/S ratios observed at 300 rain in all brain regions of the 5-day-old TP-treated rats and in certain brain regions of the 10-day-old TP-treated rats at 60 and 300 rain could be due to either stimulation of the influx mechanism and or inhibition of the efflux mechanism. Similarly, the significantly lower accumulation of AIB in most brain regions of the 5-day-old TP-treated rats at 60 rain could be due to either stimulation of the efflux system or inhibition of the influx mechanism, or both. The transient contradictory effects of TP on the accumulation of AIB seen at 60 rain in the 5- and 10-dayold rats may represent differences in the relative degree of maturation of the influx and efflux mechanisms at these ages. The inability of neonatally administered TP to alter the accumulation of AIB in brain regions of the 17-day-old rat suggests that the effectiveness of the steroid decreases with maturation of the transport mechanisms for AIB. The current status of knowledge concerning the transport of amino acids into the brain does not allow us to explain the facilitating effect of neonatally administered TP on the active transport of AI B into specific brain regions. It could be hypothesized
297 that TP increased the synthesis or availability of the carrier protein(s) or cofactors required for the transport of AIB. The development or availability of a larger number of carriers could then explain the failure of TP to increase the transport of AIB into the brain of the 17-day-old rat. Cycloheximide (an inhibitor of protein synthesis) increased the permeability of the BBB to cocaine in the intact rat 1. This effect on the BBB was prevented by prior treatment with the adrenal steroid hydrocortisone 1. The fact that this one steroid has such an effect suggests that other steroids may act in a similar manner. The fact that insulin, a non-steroidal hormone, has been shown to increase the transport of AIB across the isolated diaphragm s and bone (in vitro) 11 suggests a more generalized extension of the data presented here for a steroidal hormone. It has been reported that c-AMP (by an unknown mechanism) increased the transport of several amino acids across the BBB without influencing amino acid metabolism "a. It remains to be determined if neonatally administered TP is capable of activating those aspects of the c-AMP system responsible for the increase in amino acid transport across the brain. Alternatively, it is recognized that the effects of neonatally administered TP on the transport of AI B into specific brain regions may not be due to the direct action of the steroid on some component of the BBB or carrier mechanism for AIB. Alterations in the transport of AIB could be secondary to steroid induced metabolic changes in the brain. The oxidative metabolism of the anterior hypothalamus was elevated in female rats treated neonatally with TP 1,~. it is not known if neonatally administered TP affects the oxidative metabolism of other brain areas; nevertheless, alterations in any metabolic activity of the brain could secondarily influence the activity or synthesis of carrier transport systems. Although the mechanisms remain obscure, the data presented in this study definitively link the elevated active transport of AIB into specific brain regions of the 5- and 10-day-old rat to the administration of TP. Similar results have been obtained in neonatally estrogenized rats (in preparation) 17. The ability of neonatally administered TP to increase the transport of a small inert amino acid into the brain suggests that exposure of the developing human fetus to excessively high levels of androgens might influence the transport of other amino acids across the BBB and, hence, affect the development of the brain. ACKNOWLEDGEMENTS 1 am indebted to Dr. Melvin W. Thorner and Dr. Lidia Vitello for their many helpful suggestions during the preparation of this manuscript, i thank Chudomir G. Popoff for technical assistance and Patricia Downing for preparation of the manuscript. This study was supported by the Medical Research Service of the Veterans Administration and in part by USPHS Research Grant NSI 1501-02 from the National Institute of Neurological Diseases and Stroke.
298 REFERENCES l Angel, C. and Burkett, M . L . , Effects of hydrocortisone and cycloheximide on blood-brain barrier function in the rat. Dis. nerv. Syst., 32 (1971) 53-58. 2 Bands, G.. Daniel. P. M.. Moorhouse, S. R and Pratt. O. E.. The influx of amino acids into the brain of the rat in vivo: the essential compared with the non-essential amino acids. Proe, roy. Soc+ B, 183 (1973) 59-70. 3 Barraclough, C. A., Modification in the CNS regulation of reproduction after exposure of prepuberal rats to steroid hormones, Recent Progr. Hormone Res., 22 (1966) 503-539. 4 Battistin. L. and Lajtha, A., Regional distribution and movement of amino acids in the brain. J. Neurol. Sci.. 10 (1970) 313-322. 5 Cherayit, A., Kandera, J. and Lajtha, A.. Cerebral amino acid transport in vitro .... IV. The effect of inhibitors on exit from brain slices. J. Neurochem.. 14 (1967) 105-+115. 6 Cohen. S. R. and Lajtha, A.. Amino acid transport. In A. Lajtha (Ed.), Handbook o f Neurochemistry, Vol. 7. Plenum Press. New York. 1972, pp. 543-572. 7 Droz, B. and Koenig, H. L.. Localization of protein metabolism in neurons. In A. Lajtha (Ed+). Protein Metabolism o f Nervous System, Plenum Press. New York. 1970. pp. 93--t08. 8 Elsas. L. J.. Albrecht. I. and Rosenberg, L. E.. Insulin stimulation of amino acid uptake in rat diaphragm, J. biol. Chem., 243 (19681 1846-1853. 9 Gordon. M. W., Sims, J. A.. Hanson, R. K. and Kuttner. R. E.. The effects of some psychopharmacological agents and other drugs on the uptake of an amino acid by brain, J. Neuroehem.. 9 (1962) 477--486. 10 Gorski, R . A . and Shryne, J.. lntracerebral antibiotics and androgenization of the neonatal female rat. Neuroendocrinology, 10 (1972) 109-120. II Hahn. T. J.. Downing, S. J. and Phang, J. M.. Insulin effect on amino acid transport in bone: Dependence on protein synthesis and Na ~. Amer. J. Physiol., 220 (1971l 1717-1723. 12 Kawakami. M. and Terasawa. E.. A possible rote of the hippocampus and amygdala in the androgenized rat : Effect of electrical or electrochemical stimulation of the brain on gonadotropin secretion. Endocr. jap., 19 <1972) 349-358. 13 Kuttner. R.. Sims. J. A. and Gordon. M. W., The uptake of a metabolically inert amino acid by brain and other organs. J. Neurochem.. 6 (19611 311 317. 14 Lajtha, A., Lahiri, S. and Toth. J., The brain barrier system - - IV Cerebral amino acid uptake in different classes. J. Neurochem., 10 (1963) 765-773, 15 Lajtha, A. and Toth. J.. The brain barrier system -- II1. The eMux of intracerebrally administered amino acids from the brain, J. Neuroehem.. 9 (1962) t99+-212, 16 Litteria. M., Inhibitory action of neonatal androgenization on the incorporation of :JH-tysine into specific hypothalamic nuclei of the adult female rat, Exp. Neurol.. 41 (1973) 395-401. 17 Litteria. M.. Effects of neonatal estrogenization on the transport of alpha-aminoisobutyric acid into brain. Fed. Proc.. 35 (1976) 799. t8 Litteria, M. and Thorner, M. W.. Inhibition in the incorporation of [:3H]tysine in the Purkinje cells of the adult female rat after neonatal androgenization, Brain Research. 69 (19741 170-173. 19 Moguilevsky, J A. and Rubinstein. L.. Gtycolytic and oxidative metabolism of hypothalamic areas in prepuberal androgenized rats, Neuroendocrinology, 2 (19671 213-221. 20 Nadler. R. D.. Intrahypothalamic locus for induction of androgen sterilization in neonatal female rats, Neuroendocrinology, 9 ~1972) 349-357, 21 Navon. S. and Lajtha, A., The uptake of amino acids by particulate fractions from brain. Biochhll. Biophys. Acta Amst. . 173 (1969) 518-531. 22 Oldendorf. W. H. and Szabo. J., Amino acid assignment to one of three blood-brain ha trier amino acid carriers, Amer. J. Physiol.. 230 (t976)94-98. 23 Orlowski. M.. Sessa. G. and Green. J. P.. ~,-Glutamyl transpeptidase in brain capillaries: Possible site of a blood-brain barrier for amino acids, Science, t84 (1974) 66-68. 24 Petkov. V.. Usunov, P. and Kushev. V.. The role of 3',5~-AMP system in amino acid transport across the blood-brain barrier. ~Iota biol. reed+ germ., 33 11974) 371--383. 25 Riggs, T. R. and Walker. L. M.. Sex hormone modification of tissue levels and urinary excretion of a-aminoisobutyric acid in the rat, Endocrinology, 73 (1963"t 781-788. 26 Salaman, D . F . , RNA synthesis in the rat anterior hypothalamus and pituitary: Relation to neonatal androgen and the oestrus cycle, J. Endocr.. 48 (1970) 125-137. 27 Schain. R. J. and Watanabe. K. S.. Distinct patterns of emry of two non-metabolizable amino acids into brain and other organs of infant guinea pigs, J. Neuroehem.. 19 <1972) 2279--2288.
299 28 Seta, K., Sershen, H. and Lajtha, A., Cerebral amino acid uptake in vivo in newborn mice, Brain Research, 47 (1972) 415~!-25. 29 Shimada, H. and Gorbman, A., Long lasting changes in RNA synthesis in the forebrains of female rats treated with testosterone soon after birth, Biochem. biophys. Res. Commun., 38 (1970) 423-430. 30 Soriero, O. and Ford, D. H., The composition of different regions of the neonatal rat brain in relation to sex. In D. H. Ford (Ed.), Neurobiological Aspects ~[" Maturation and Aging, Progr. Brain Res., Vol. 40, Elsevier, Amsterdam, 1973, pp. 309 319.
287
T H E EFFECTS OF N E O N A T A L A N D R O G E N I Z A T I O N ON T H E IN VlVO T R A N S P O R T OF A L P H A - A M I N O I S O B U T Y R | C A C I D I N T O SPECIFIC R E G I O N S OF T H E R A T BRAIN
M. LITTERIA Nenroh)gy Research, Veterans Admhtistration Hospital, North Chicago, Ill. 60064 (U.S.A.)
(Accepted December 10th, 1976)
SUMMARY The purpose of the present study was to determine if the administration of testosterone propionate (TP) to neonatal rats is followed in vivo by alterations in the transport of the non-metabolizable amino acid, a-aminoisobutyric acid (AIB), into specific regions of the brain. Forty-eight hours after birth, male and female rats were injected s.c. with either 1.25 mg TP or an equivalent volume of vehicle. Five, 10 and 17 days after birth, control and TP-treated rats were decapitated at intervals of 2, 5, 60 and 300 rain after the i.p. injection of 0.25 #Ci [1-14C]a-aminoisobutyric acid/g body weight. Twelve brain regions, i.e., amygdala, cerebellum, corpora quadrigemina, frontal cortex, hypothalamus, medulla, occipital cortex, olfactory bulbs, olfactory tubercles, parietal cortex, pons, pyriform cortex and samples of serum were analyzed in terms of disint./min/mg tissue and as tissue/serum (T/S) ratios. At the end of 300 min there was a significant increase in the active transport of AIB in all brain regions of the 5-day-old TP-treated rats. Similarly, by 300 rain, the active transport of AIB was significantly increased in all brain regions except cerebellum and pons of the 10-dayold TP-treated rats. The administration of TP to neonatal rats did not alter the accumulation and/or active transport of AI B in brain regions of the 17-day-old rat at any of the tested intervals. These data indicate that (I) neonatally administered TP enhanced (either directly or indirectly) the transport and/or accumulation of AIB in specific brain regions of 5- and 10-day-old rats and (2) the effectiveness of the steroid decreased with the age of the rat.
INTRODUCTION The administration of testosterone propionate (TP) to neonatal female rats during the critical period of brain differentiation is followed postpuberally by a syn-
288 drome characterized by anovulation, polycystic ovaries, persistent vaginal cornification and masculinization of sexual behavior a. This syndrome is probably related to TP-induced alterations in hypothatamic 2° and timbic 1~ structures associated with the synthesis and/or release of the pituitary gonadotropins. Although the biochemical nature of these alterations Is unknown, they may be directly or indirectly related to changes in brain nucleic acid z6,29 and or protein synthesis 10 following neonatatly administered TP. Quantitative autoradiographic studies from this laboratory have localized some of the neuroanatomic sites metabolically responsive to neonatallv administered TP u~.ls. The incorporation of [aH]lysine into proteins of specific hypothalamic nuclei (arcuate. periventricular, paraventricular and supraoptict was sign> ficantly inhibited in adult female rats treated neonatally with TP 16. Similarily, the Purkinje cells, but not the granule or stellate cells of the cerebellar cortex incorporated less [aH]lysine into proteins ~s Autoradiography is an invaluable technique for localizing newly synthesized protein 7 However. differences in silver grains between the TP-treated rats and their controls does not imply that the steroid inhibited protein synthesis per se. For example, the steroid might have affected the transport of [aH]lysine into the brain, or produced alterations in the endogenous free lysine paol and or nucleic acid metabolism. The purpose of the present study was to determine if the administration of TP to neonatal rats during the critical period of brain differentiation is followed by alterations in the transport of an amino acid across the blood-brain barrier into specific regions of the brain. Alpha-aminoisobutync acid {AIB} is a small neutral amino acid that enters the brain via career mediated processes z7, but is neither incorporated into proteins nor metabolized by either the brain or other organs ~. AIB was selected ['or this study in order to avoid the problems associated with amino acids that are metabolized by the brain. MATERIALS AND METHODS Three month old virgin Sprague-Dawley rats (Madison. Wisc.) were exposed to a 14:10 h light/dark schedule and fed Purina Rat Chow and water ad libitum in our animal facilities which are maintained at approximately 72 °F. These animals served as our breeding stock. Litter size was reduced to 8 pups (4 males and 4 females whenever possible) within 24 h after birth. The day of birth is called day one. Forty-eight hours after birth, male and female rats were mjected s.c. with either 1.25 mg testosterone propionate (TP) dissolved in sesame oil or with an equivalent volume of vehicle. At 5, 10 and 17 days after birth, a minimum of 8 control and 8 TP-treated rats were each injected i.p. with 0.25 uCi [I-14C]a-aminoisobutyric acid/g body weight (sp. act., 9.0 mCi/mmole. New England Nuclear). Prior to sacrifice, the pups were placed in a small beaker (warmed by a lampl containing bedding from the home cage. The rats were decapitated at intervals of 2.5. 60 and 300 rain following the injection of the [I-t4C]a-aminoisobutyric acid (AIB). The brain was rapidly removed, placed on a Petri dish over ice and freed of adhering meninges and blood. Twelve brain regions, i.e., amygdala (with overlying cortexl (AMY), cerebellum (CB), corpora
289 quadrigemina (CQ), frontal cortex (FCX), hypothalamus (HYPO), medulla (MED), occipital cortex (OCX), olfactory bulbs (OB), olfactory tubercles (OT), parietal cortex (PCX), pons and pyriform cortex (PYCX) were placed in a Petri dish resting on a block of dry ice. It has been shown that only 5 ~o of whole brain radioactivity is due to plasma AIB la. Therefore, it was not necessary to make corrections for tile amount of radioactivity contributed by the blood present in the individual brain regions la. Each frozen tissue was weighed and transferred while still frozen into scintillation vials containing 1.0 ml of solubilizer (Soluene-350, Packard Instrument Co., Inc., Downers Grove, lll.). Blood collected at the time of sacrifice was allowed to clot and serum was separated following centrifugation at 2 C for 30 rain at 600 × g. Ten to 50 #1 aliquots of serum (S) were added to 1.0 ml Soluene-350. Fifteen ml of liquid scintillation cocktail (Dimilume TM-30, Packard Instrument Co., Inc.) was added to the vials following the overnight solubilization of tissues at room temperature. Counts/min (CPM) were determined in a Packard Tri-Carb Liquid Scintillation Spectrometer, Model 3385, following temperature and dark adaptation. Quench corrections were made using the external standardization and least squares methods. Data were expressed in terms of disint./min (DPM)/mg tissue, DPM/I,I serum and as tissue/serum (T/S) ratios. In a separate experiment, the total water content of each of the 12 brain regions was determined for the 5-, 10- and 17-day-old control and TP-treated rats. The removal and handling of tissues is described above. The tissues were dried to constant weight and the amount of water was calculated from the difference between the weights of the frozen and dried tissues. The significance of differences between the control and TP-treated groups was determined by Student's t-test. P --< 0.05 was considered significant. RESULTS A IB dose response
The in vivo uptake and accumulation of AIB by the 12 brain regions and serum were determined at three different concentrations of AIB in order to insure that the dose selected for this study would be below saturation kinetics. Five- and 10-day-old control rats were each injected i.p. with C1 0.125 #Ci; C2 -- 0.25 ~Ci; Ca = 0.40 /~Ci of AIB/g body weight. The rats were sacrificed 5 rain after the injection of the isotope. DPM/mg tissue and DPM/#I serum were twice as great for Cz than Ci, and approximately 1.6 times higher for Ca than 02. There were no significant differences between T/S ratios for any given tissue for either the 5 or 10 day old rats at C1, C2 and Ca. These concentrations of AIB were, therefore, below saturation kinetics and 0.25 /zCi/g body weight was chosen for this study. T/S ratios rather than tissue concentration ( D P M / m g tissue) are an accurate measure of AIB transport. Ratios greater than unity' indicate the occurrence of active transport. Tissue water content
In agreement with others a0, normal neonatal rats showed no sex differences in
290 the water c o n t e n t o f specific b r a i n regions. Similarly, there were n o sex :differences in the water content o f the 12 b r a i n regions o f the 5-, 10- a n d 17-day-old r a t f o l l o w i n g the a d m i n i s t r a t i o n o f TP. D a t a f r o m b o t h sexes were, therefore; combined: for the c o n t r o l or T P - t r e a t e d groups. Differences in water content were f o u n d between control a n d T P - t r e a t e d rats for some tissues at 5, 10 a n d 17 days o f age. However, these changes were so small that corrections m a d e for these differences did n o t alter the significance o f the T/S ratios between c o n t r o l a n d T P - t r e a t e d groups, E f f e c t s o f T P on the transport o f A I B S e x differences. There were no sex differences in the c o n c e n t r a t i o n o f A I B in serum or tissues o f control or T P - t r e a t e d 5-, 10- a n d 17-day-old rats at any o f the tested time intervals. D a t a from both sexes were, therefore, c o m b i n e d in the control o r TPtreated groups. Serum. In all age groups the c o n c e n t r a t i o n o f A I B was highest in the serum o f b o t h c o n t r o l a n d T P - t r e a t e d rats 5 minutes after a d m i n i s t r a t i o n o f t h e motope (Table 1). In pilot studies, A I B levels were lower t h a n t h e 5 min values in samples o f serum from rats sacrificed 15 and 30 min after t h e injection o f the isotope. This indicates t h a t the actual p e a k in serum A I B was between 2 a n d 15 min. Serum A I B levels for 5-day-old c o n t r o l or T P - t r e a t e d rats were m a r k e d l y lower t h a n those o f the 10- and 17-day-old rats 2 a n d 5 min after a d m i n i s t r a t i o n o f the i s o t o p e (Table 1). O t h e r workers have r e p o r t e d that the low c o n c e n t r a t i o n o f A t B p r e s e n t in the p l a s m a o f n e w b o r n rats could not be a t t r i b u t e d to a greater a c c u m u l a t i o n o f the i s o t o p e in the neonatal liver a n d kidney aa. Differences in serum A I B levels between c o n t r o l a n d T P - t r e a t e d groups occurred only in the 5-day-old rats 2 and 60 min after a d m i n i s t r a t i o n o f the isotope (Table I). S e r u m A I B levels in the TP-
TABLE 1 Concentration of AIB in rat serum 2. 5, 60 and 300 min after admin&tration
Number of rats used is listed in parentheses. C, control: TP, treated with testosterone prop~onate. Age
DPM/ttI* ( Min after injection o1 AIB ) 2
5-day C TP 10-day C TP 17-day C TP
5
604.9±17.3(8) 766.9 -L 36.3 (81"*
60
797.3 ~ 64.6(10) 818.4 m 36.1 (10)
30o
255.1r~ 10.8(10) 174.7~ 12.6(101 331.0-t 21.7 (10~*** 161.1 11.8 (10/
1128.0±25.3(101 1091.7 + 29.9 ( 1 0 )
1177.8~: 38.2 (10) 1163.0:, 32.5 (10~
228.6 j : l l . 4 ( 1 0 ) 230.7• 1t.9 (101
153.7 i 15.4(10) 141.5 : 4.0 (10~
1115.1 ~39.2(10) 1096.4±69.2(101
1232.1 ~ 51.2(8) 1175.23 29.8(81
277.3-~ 9.4(10) 250.9 ~_ 10.5(10)
163.2-~ 3.8(10~ 156.3 ~. 5.6(i0)
* Mean ± S,E.M. ** P <~ 0.005. *** P -< 0.01.
291 TABLE 1l
Effects of neonatal androgenization on T/S ratios o[' A IB in brain regions o]' the 5-day-oM rat T/S ratios are expressed as mean ± S.E.M. Number of rats used is listed in parentheses. C, control; TP, treated with testosterone propionate. Abbreviations for tissues are listed in the Materials and Methods section. P values are indicated as * < 0.05; ** < 0.025; *** < 0.02; § < 0.01 ; §§ < 0.005 ; ~§~ -< 0.001.
Braitt
T/S (Mitt aJ?er injection o / A I B )
regions
2
5
60
300
0.0400±0.0015~§§ (8) 0.0250~0.0025 (8)
0.0685-20.0038 (10) 0.0750±0.0062 (10)
0.6994±0.0303§§§ (10) 0.4495±0.0332 (10)
1.3044 ±0.0661.~§¢ (10) 2.3523i0.0450 (10)
0.1074±0.0087"** 0.0751 ±0.0070
0.1246±0.0045 0.1235±0.0034
1.3262 ±0.0460§~ 0.7930~0.0381
2.3920i 0.0561§§§ 4.0913 ±0.0578
0.0543±0.0040*** 0.0400±0.0037
0.0545 ~¢O.0064 0.0568±0.0054
0.5490 ±0.0290§,~ 0.3515:£0.0513
1.1051 ~0.0492,~ 1.8904±0.0786
0.0540£=0.0037*** 0.0390±0.0036
0.0831 ±0.0068 0.0986i0.0137
0.9444 £0.0502§~,~ 0.5410±0.0194
1.6293±0.1060~§ 3.3321 ±0.0507
0.0342 ±0.0008~ 0.0273±0.0015
0.0459±0.0040 0.0555±0.0047
0.5522 ~:0.0209§,~ 0.4042i0.0200
1.2711 ±0.0586,~ 1.9880i0.0405
0.1118 :~ 0.0074"** 0.0806-~ 0.0090
0.0965±0.0066 0.0990 ~ 0.0095
0.7851 i 0 . 0 4 7 7 ~ 0.4578 :~0.0244
1.7015 ±0.0754§§~ 2.5506 ~0.0792
0.0548 ~0.0040"** 0.0420±0.0027
0.0751 -E0.0045 0.0739±0.0066
0.8503±0.03080§§ 0.4751 i0.0098
1.7351 ±0.0814§~ 2.9699±0.0762
0.1098 ±0.0081 ~ 0,0733+0.0090
0.1069 i: 0.0111 0.1044 ~0.0070
1.3127±0.0537§§~ 0.8942:~ 0.0319
2.6491 ±0.0877§§~ 3.2842i0.0322
0.0331 ±0.0008* 0.0281 ±0.0020
0.0520~0.0074 0.0553 tO.0053
0.5301 i0.0308§§~ 0.2917 f 0.0140
1.1577 t 0.0601 §§,~ 1.8408 ±0.0213
0.0529±0.0047* 0.0400~0.0037
0.0752 i0.0062 0.0811 50.0036
0.8602 ±0.0356,~§§ 0.4772 t:0.0328
1.7883 i 0 . 1 0 0 4 - ~ 3.0000 ~:0.0609
0.0706+0.0065*** 0.0506£0.0038
0.0742 ~0.0077 0.0819 %0.0082
0.7826±0.0291 ~§~ 0.4722 90.0156
1.9387±0.1101 §§~ 2.7196 ~0.0731
0.0560±0.0022~§ 0.0410i0.0037
0.0788 ~0.0050 0.0884~0.0077
0.8818±0.0232§§8 0.55101 0.0135
1.5932 ±0.1079§§,~ 2.5635 i0.0553
AMY C TP CB C TP CQ C TP FCX C TP HYPO C TP MED C TP OCX C TP OB C TP OT C TP PCX C TP PONS C TP PYCX C TP
treated rats were significantly greater than the control values at both intervals. This f i n d i n g m a y p o s s i b l y be a t t r i b u t e d t o t h e a b i l i t y o f T P t o d e c r e a s e t h e r e n a l c l e a r a n c e o f A I B in r a t s 2'5.
Brain regions. T h e t r a n s p o r t o f A I B i n c r e a s e d w i t h t i m e in all b r a i n r e g i o n s o f t h e 5-, I0- a n d 1 7 - d a y - o l d c o n t r o l a n d T P - t r e a t e d r a t s ( T a b l e s II, I I | a n d IV). T h e s e
292
results are in agreement with the m vivo study by Kuttner et al.~% in which the uptake of AIB by the whole brain of adult rats increased slowly with time and reached a maximum at approximately 20 h after administration. There is considerable regional variation in the uptake of AIB by the brain of both control and TP-treated rats. However, the accumulation of AIB was consistently higher in the CB. OB and P O N S T A B L E I11
Effects o[ neonatal androgenization on T/S rath~s o f AIB in brain regions oJ the lO-day-old rat T/S ratios are expressed as mean _~ S.E.M. N u m b e r of rats used is listed in parentheses, C. control: TP, treated with testosterone propionate. Abbreviations for tissues are listed in the Materials and Methods section. P values are indicated as * - 0.05 : ** --~ 0.025 : *** - 0.02 : ~ . 0.01 ; ~ - " 0.005 : §§,~ .: 0.001.
Brain
T/S ( Min after injection o f AIB)
regkms
2
5
60
300
AMY C
0.0283±0.0012 (10)
0.0513 -~ 0.0011 I101
0.0289 ~O.0016 (10)
0.0567 :~: 0.0037 ~10)
0.6059 ~ 0 . 0 3 9 0 . ~ (10} 0.9347 3:0,0348 ( 1 0 1
2.1764 0,0757~§~ (10p 2,9205 }0,1118~10]
0.0573 ~0.0029 0.0558-2.0.0030
0,0916 i0,0041 0.1011 ~0.0031
1.3774. 0.0473 1.4455 5 0.0646
4.2221 0.0082 4.4810-k O, 1879
0.0295_+0.001 I 0.0328 ~ 0.0031
0.0488-;0.0008 0.0488 -r 0.0009
0.6599 T 0.0480" 0.7817 -v0.0307
1.9721 0,0563§§6 2.6060 ~ 0.1248
0.0362 :E0.0020 0.0370+0.0018
0.0737d 0.0021 0.0806 3 0.0033
0,9300s 0.0419~ 1.2115-c 0.0714
2.712t-: 0 1069~ 3.8881 0.3608
0.0235 ~:0.0009 0.0231 ~ 0.0013
0.0384 ~ 0.0014 0.0380-1 0.0023
0.5884 _: 0.0364 0.5981- 0.0222
1.7892 i 0.0440.~ 2.3518 : 0.0832
0.0437 3 0.0045 0.0089 ~0.0031
0.0777 ~0.0040 0,0745 ~ 0.0023
0.8012~ 0.0339§§~ 1.0253 ~ 0.0332
2.8329 0.0514,~.~,~ 3.5890 ~O.1367
0.0351 t 0 . 0 0 1 4 0.0353.3 0.0030
0.0692 -c 0.0022 0,0699,0.0025
0.8077-L0.0329 §~,~ 1.0615 -i 0.0472
2.6827 -_0.0311 ,~§ 3.4031 40.1163
0.0476 ~0.0016 0.0460 ~ 0.0031
0.0823 --0.0031 0.0945-50.0076
1.1019:a 0.0045"** I 3246 m0.0640
3.0127 r_0.0585~,~ 3.9640~ 0.1161
0.0245 _k0.0013 0.0260%O.0019
0.0444T0.0014 0.0498::-0.0041
0.5405 _L0.0232 ~'~'~ 0.71013 :~ 0.0314
1.8723 ~. 0.648 ~i§~ 2 4154 £0,1040
0.0364 3.0.0023 0.0389~ 0.0024
0.0682 - 0.0020 0.0641 ~ 0.0025
0 8554~0.0469"** 1.0664 ~ 0.0619
2.6287 ~ t),0600§§-~ 3.4468" 0.1236
0.0452~_0.0032 0.0068+0.0040
0.0718 T0.003 I 0.0714~ 0.0036
0 8260=. 0.0054 0.8677 ~ 0.0000
3 0573 :O.1200 3.2065 -0.2231
0.0335-t0.0018 0,0345 j-0.0023
0.0655 ~_0.0020 0.0726-c0.0035
0.9378 ~0.0041 1.0384 7 0 0854
2,5827 ~ 0 . 0 6 6 9 ~ 3,3765 <0.1377
TP CB C TP
CQ C TP FCX C TP HYPO C TP MED C TP OCX C TP OB C TP OT C TP PCX C TP PONS C TP PYCX C TP
293
of the 5- and 10-day-old control rats 300rain after administration of the isotope (Tables ii and lII). The uptake of AlB was also highest in the CB and PONS of the 17-day-old control rats at the 300 min interval (Table IV). By 60 rain, AIB had been actively transported into the CB and OB of the 5- and 10-day-old control rats and into the CB, FCX, MED, OCX, OB, PCX and PYCX of the 10-day-old TP-treated rats (Tables 1I and l If). AI B was actively transported into T A B L E IV
Ej]~wts qf neonatal androgenization
on
T/S ratios of A IB in brain regions o]' the l 7-day-old rat
T/S ratios are expressed as m e a n ± S.E.M. N u m b e r o f rats used is listed in parentheses. C, control; TP, treated with testosterone propionate. Abbreviations for tissues are listed in the Materials and Methods section.
Brahl
T/S (minutes t~?er injection of AIB)
re~ions
2
5
60
300
0.0227 ± 0.0014 (10) 0.0231 ~ 0.0008 (10)
0.0362 i 0.0013(8) 0.0363 i 0.0019 (8)
0.5401 ± 0.0196 (10) 0.5464 ~ 0.0352 (10)
1.7814 L 0.0469 (10) 1.7765 ± 0.0521 (10)
0.0444 :~z 0.0028 0.0421 ~ 0.0036
0.0560 i: 0.0032 0.0602 ± 0.0033
0.7402 ~_ 0.0457 0.7624 ~ 0.0355
2.5268 ~ 0.0762 2.5559 i: 0.0597
0.0381 _L 0.0016 0.0368 ~ 0.0014
0.0454 ± 0.0014 0.0485 +_ 0.0028
0.4708 k 0.0150 0.5115 i 0.0187
1.7221 i 0.0436 1.7170 ± 0.0430
0.0410 i 0.0029 0.0425 ± 0.0024
0.0543 i 0.0021 0.0554 ± 0.0046
0.6400 ~ 0.0140 0.6542 ~ 0.0217
2.1969 £ 0.0472 2.2035 ± 0.0559
0.0212 ± 0.0013 0.0216 ± 0.0008
0.0300 ± 0.0009 0.0310 i 0.0005
0.4547 -- 0.0162 0.4639 5z 0.0129
1.6123 ± 0.0400 1.6722 -- 0.0340
0.0499 ~ 0.0056 0.0534 ± 0.0047
0.0904 ± 0.0082 0.0922 ± 0.0106
0.6124 Az 0.0207 0.6758 ~ 0.0325
2.4321 ± 0.0625 2.4886 ± 0.0479
0.0327 ± 0.0014 0.0344 { 0.0013
0.0493 ± 0.0013 0.0501 ~ 0.0035
0.5810 & 0.0215 0.5914 ~ 0.0248
2.0136 i 0.0576 2.0665 ± 0.0896
0.0367 ± 0.0027 0.0383 i 0.0031
0.0653 t: 0.0027 0.0670 -- 0.0015
0.7228 ~ 0.0483 0.7547 ~ 0.0175
2.0787 ± 0.0656 2.1318 ± 0.0559
0.0224 ~ 0.0015 0.0228 ± 0.0010
0.0391 ± 0.0014 0.0412 i 0.0029
0.4606 ± 0.0241 0.4882 -- 0.0224
1.4372 t~ 0.0342 1.4670 i 0.0255
0.0298 ± 0.0015 0.0310 ± 0.0034
0.0512 ± 0.0035 0.0502 i 0.0032
0.5718 £ 0.0278 0.5852 ± 0.0212
2.1071 ± 0.0605 2.1660 ± 0.0560
0.0378 ~ 0.0018 0.0379 ± 0.0019
0.0600 ± 0.0029 0.0634 £ 0.0023
0.7600 ± 0.0327 0.7693 t 0.0494
2.7681 ± 0.0890 2.8150 ± 0.0664
0.0297 ± 0.0019 0.0299 i 0.0021
0.0489 % 0.0018 0.0524 ~ 0.0023
0.5702 k 0.0114 0.5743 ~ 0.0243
1.9128 t 0.0455 1.8856 k 0.0428
AMY C TP CB C TP
co C TP FCX C TP HYPO C TP MED C TP OCX C TP OB C TP OT C TP PCX C TP PONS C TP PYCX C TP
294 all brain regions of both control and TP-treated 5-. 10- and 17-day-old rats 300 min following administration of the isotope (Tables I1.11t and IV). Administration of TP to neonatal rats did not affect the absolute concentration of AIB (DPM/mg tissue) in brain regions of the 5-day-old rat 2 min after injecnon of the isotope. Therefore, the lower T S ratios observed at this interval are due to the higher concentration of AIB in the serum of the TP-treated rats (Tables i and i[). There were no differences in either the absolute concentrations or transport of AIB into brain regions of the control or TP-treated 5-day-old rats at the 5 min interval (Table I!). With the exception of the CQ and HYPO. the absolute concentrations of AIB in the remaining 10 brain areas of the 5-day-old TP-treated rats were significantly lower than their controls at the 60 min interval. The lower T S ratios observed for the CQ and HYPO of the 5-day-old TP-treated rats are due to the higher level of AIB in the serum of these animals at 60 min. Two factors contributed to the lower T/S ratios observed in the remaining 10 brain regions of the 5-day-old TP-treated rats. i.e., significantly lower absolute concentrations (DPM/mg tissue)of A1B present in these brain areas and the higher level of isotope in the serum of the steroid treated rats (Tables 1 and IlL By 300 min. T/S ratios tbr all brain regions of the 5-day-old TP-treated rats were significantly increased above those of the controls (Table I11. Serum levels of AIB were the same for both control and TP-treated rats at 300 min (Table I). Therefore, the higher T'S ratios are entirely attributable to the greater concentration of isotope in the brain regions of the steroid treated rats (Table I1). The administration of TP to neonatal rats did not affect either the absolute concentrations or the transport of AIB into brain regions of the 10-day-old rats 2 and 5 min after the injection of the isotope (Table 111). With the exceptions of CB. HYPO, PONS and PYCX. the T S ratios for brain regions of the 10-day-old TPtreated rats were significantly higher than the controls at the 60 min interval. By 300 min, T S ratios were significantly greater in all brain regions of the steroid treated rats with the exception of the CB and PONS (Table !11). There were no differences in serum AIB levels between the 10-day-old control and TP-treated rats (Table t). Therefore. the higher T/S ratios for certain brain regions of the TP-treated rats are entirely due to the higher absolute concentrations of isotope in these regions at 60 and 300 rain. The administration of TP to neonatal rats did not alter the transport of AIB into specific brain regions of the 17-day-old rat at any of the tested intervals ITables 1 and IVI. In general, the ability of anaino acids to enter the brain in vivo ~s greater in younger animals than in the adult 28. Therefore. it was of interest to determine if the transport of AIB into individual brain regions followed this pattern. A comparison of the data for the control groups in Tables 11, I11 and IV indicate that there are numerous exceptions to the general rule. For example, the transport of AIB into CQ, FCX and M E D at the 2 min interval is greater for the 17-day-old rats in comparison to the 10-day age group, and at 5 rain, transport of AIB into the MED of the 17-dayold age group exceeded that of the 10-day-old rats. In view of these variations, an
295 TABLE V
Calculated mean values (T/S ratio ± S.E.M.) for control and TP-treated brain regions at each age groltp 2, 5, 60 and 300 rain after injection o I A I B Number of rats used is listed in parentheses.
Min
Z/S (age in days) 5
Significam'e levels (P) lO
17
5vs. lO
IOvs. 17 5vs. 17
0.0367±0.0020 (10) 0.0378±0.0026 (10)
0.0339±0.0022 (10) 0.0345 r:0.0022 (10)
< 0.001 NS* NS NS
0.0779~ 0.0062 (10) 0.0828±0.0068 (10)
0.0652±0.0023( 10) 0.0685±0.0033 (10)
0.0522±0.0026 (8) 0 . 0 5 4 0 i 0 . 0 0 3 3 (8)
NS NS
0.8395 ~ 0.0356 (10) 0.5132 ~ 0.0253 (10)
0.8360 c0.0403 (10) 1.0055-c0.0489(10)
0.5936-~ 0.0248 (10) 0.6148±0.0263(10)
NS 0.001 ": 0.001 0.001 , 0.001 , 0.02
1.6888~0.0799 (10) 2.7152:50.0559 (10)
2.6284±0.0653 (10) 3.3041 ± 0 . 1 5 2 2 ( 1 0 )
2.0491 ±0.0557 (10) 2.0788:L0.0524(10)
0.001 ~: 0.001 0.005 ~-- 0.005 0.001 • 0.001
2 C 0.0649J 0,0044 (8) TP 0.0468±0,0044 (8)
~ 0,001 0.025
5 C TP 6O C TP 3O0 C TP
0.005 - 0.005 < 0.01 ~: 0.005
* P is not significant.
average T/S ratio +: S.E.M. was calculated for the 12 brain regions of control and TP-treated groups for each age group at 2, 5, 60 and 300 rain (Table V). It can be seen that 2, 5 and 60 rain after injection, the average transport of AIB in all brain regions of the controls followed the expected pattern and progressively decreased with maturation (Table V). However, by 300 rain, the lowest average T/S ratio for AIB occurred in the brain regions of the 5-day-old rats and the highest ratio was observed in the 10-day-old age group. Two and 5 rain after injection, the average transport of AIB into all brain regions of the TP-treated rats followed the expected pattern and progressively decreased with age. However, variations from this pattern were observed at the 60 and 300 rain intervals (Table V). During the interval between 2 and 300 rain, the average uptake of AIB by all brain regions of the 5-day-old TP-treated rats was approximately 2.2 times greater than that of the controls, whereas the average transport into brain regions of the 10day-old TP-treated rats was only 1.2 times greater than their controls (Table V). There were no differences in transport between brain regions of control and TPtreated 17-day-old rats. DISCUSSION
Recent in vivo studies indicate that amino acids cross the blood-brain barrier (BBB) by specific carrier mediated transport systems presumed to be localized in plasma membranes of the endothelium of the cerebral capillaries'),~,2z,23,27, 2s. Stereospecific and saturable carrier mediated transport systems have been described for the neutral, basic and acidic amino acids~,2z, zT. The in vivo transport of amino acids out
296 of the brain against a concentration gradient also suggests the presence of carrier mediated efltux systems 15. In general, the ability of amino acids to cross the BBB is greater in the younger animal than in the adult ~8. This has been attributed to incomplete maturation of bidirectional carrier mediated transport systems z3,~s. Although important discrepancies exist between the transport of amino acids into brain slices and their transport across the BBB 2,6. the in vitro active transport system described for AIB~, a4 may have a distinct in vivo counterpart 6.27. in vitro. the uptake of neutral amino acids by brain slices has been divided into somewhat arbitrary and overlapping transport classes ~. These include one ['or large neutral amino acids (e.g., leucine, valine and phenylalanine), one for small neutral amino acids (e.g., AIB. alanine, serine and glycine), and a separate transport system for G A B A 6. In vivo. AIB and cycloteucine (a non-metabolizable neutral amino acid) were found to enter the brain of the infant guinea pig by separate transport mechanisms z7. The regional heterogeneity observed in the transport of AIB by the brain is probably due (among others) to differences in the concentration or availability of carrier molecules in the cell membranes of neurons and]or subceltular particles 4,21. Nuclei and mitochondria prepared from neonatal or adult rat brains actively transported AIB against a concentration gradient in vitro ~1. However. the failure to demonstrate radioactivity in brain nuclei and mitochondria 3 h after the rejection of AIB in rats argues against the subcellular compartmentation of the isotope m vivo ~:~. Separate carrier mediated influx and efflux systems have been proposed for AIB both in vivo 9,t:~ and in vitro'5, ~.5. The differential responses of the influx and efftux systems to drugs 9,15 are of some value in attempting to explain the effects o f TP on the accumulation of AIB in specific brain regions. At any gtven moment the accumulation of AIB is dependent upon the activities of the influx and efflux systems: therefore. it is reasonable to propose that TP preferentially or predominantly influenced a structural, enzymic or metabolic component of one of the two systems. Thus. the significantly higher T/S ratios observed at 300 rain in all brain regions of the 5-day-old TP-treated rats and in certain brain regions of the 10-day-old TP-treated rats at 60 and 300 rain could be due to either stimulation of the influx mechanism and or inhibition of the efflux mechanism. Similarly, the significantly lower accumulation of AIB in most brain regions of the 5-day-old TP-treated rats at 60 rain could be due to either stimulation of the efflux system or inhibition of the influx mechanism, or both. The transient contradictory effects of TP on the accumulation of AIB seen at 60 rain in the 5- and 10-dayold rats may represent differences in the relative degree of maturation of the influx and efflux mechanisms at these ages. The inability of neonatally administered TP to alter the accumulation of AIB in brain regions of the 17-day-old rat suggests that the effectiveness of the steroid decreases with maturation of the transport mechanisms for AIB. The current status of knowledge concerning the transport of amino acids into the brain does not allow us to explain the facilitating effect of neonatally administered TP on the active transport of AI B into specific brain regions. It could be hypothesized
297 that TP increased the synthesis or availability of the carrier protein(s) or cofactors required for the transport of AIB. The development or availability of a larger number of carriers could then explain the failure of TP to increase the transport of AIB into the brain of the 17-day-old rat. Cycloheximide (an inhibitor of protein synthesis) increased the permeability of the BBB to cocaine in the intact rat 1. This effect on the BBB was prevented by prior treatment with the adrenal steroid hydrocortisone 1. The fact that this one steroid has such an effect suggests that other steroids may act in a similar manner. The fact that insulin, a non-steroidal hormone, has been shown to increase the transport of AIB across the isolated diaphragm s and bone (in vitro) 11 suggests a more generalized extension of the data presented here for a steroidal hormone. It has been reported that c-AMP (by an unknown mechanism) increased the transport of several amino acids across the BBB without influencing amino acid metabolism "a. It remains to be determined if neonatally administered TP is capable of activating those aspects of the c-AMP system responsible for the increase in amino acid transport across the brain. Alternatively, it is recognized that the effects of neonatally administered TP on the transport of AI B into specific brain regions may not be due to the direct action of the steroid on some component of the BBB or carrier mechanism for AIB. Alterations in the transport of AIB could be secondary to steroid induced metabolic changes in the brain. The oxidative metabolism of the anterior hypothalamus was elevated in female rats treated neonatally with TP 1,~. it is not known if neonatally administered TP affects the oxidative metabolism of other brain areas; nevertheless, alterations in any metabolic activity of the brain could secondarily influence the activity or synthesis of carrier transport systems. Although the mechanisms remain obscure, the data presented in this study definitively link the elevated active transport of AIB into specific brain regions of the 5- and 10-day-old rat to the administration of TP. Similar results have been obtained in neonatally estrogenized rats (in preparation) 17. The ability of neonatally administered TP to increase the transport of a small inert amino acid into the brain suggests that exposure of the developing human fetus to excessively high levels of androgens might influence the transport of other amino acids across the BBB and, hence, affect the development of the brain. ACKNOWLEDGEMENTS 1 am indebted to Dr. Melvin W. Thorner and Dr. Lidia Vitello for their many helpful suggestions during the preparation of this manuscript, i thank Chudomir G. Popoff for technical assistance and Patricia Downing for preparation of the manuscript. This study was supported by the Medical Research Service of the Veterans Administration and in part by USPHS Research Grant NSI 1501-02 from the National Institute of Neurological Diseases and Stroke.
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