α1- and α2-noradrenergic receptors in steroid-sensitive brain areas: development and response to estradiol-17β benzoate in neonatal guinea pigs

Developmental Brain Research, 42 (1988) 247-252 Elsevier

247

BRD 50787

a 1- and a2-noradrenergic receptors in steroid-sensitive brain areas: development and response to estradiol-17fl benzoate in neonatal guinea pigs Allan E. Johnson 1, Bruce Nock 2, Bruce S. McEwen 3 and Harvey H. Feder 4 t Section on Comparative Studies of Brain and Behavior, Laboratory of Clinical Sciences, National Institute of Mental Health, Poolesville, MD 20837 (U.S.A.), 2Departments •f Psychiatry/Anat•my and Neur•bi•l•gy and McD•nneu Center f•r Studies •f Higher Brain Function, Washington University School of Medicine, St. Louis, MO 63110 (U.S.A.), 3The Rockefeller University, New York, NY 10021 (U.S.A.) and ~Department of Biological Sciences and Institute of Animal Behavior, Rutgers University, Newark, NJ 07102 (U.S.A.)

(Accepted 5 April 1988) Key words: al-Receptor; a2-Receptor; Prazosin; p-Aminoclonidine; Quantitative autoradiography; Ventromedial hypothalamic nucleus; Guinea pig; Sexual behavior

Immature female guinea pigs are relatively insensitive to the effects of ovarian steroids on certain reproductive processes including the facilitation of sexual receptivity and the induction of hypothalamic progestin receptors by estrogen. In adult guinea pigs, a-receptor-mediated noradrenergic transmission is known to affect both of these processes. In the experiments reported here, we investigated the possibility that age-related differences in al- and a2-noradrenergic receptors might underlie the insensitivity of neonates to ovarian steroids. With tritium-sensitive film autoradiography, the distribution of a : and a2-receptors in neonatal and adult female guinea pigs was compared and the effects of exogenous estradiol-17fl on a-receptor binding was examined in neonatal guinea pigs. The results of these experiments showed that in animals not treated with estrogen the binding of both a-receptor subtypes differed between adults and neonates in several brain regions including areas known to be involved in the regulation of reproduction. In all regions where differences occurred, a-receptor levels were higher in neonatal females than in adult females. In addition, in contrast to previously reported results in adults, estrogen did not affect a-receptor binding in any region of neonatal guinea pig brain. INTRODUCTION In adult guinea pigs, a-receptor-mediated noradrenergic transmission is known to interact with ovarian steroids to regulate a n u m b e r of reproductive processes 16. For example, the steroid-dependent lordosis response can be blocked by specific a t- and a2-receptor antagonists 14'28 and potentiated by a-receptor agonists 5. Similarly, a-receptor activation appears to influence nuclear estrogen receptor accumulation 4 and the induction and/or redistribution of progestin receptors in certain brain areas (including the hypothalamus) that are involved in the regulation of sexual receptivity 2A6'17. Conversely, estradiol-17fl benzoate has been shown to alter the binding of a 1- and a2-receptors in

many steroid-concentrating regions of adult guinea pig brain l°'lt. A m o n g the areas where a-receptors are affected by estradiol-17fl benzoate is the ventromedial hypothalamic nucleus which is a principal site of steroid action for the induction of sexual receptivity in guinea pigs 6A3. Steroid-induced changes in a-receptor mediated noradrenergic transmission, therefore, may be involved in the regulation of certain reproductive processes in adult guinea pigs. Compared to adult females, immature guinea pigs are relatively insensitive to the behavioral and biochemical effects of ovarian steroids 23. Similar to adults treated with a-receptor blockers, immature females do not display the lordosis response when treated with ovarian steroids 1"3'7'23"3°. Furthermore, the degree of induction of hypothalamic progestin re-

Correspondence: A.E. Johnson, NIH Animal Center, P.O. Box 289, Poolesviile, MD 20837, U.S.A.

0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

248 ceptors by estradiol-17fl benzoate is lower in immature guinea pigs than in adults ~4'25. Because a-receptor-mediated noradrenergic transmission and ovarian steroids interact to regulate steroid-facilitated sexual receptivity and the induction of hypothalamic progestin receptors in adults, it is possible that the insensitivity of immature guinea pigs to steroids may be due to an age-related difference in the interaction of these two systems. In the following experiments, we examined the possibility that age-dependent changes in a-receptor binding or in the sensitivity of hypothalamic a-receptors to estradiol-17fl benzoate modulation might in part underlie the insensitivity of neonates to ovarian hormones. In the first experiment, in vitro quantitative receptor autoradiography was used to compare the distribution and concentration of a-receptors in neonatal and adult female guinea pig brains. Particular attention was paid to regions involved in the regulation of sexual receptivity and to regions where ~zreceptor activation affects estrogen and progestin receptors. In a second study, the effect of exogenous estradiol-17fl benzoate treatment in neonates was examined to determine if a-receptor binding is sensitive to steroid modulation in immature guinea pigs as it is in adults. MATERIALS AND METHODS

Animals Female, Hartley strain guinea pigs (from stock originally purchased from Camm Research Laboratories, Wayne, N J) were bred in our laboratory. After parturition, mothers and their litters were housed in cages measuring 56 x 51 x 38 cm. Adults were group-housed in cages measuring 90 x 60 x 38 cm. Purina guinea pig chow-and water were available at all times and cabbage was supplied weekly. Lights in the colony room were on from 06.00 to 20.00 h and temperature was maintained at about 22 °C. Adult females were ovariectomized under a combination of Innovar-Vet (Pitman-Moore, Washington Crossing, NJ; 0.08 ml/animal, s.c.) and Chloropent (Fort Dodge Laboratories Inc., Fort Dodge, Iowa; 1.7 ml/kg, i.p.) one week prior to experimentation. Newborn infant females were ovariectomized under a combination of ketamine hydrochloride (80 mg/kg, Bristol Laboratories, Syracuse, NY) and Rompun (4

mg/kg, Miles Laboratories, Shawnee. KS) and treated with either estradiol-17fi benzoate (10 ugA). 1 ml sesame oil) or vehicle 24 and 48 h later. Animals were sacrificed 48 h after the second injection.

a-Receptor assays Animals were killed by decapitation and brains were rapidly removed and frozen on dry ice. Brain slices (30 ~tm thick) through preoptic area and hypothalamus (Plates 15-21 in ref. 19) were cut with a cryostat at -12 °C, thaw-mounted onto subbed slides and stored at -60 °C. Alternate pairs of slices were assigned to a~- or as-receptor assays. a-Receptors were labeled as previously described 11'15'1s'2° with either 1.0 nM [3H]prazosin for a~-receptors (New England Nuclear, spec. act. = 80.9 Ci/mmol) or 2.5 nM [3H]p-aminoclonidine for a2-receptors (New England Nuclear; spec. act. = 40.5 Ci/mmol) as the radiolabeled ligands. Phentolamine hydrochloride (10uM) was used to define nonspecific binding for both receptor subtypes. Autoradiograms were generated by exposing tritium-sensitive LKB Ultrofilm (LKB, Gaithersburg, MD) to labeled slices at room temperature for 6 (ai) or 10 (a2) weeks. Receptor binding was quantified using a computer-assisted spot-densitometer that converted optical density to tool/rag protein using a standard curve derived from coexposed tritiated brain-mash standards. RESULTS

Distribution of a t- and a2-receptors in brain of adult and neonatal female guinea pigs In this experiment, the concentration and distribution of a~- and a~-receptors were compared in intact newborn female (i.e. less than 24 h old; n = 4) and ovariectomized adult (60 day old; n = 4) guinea pigs. Differences were found in both [3H]prazosin and [3H]p-aminoclonidine binding (Table I). In all cases where differences occurred, binding was higher in neonates than in adults. Differences in a~-receptor binding were found in the medial, lateral and periventricular regions of the preoptic area, the bed nucleus of the stria terminalis, fundus striati, the anterior, supraoptic and suprachiasmatic hypothalamic nuclei, lateral hypothalamic area, median eminence and the medial amygdaloid nucleus. Differences in

249 TABLE I

[JH]Prazosin (al-receptors; n = 4) and f Hlp-aminoclonidine (ct2-receptors;n = 4) binding (fmol/mg protein) in newborn and ovariectomized adult females. Values are means + S.E.M. MPO, medial preoptic area; LPO, lateral preoptic area; PePO, periventricular preoptic area; AHN, anterior hypothalamic nucleus; SCh, suprachiasmatic nucleus; PeAH, periventricular area of the anterior hypothalamus; VMN, ventromedial hypothalamic nucleus; LH, lateral hypothalamic area; Arc-ME, arcuate-median eminence; LD Thai, lateral-dorsal thalamic area; VL Thai, ventrolateral thalamic area; MeAmg, medial amygdaloid nucleus.

Brain area

MPO ÷ LPO + PePO + BStr + FStr CPu AHN ÷ SCh SO + PeAH + VMN ÷ LH + Arc-ME + PeVMH ÷ LD Thai VL Thai MeAMG ÷

Prazosin

p-Aminoclonidine

Neonates

Adults

Neonates

Adults

424.4 213.0 176.8 267.0 248.3 101.8 226.5 141.4 275.2 301.9 299.6 301.7 260.5 352.2 372.3 336.6 275.6

336.1 + 22.2** 146.1 + 7.1"** 128.9 + 9.5** 223.5 _+ 6.0** 160.7 + 8.5* 99.8 + 6.7 186.0 + 6.4** 127.7 + 3.1" 245.7 +- 9.5 277.0 _+ 19.6 314.5 + 16.0 235.2 + 8.0*** 226.1 + 4.0** 332.7 + 16.0 409.9 + 26.0 380.2 + 28.3 231.9 + 7.0*

866.6 505.8 536.2 927.5 788.3 336.2 989.2 387.3 321.1 806.3 485.9 828.2 689.3 886.9 554.4 231.1 792.2

661.0 282.9 362.1 863.1 749.3 324.1 712.5 356.4 309.6 771.2 380.4 607.4 621.7 725.9 517.5 234.6 656.1

+ 7.5 + 5.4 _+ 4.7 + 6.7 + 22.3 + 9.7 + 7.9 + 2.3 _+ 19.5 + 11.6 _+ 11.0 +- 2.8 + 4.5 + 14.1 + 15.8 + 15.1 + 10.6

+ 39.2 + 45.4 + 50.1 + 35.1 + 39.4 +- 36.8 + 40.4 + 37.7 + 49.5 _+ 71.0 +_ 24.1 _+ 20.8 + 60.1 + 62.5 +- 28.0 _+ 12.2 + 21.2

+ 61.6" + 2.4** + 27.1" + 38.8 + 62.7 +_9.6 + 63.2* + 39.4 + 29.6 + 76.3 + 16.3" + 41.0"* +- 37.1 + 38.7 + 40.8 + 15.6 +- 27.2**

*P < 0.05; **P < 0.01, ***P < 0.001 vs neonates (t-test, two-tailed, df = 6). ÷ Estradiol-concentrating brain region 26.

a 2 - r e c e p t o r b i n d i n g w e r e d e t e c t e d in t h e m e d i a l , lat-

known to influence sexual receptivity and estrogen

eral and periventricular regions of the preoptic area,

a n d p r o g e s t i n r e c e p t o r s in a d u l t s . I n t h e first s t u d y ,

the anterior and ventromedial hypothalamic nuclei,

we c o m p a r e d

l a t e r a l h y p o t h a l a m i c a r e a a n d in t h e m e d i a l a m y g d a -

o v a r i e c t o m i z e d adults. D i f f e r e n c e s in b o t h a~- a n d

loid n u c l e u s .

a z - r e c e p t o r s w e r e f o u n d in c e r t a i n b r a i n a r e a s . I n all cases where

Effects o f estradiol-17fl benzoate on al- and a2-receptor binding in neonatal female guinea pigs T h e p u r p o s e o f t h i s e x p e r i m e n t w a s to d e t e r m i n e if estradiol-17fl benzoate

affects a l- a n d a 2 - r e c e p t o r

a-receptor

b i n d i n g in n e w b o r n

differences occurred,

receptor

and

levels

w e r e h i g h e r in n e w b o r n a n i m a l s t h a n in a d u l t s . I n a n u m b e r o f s t e r o i d c o n c e n t r a t i n g a r e a s 26 a g e - r e l a t e d d i f f e r e n c e s in a l - r e c e p t o r

binding were

detected.

These areas include the medial, lateral and periven-

b i n d i n g in n e o n a t e s as it d o e s in a d u l t s . T h e r e s u l t s

t r i c u l a r r e g i o n s of t h e p r e o p t i c a r e a , b e d n u c l e u s o f

a r e s h o w n in T a b l e II. E s t r a d i o l - 1 7 f l b e n z o a t e d i d n o t

the stria terminalis, anterior hypothalamic nucleus,

affect a~- a n d a 2 - r e c e p t o r b i n d i n g in a n y o f t h e b r a i n

lateral hypothalamic

regions examined.

cleus and arcuate-median

area, medial amygdaloid nueminence;

a n d also t w o

brain regions which do not concentrate steroids, the s u p r a c h i a s m a t i c n u c l e u s a n d f u n d u s striati. D i f f e r -

DISCUSSION

e n c e s in a 2 - r e c e p t o r b i n d i n g w e r e l i m i t e d t o t h e m e I m m a t u r e f e m a l e g u i n e a pigs a r e less s e n s i t i v e t o o v a r i a n s t e r o i d s t h a n a d u l t s 23-25. T h e s t u d i e s re-

dial, l a t e r a l a n d p e r i v e n t r i c u l a r r e g i o n s o f t h e p r e o p -

ported here examined the possibility that this insensi-

nuclei, lateral hypothalamic

tivity t o e s t r o g e n m i g h t b e a t t r i b u t a b l e in p a r t t o a n

a m y g d a l o i d n u c l e u s all o f w h i c h c o n c e n t r a t e s t e r o i d s

immaturity of brain a-receptor

to some extent.

systems which are

tic a r e a , t h e a n t e r i o r a n d v e n t r o m e d i a l h y p o t h a l a m i c area and the medial

250 TABLE II FH]Prazosin (ctl-receptors) and ['~H]p-aminoclonidine (a2-receptors) binding (fmol/rng protein) in ovariectornized neom~tal guinea pigs treated with either estradiol (n = 5) or oil {n = 5 ~

Values are means _+ S.E.M. Abbreviations as in Table 1. Brain area

Prazosin

p-Arninoclonidine

Estradiol

Oil

Estradiol

Oil

MPO + LPO + PePO ÷ BStr + FStr CPu AHN ÷ SCh SO + PeAH + VMN + LH + Arc-ME + PeVMH + LD Thai VL Thai MeAMG +

249.3 + 16.6 168.7 _+ 12.8 149.1 +_ 11.7 268.2 ___14.2 212.7 _+, 11.1 72.3 _+ 5.8 250.4 _+6.3 139.1 __+4.9 300.8 _+ 15.2 278.3 ___12.9 306.3 _+ 11.9 188.7 _+ 8.1 118.0 _+4,7 214,1 _+ 7,6 583.4 _+ 7.8 297.8 __+23.3 182.1 _+ 16,3

248,5 + 17.4 167.3 -+ 12.8 165,8 + 17.2 246.1 --+ 16.6 2(1//.3 __+8.8 69.3 -+ 3.9 249.8 __+14,1 125.5 + 8.2 296.9 -+ 39.7 307,4 __+24,4 311.5 -+ 11.9 191.2 __+11.0 111.4 + 9.1 214.5 +- 8.4 598.4 __+30.0 300.2 -+ 19.6 195.2 _+ 11.1

584.0 _+ 43.0 45(I.2 + 59.3 595.2 +-+-55,9 843.1 + 68.3 899.9 -+ 18.3 383.6 __+16.0 893.6 +- 94.9 427,4 __+71.1 472.7 + 35.7 716.6 +- 88.7 449.4 + 54.0 822.7 -+ 62.4 576.6 __+76.3 710.9 + 75.9 638.0 + 32.0 182.2 -+ 14.1 644.4 -+ 18.5

632.2 _+ 34,0 368.1 __+23.6 496.2 + 45.0 690.3 -+ 42,6 842.7 __+20.1 385.3 + 35.~ 776.2 + 63,8 365.7 __+55.(I 413.8 __+40.1 571,4 __+55.4 383.8 +- 22.0 72(I.8 __+33,1 486.1 + 19,2 652.8 -+ 35,1 664.2 + 28,0 179.2 + 19.0 601.2 _+ 36.4

+ Estradiol-concentrating brain regions- . "

"

~6

A t p r e s e n t , we c a n n o t c o m p l e t e l y rule o u t the possibility that differences in tissue d e n s i t y d u e to aged e p e n d e n t changes in lipid c o n t e n t s c o n t r i b u t e d to

of r e p r o d u c t i o n 6, a n d are regions w h e r e a - r e c e p t o r

the o b s e r v e d differences in r e c e p t o r b i n d i n g 21. H o w ever, several factors argue against a significant cont r i b u t i o n of this p o t e n t i a l p r o b l e m . In p a r t i c u l a r , similar p o s t n a t a l c h a n g e s in a - r e c e p t o r b i n d i n g have previously b e e n d e m o n s t r a t e d in tissue blocks of hypot h a l a m u s a n d p r e o p t i c area 9,12 using c o n v e n t i o n a l

particular interest in view of the relative insensitivity of i m m a t u r e g u i n e a pigs to o v a r i a n steroids. It is t e m p t i n g to speculate o n the r e l a t i o n s h i p b e t w e e n n o r a d r e n e r g i c activity a n d b r a i n steroid receptors at

m e m b r a n e b i n d i n g t e c h n i q u e s which are n o t affected by lipid c o n t e n t . F u r t h e r m o r e , a g e - r e l a t e d differences in a 1- a n d in a2-receptor b i n d i n g in the p r e s e n t study did n o t always occur in the s a m e b r a i n areas. F o r e x a m p l e , in the b e d n u c l e u s of stria t e r m i n a l i s , f u n d u s striati, s u p r a c h i a s m a t i c n u c l e u s a n d arcuatem e d i a n e m i n e n c e , differences were f o u n d o n l y in a Ireceptors. In the v e n t r o m e d i a l h y p o t h a l a m i c n u c l e u s differences were f o u n d o n l y in a2-receptors. It is u n likely that differences in tissue density c o n t r i b u t e d significantly to the a g e - r e l a t e d differences in b i n d i n g

tor b l o c k a d e has b e e n s h o w n to m a r k e d l y decrease n u c l e a r e s t r o g e n r e c e p t o r c o n c e n t r a t i o n in h y p o t h a l amus a n d p r e o p t i c area of adult g u i n e a pigs 4. In the p r e s e n t study, b o t h a l - a n d a2-receptor c o n c e n t r a tions were f o u n d to be h i g h e r in h y p o t h a l a m i c a n d preoptic areas in i m m a t u r e a n i m a l s t h a n in adults. It is possible that high a - r e c e p t o r levels in n e o n a t e s reflect an i n c o m p l e t e f u n c t i o n a l d e v e l o p m e n t of the n o r a d r e n e r g i c system. In s u p p o r t of this possibility are data that d e m o n s t r a t e low n o r e p i n e p h r i n e levels a n d n o r e p i n e p h r i n e activity ( c o m p a r e d to adults) d u r i n g early p o s t n a t a l d e v e l o p m e n t of the g u i n e a pig in whole h y p o t h a l a m i c a n d p r e o p t i c area p r e p a r a tions 12. T h u s , n a t u r a l l y o c c u r r i n g low n o r a d r e n e r g i c activity in i m m a t u r e a n i m a l s might be v i e w e d as analogous to the p h a r m a c o l o g i c a l l y i n d u c e d r e d u c t i o n in

in these areas, since t r i t i u m q u e n c h i n g s h o u l d be identical for b o t h ligands. It is i n t e r e s t i n g to n o t e that a n u m b e r of the areas where a g e - r e l a t e d differences in b i n d i n g were f o u n d are k n o w n to play an i m p o r t a n t role in the r e g u l a t i o n

blockade has b e e n s h o w n to i n f l u e n c e estrogen 4 a n d progestin 27 receptors in adults. T h e s e results are of

different stages of d e v e l o p m e n t . F o r e x a m p l e , interference with n o r a d r e n e r g i c t r a n s m i s s i o n by a - r e c e p -

251 noradrenergic activity in adults that affects behavioral and biochemical sensitivity to steroids in adults 16. In the specific case of the ventromedial hypothalamic nucleus an additional factor might contribute to the behavioral insensitivity of neonatal guinea pigs to ovarian steroids. In the ventromedial hypothalamic nucleus, age-related differences were found only in a2-receptors; al-receptor levels were similar in immature and adult animals (Table I). Because both a 1and a2-receptors are involved in lordosis regulation 28, an imbalance between the two receptor systems in immature animals might also contribute to the behavioral insensitivity of neonatal guinea pigs to steroids. A similar imbalance of adrenergic receptor systems has been shown to play an important role in determining the responsiveness of the uterus to norepinephrine 22.29. In addition to comparing the distribution of a-receptors in adults and neonates, the effect of estradiol17fl benzoate on a-receptor binding was examined in immature female guinea pigs. Estradiol-17fl benzoate has been shown to alter both a 1- and a2-receptor binding in adults 1°'11. In the ventromedial hypothalamic nucleus of adult females, estradiol-17fl benzoate decreased the binding of both a 1- and a2-rece ptors relative to oil-treated controls. In the present study, we found no effect of estradiol-17fl benzoate on a l- or a2-receptors in ventromedial hypothalamic nucleus or any other brain area examined in neonatal females. The absence of a steroid effect on a2-rece pREFERENCES 1 Beach, F.A., Ontogeny of 'coitus-related' reflexes in the female guinea pig, Proc. Natl. Acad. Sci. U.S.A., 56 (1966) 526-532. 2 Blaustein, J.D., Noradrenergic inhibitors cause accumulation of nuclear progestin receptors in guinea pig hypothalamus, Brain Res., 325 (1985) 89-98. 3 Boling, J.L., Blandau, R.J., Wilson, J.G. and Young, W.C., Postparturitional heat responses of newborn and adult guinea pigs. Data on parturition, Proc. Soc. Exp. Biol. Med., 42 (1939) 128-132. 4 Clarke, A., Nock, B., Feder, H.H. and Roy, E.J., at-Noradrenergic receptor blockade decreases nuclear estrogen receptor binding in guinea pig hypothalamus and preoptic area, Brain Res., 330 (1985) 197-199. 5 Crowley, W.R., Nock, B.L. and Feder, H.H., Facilitation of lordosis behavior by clonidine in female guinea pigs, Pharmacol. Biochem. Behav., 8 (1978) 207-209. 6 Feder, H.H., Hormones and sexual behavior, Annu. Rev. Psychol., 35 (1984) 165-200. 7 Goy, R.W., Phoenix, C.H. and Meidinger, R., Postnatal

tors in the neonatal ventromedial hypothalamic nucleus is particularly interesting since in a previous study estradiol-17fl benzoate also did not affect a2receptors in adult males 11 which, like immature females, do not display sexual receptivity when treated with steroids. Thus in neonatal females, adult males and in adult females treated with a-receptor blockers, deficits in a-receptor-mediated noradrenergic transmission are correlated with insensitivity to steroids as measured by lordosis responses. These studies demonstrate that (1) differences exist in the binding of a-noradrenergic receptors in the brain of adult and newborn female guinea pigs and (2) unlike the situation in adults, a-receptors in neonatal guinea pig brain are not modulated by exogenous estradiol-17fl benzoate. These age-related differences in a - r e c e p t o r - m e d i a t e d noradrenergic transmission may be related to the insensitivity of immature guinea pigs to the facilitative effects of ovarian steroids of ovarian steroids on female reproductive processes.

ACKNOWLEDGEMENTS Supported by Grants NS07911 (A.E.J.), NS07080 (B.McE.), NIHD-04467 and a Busch Bequest grant (H.H.F.). Contribution number 478 of the Institute of Animal Behavior and 59 of the Department of Biological Sciences, Rutgers-Newark. development of sensitivity to estrogen and androgen in the male and female pseudohermaphroditic guinea pigs, Anat. Rec., 157 (1967) 87-96. 8 Jacobson, S., Sequence of myelination in the brain of the albino rat. A. Cerebral cortex, thalamus, and related structures, J. Comp. Neurol., 121 (1963) 5-29. 9 Johnson, A.E., Nock, B., Ryer, H.I. and Feder, H.H., Hypothalamic and preoptic area al-receptor concentrations decrease, and cerebral cortical a~-receptor concentrations increase during postnatal maturation of male guinea pigs, Neuroendocrinology, 38 (1984) 243-247. 10 Johnson, A.E., Nock, B., McEwen, B.S. and Feder, H.H., Estradiol modulation of az-noradrenergic receptors in guinea pig brain assessed by tritium-sensitive film autoradiography, Brain Res., 336 (1985) 153-157. 11 Johnson, A.E., Nock, B., McEwen, B.S. and Feder, H.H., a t- and a2-noradrenergic receptors in guinea pig brain: sex differences and effects of estradiol, Brain Res., 442 (1988) 205-213. 12 Johnson, A.E., Renner, K.L., Allen, D., Luine, V., Nock, B. and Feder, H.H., Noradrenergic regulation of at-rece ptors during the postnatal development of the guinea pig,

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