Brain Research, 86 (1975) 293-306

293

©Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

EFFECT OF GONADAL STEROIDS ON ACTIVITIES OF MONOAMINE OXIDASE AND CHOLINE ACETYLASE IN RAT BRAIN

VICTORIA. N. LUINE, R A D A I. KHYLCHEVSKAYA* AND B R U C E S. M c E W E N

The Rockefeller University, New York, N.Y. 10021 (U.S.A.) (Accepted October 7th, 1974)

SUMMARY

Gonadectomized male and female rats were treated with equimolar doses of estradiol benzoate (EB) and testosterone propionate (TP) daily for periods of 3 days to 1 week and activities of monoamine oxidase (MAO) and choline acetyltransferase (ChAc) were measured in the cortex, hippocampus, basomedial hypothalamus, corticomedial amygdala and medial preoptic areas. After hormone treatment, changes in enzyme activities were found in those brain regions where gonadal hormones are known to affect sexual behavior and/or gonadotropin release and which contain putative hormone receptor sites. More specifically, EB administration to females resulted in decreased activity of MAO in the corticomedial amygdala and basomedial hypothalamus and an elevation of ChAc activity in the medial preoptic area and corticomedial amygdala while TP administration did not alter enzyme levels in any brain region. In contrast, EB administration to castrated males was without significant effect on enzyme activities while TP administration resulted in increased activity of MAO and ChAc in the medial-preoptic area. The estrogen antagonist, MER-25, given concomitantly with EB, effectively blocked EB-dependent changes in both enzymes in ovariectomized female rats. EB treatment to hypophysectomized females led to similar enzymatic changes as in ovariectomized females in all areas except the basomedial hypothalamus. Estradiol added directly to the enzyme incubation medium did not result in altered enzyme activities. Results obtained are discussed in relation to sexual differentiation of the brain, metabolism of gonadal hormones, and possible mechanism of gonadal hormone regulation of enzyme activities.

* Present address: Institute of General Genetics, USSR Academy of Sciences, Moscow 117312, USSR.

294 INTRODUCTION

We have reported that exogenously administered estradiol increases the activity of a number of enzymes in the pituitary and in areas of the brain where putative estradiol receptors are present in high concentrations26, 27. Previously measured enzymes were ones involved in glucose metabolism and in providing reducing equivalents for reductive synthesis. In this study, we have considered whether the effects of gonadal hormones on enzymes may also include enzymes which are more closely involved with neuronal activity, namely, enzymes of neurotransmitter metabolism. We have also studied the effects of androgen and estrogen in both males and females to determine if sexual differentiation of the brain has an effect on enzymatic responsiveness to gonadal hormones. Finally, we have completed a number of experiments to determine whether the changes in enzyme levels after gonadal steroid treatment may be causally related to the presence of putative steroid receptors in specific brain regions. One enzyme selected for study was monoamine oxidase (MAO), an enzyme present in the mitochondria of neural tissue and responsible for deactivation of catecholamines and serotonin by the formation of deaminated metabolites3, 48. This enzyme was chosen for study because several laboratories have shown that levels of MAO in the hypothalamus and amygdala fluctuate during the estrus cycle when levels of ovarian and pituitary hormones are changing13,15,e0, 53. Activity of another enzyme, choline acetyltransferase (ChAc), which is responsible for the synthesis of the neurotransmitter acetylcholine, was also measured as a function of gonadal hormone treatment. This enzyme was chosen because Libertun e t al. 23 found differences between males and females in levels of ChAc in the pre-optic area and concluded that these differences were a result of the action of testosterone in sexual differentiation during neonatal development. METHODS

Sexually mature, male and female Sprague-Dawley rats (Charles River Co., Wilmington, Mass.) were castrated, ovariectomized, or hypophysectomized; one week later, daily, subcutaneous injections of testosterone propionate (TP) or estradiol benzoate (EB), dissolved in sesame oil, were begun. Doses of hormone were given per 220 g body weight, the average weight of a 70-80-day-old female rat. TP doses were equimolar to EB doses. After 3-7 days of treatment, the animals were decapitated and subregions of the hypothalamus, amygdala and pre-optic area were dissected along with the whole hippocampus and portions of the cerebral cortex as previously described 2~. Brain regions from each animal were homogenized individually in 0.05 N Tris-HCl buffer, pH 8.0, an aliquot was taken for protein analysis 25, and assays of monoamine:O2 oxidoreductase (E.C. 1.4.3.4.) and acetyl-CoA:chotine O-acetyltransferase (E.C. 2.3.1.6.) were immediately begun. Triplicate determinations were made on each enzyme in each area, and then the average of the triplicates was used to calculate the enzyme rates. Microradiochemical techniques enabled measurement of both enzymes in a single brain region.

295 Monoamine oxidase was assayed by the radiochemical method of McCaman et al. 2s except that tritium labeled 5-hydroxytryptamine (serotonin) was used instead

of 14C-labeled serotonin. In the incubation mixture, serotonin creatinine sulfate complex (Sigma Chemical Co., St. Louis, Mo.) was present at a concentration of 2 mM and ZH-labeled serotonin binoxalate (New England Nuclear, Boston, Mass.) was added to give a specific activity of approximately 10 mCi/mmole. Ten #1 of the enzyme incubation mixture was added to microconical tubes (VWR Scientific, Buffalo, N.Y.) in an ice bath, then 1 #1 of homogenate was added to start the reaction. After 20 min incubation at 38 °C, the reaction was stopped with HC1. The labeled products were extracted with ethyl acetate and counted in 10 ml of toluene-based scintillation fluid (160 ml Liquifluor, New England Nuclear, in 8 pints toluene, Merck, A.R.) in a Packard Model 3375 scintillation counter with an efficiency of 50 ~. Choline acetylase was assayed by the radiochemical method of Glover and Green 12 except that tritium labeled acetyl-CoA was used instead of 14C-labeled substrate. Acetyl-CoA (Sigma) was present at a concentration of 140/zM in the enzyme incubation mixture and acetyl-[3H]CoA (New England Nuclear) was added to give a specific activity of approximately 50 mCi/mmole. Triton X-100 was added to the homogenates to give a concentration of 0.1 ~ 5 min before the assay was begun. Without Triton X-II30, activity of ChAc was approximately 60 ~ lower. Incubation with 10/~1 of enzyme reagent and 5 #1 of homogenate was carried out at 38 °C for 10 rain. The reaction was stopped with formic acid, and the labeled acetylcholine was extracted and counted in 10 ml of toluene-based scintillation fluid (50 ml methanol, Eastman, Spectro A.C.S. and 2 g PPO, New England Nuclear in 450 ml toluene, Merck, A.R.) with an efficiency of 10~. For both enzyme reactions, activity was expressed as/~moles product formed/g protein/h. In studies utilizing MER-25, females received 4 mg/220 g body weight in a sesame oil suspension. On day 8 after gonadectomy, females received MER-25, on days 9-11 they received MER-25 and EB (5/~g/220 g body weight), and on day 13 they were sacrificed and brain regions analyzed for enzyme activity. For studies of the in vitro effects of estradiol, 17fl-estradiol was dissolved in ethyl alcohol. The alcohol solution was added directly to the enzyme incubation reagent and the enzyme analysis was completed as described above. An equal volume of alcohol was added to the control incubation mixture. Estradiol benzoate was purchased from Mann Research Laboratories (New York, N.Y.) testosterone propionate from Calbiochem (La Jolla, Calif.). MER-25 was obtained from Dr. A. Richardson of Merrell National Laboratories (Cincinnati, Ohio). Differences between gonadectomized and hormone treated groups were tested using the t-statistic. When comparisons between two groups were made, the Student's t-distribution was utilized. When comparisons involved more than two groups, the Dunnett multiple comparison t-statistic was used 51. The Dunnett t-test has the same distribution as the Student's t-test when only two comparisons are made, but with more than two groups, the critical values are higher for the Dunnett t-distribution, leading to a more conservative test.

296 RESULTS

Regional and dose-response studies The effect of estradiol benzoate (EB) administration (30/~g/animal/day for 7 days) on the activity of monoamine oxidase (MAO) in a number of brain regions in ovariectomized female rats is shown in Table I. In the cortex, medial pre-optic area (MPOA) and the hippocampus, administration of EB did not lead to significant changes in the activity of MAO, while in the basomedial hypothalamus (BM-hypothalamus) and corticomedial amygdala (CM-amygdala) activity of M A O was depressed by 30 and 50 ~/o respectively after EB treatment. Whether changes in enzyme activity could be obtained with lower doses of EB was investigated in ovariectomized females given 5 and 30/~g of EB for 3 days. In Fig. 1 is shown the per cent change in M A O activity at these doses of EB and also at the 30-/~g dose level (data from Table 1). In the amygdala, lower doses of EB also led to significant decreases in M A O activity, and more importantly, the quantitative decrease in activity was related to the amount of estrogen given. M A O activity in the BM-hypothalamus was significantly lowered by all estrogen doses; however, in contrast to the CM-amygdala, the per cent change in activity did not appear to be dependent on the dose of estrogen given. Activity in the M P O A was not significantly changed at high or low doses of EB. In the same EB-treated ovariectomized females another enzyme, choline acetyltransferase (ChAc), was also measured. Table II shows that EB administration led to opposite effects on ChAc activity as compared to M A O activity. Activity of ChAc in the CM-amygdala and M P O A was increased when EB was administered for 3 days at 5 and 30/~g. Activities of ChAc in the BM-hypothalamus were unaffected by either

TABLE I ACTIVITY OF MONOAMINE OXIDASE IN OVARIECTOMIZED ( O v x ) FEMALES GIVEN ESTRADIOL BENZOATE ( E B )

Activity is expressed as/tmoles/g protein/h and represents the average ~ S.E.M. Number of animals is given in parentheses. Ovariectomized animals were treated for 7 days with 30 #g EB/animal. Differences between Ovx and treated group were tested by Student's t-test where *P < 0.05 and **P < 0.01.

Cortex BM-hypothalamus CM-amygdala Medial pre-optic Hippocampus

Ovx + EB

Ovx

142 ~ 9 (6) 73.9 ± 6* (7) 62.6 -3=5** (6) 92.3 ~ 3 (6) 156 _-L7 (6)

135 ± 11 (6) 104 ± 11 (9) 125 ~ 14 (7) 97.3 ~ 10 (5) 164 ± 15 (6)

297

10

AMYGDALA

HYPOTHALAMUS

0

-i0

g = o -20

N -L'-

PRE-OPTIC

-30

-40

15FgEB -50

g771 90/xgEB roll 210~g EB

Fig. 1. Per cent change in monoamine oxidase activity in brain of ovariectomized (Ovx) females as a function of total administered estradiol benzoate (EB). Open bars indicate 5/zg of EB given for 3 days to 8 animals; striped bars indicate 30 ~g EB given for 3 days to 8 animals; closed bars, data from Table I. Differences were tested by Student's t-test where *P < 0.05, **P < 0.01.

d o s e o f estrogen while activity in the h i p p o c a m p u s was significantly elevated only at the highest dose o f estrogen. Nature o f enzyme changes

I n o r d e r to m o r e precisely define the relationship between EB a d m i n i s t r a t i o n a n d c o n s e q u e n t enzyme changes, several types o f experiments were p e r f o r m e d . First, the p o s s i b i l i t y t h a t enzyme changes were due to a direct i n t e r a c t i o n o f estrogen with enzyme p r o t e i n s was tested b y a d d i n g 17fl-estradiol directly to the enzyme i n c u b a t i o n m i x t u r e with h o m o g e n a t e s f r o m o v a r i e c t o m i z e d females. A s s h o w n in T a b l e I I I , e s t r a d i o l in c o n c e n t r a t i o n s f r o m 10 -6 to 10 - s M h a d no significant effect o n M A O o r C h A c activity in B M - h y p o t h a l a m u s a n d M P O A , respectively. TABLE II ACTIVITYOFCHOLINEACETYLTRANSFERASEINOVARIECTOMIZEDFEMALESGIVENESTRADIOLBENZOATE(EB) Activity is expressed as/~moles/g protein/h and represents the average 4- S.E.M. Number of animals is given in parentheses. Ovariectomized (Ovx) animals were treated for 3 days with EB at 5/zg/animal and at 30/~g/animal. Differences between Ovx and treated groups were tested by Dunnett t-test where *P < 0.05 and **P < 0.01.

BM-hypothalamus CM-amygdala Medial pre-optic Hippocampus

Ovx

Ovx 5 I~g EB

Ovx 30 ttg EB

45.1 4- 2 (5) 46.1 4- 1 (6) 49.7 4- 3 (6) 49.1 4- 3 (6)

42.6 ± 2 (5) 53.8 -4- 1" (5) 59.6 ± 3* (6) 50.9 4- 2 (6)

47.3 q(5) 56.5 4(6) 65.8 ± (6) 55.2 4(6)

3 3** 3** 1"

298 TABLE III In vitro

EFFECT OF ESTRADIOL-17t5

ON BRAIN

ENZYME ACTIVITIES

Results were obtained using the assay procedure described in Methods. 17/3-Estradiol was added to enzyme reagent in ethyl alcohol, and activities are expressed as a percentage of a control to which only alcohol was added. Each entry represents the mean of 6 determinations in 3 animals. Hypothalamic homogenates from ovariectomized females were used for measurement of MAO; medial preoptic homogenates were used for ChAc. Student's t-test indicated no significant differences between the control and the 17fl estradiol groups. Enzyme

Estradiol-I 7fi

Monoamine oxidase Choline acetyltransferase

10 ~ M

IO-T M

10 -6 M

102 107

106 102

99 108

Since estrogens are known to affect circulating levels of a number of pituitary hormones, the possibility that enzyme changes noted after EB treatment might be related to estrogen-dependent changes in other hormones was investigated using females that had been hypophysectomized for at least one week. Females received either sesame oil or EB, 5/zg/animal/day for 3 days and 30/tg/animal/day for 7 days. The levels of MAO and ChAc measured in these animals are shown in Table IV. Activity of ChAc in the MPOA and MAO in the CM-amygdala were still significantly altered by EB at both doses of EB. In contrast, activity of MAO in the BM-hypothalamus was not decreased when EB was given at either dose. High levels of estrogen are known to depress eating 47, and hypothalamic catecholamines have been implicated in eating behavior 22. Our females treated with the highest dose of EB (30/zg/day/7 days) lost an average of 6 o / o f their body weight.

TABLE IV EFFECT OF ESTRADIOL BENZOATE FEMALE BRAIN

(EB) ON

ACTIVITY OF ENZYMES IN HYPOPHYSECTOMIZED

(nYPOX)

Activity is expressed as/~moles/g protein]h. Each entry represents the mean ± S.E.M. for determinations in 11 animals in the 3-day group and 8 animals in the 7-day group. Animals were treated daily with EB at the dose indicated. Comparison of Hypox and EB-treated group by Student's t-test where *P < 0.05, **P < 0.01. Treatment

CM-amygdala MAO

BM-hypothalamus MAO

Medial pre-optic ChAc

Hypox Hypox + 30/~g EB (7 days) Hypox Hypox + 5 #g EB (3 days)

130.0 ~ 5 104.0 ~z 7*

126.0 ± 10 127.0 ~ 8

32.1 3- 1 38.4 ± 2*

88.3 ± 5 86.4 ± 7

30.0 + 8 34.5 :k_ 2*

87.4 ~_ 4 67.2 ~z 5**

299 TABLE V EFFECT OF FOOD DEPRIVATION ON ACTIVITY OF MONOAMINE OXIDASE IN OVARIECTOMIZED FEMALES

Activity is expressed as/~moles/g protein/h. Each entry represents the mean q- S.E.M. for determinations in 4 animals. The group that received food ad lib#am showed a 5 % increase in body weight while the food-deprived group showed 23 % decrease. Student's t-test indicated no significant differences in enzyme activity between groups. Brain area

+ Food

-- Food

BM-hypothalamus CM-amygdala

107.0 4- 15 77.7 -4- 10

105.0 4- 5 77.3 4- 12

Therefore, we investigated the possibility that changes in MAO levels were a result of altered eating and weight loss. Two groups of ovariectomized rats were used: one received food ad libitum while the experimental group was food deprived until they had lost 23 % of their body weight. Neither group received EB. MAO activity was measured in the BM-hypothalamus and CM-amygdala where decreased MAO activity was found after EB treatment. As shown in Table V, food deprivation and consequent weight loss did not affect MAO levels. A final experiment was concerned with defining the mechanism of EB-dependent changes in enzyme activities by determining if an antiestrogenic compound, MER-25, would block the enzyme changes. Preliminary experiments with ChAc and MAO, and measurements of other brain and pituitary enzymes showed that MER-25 alone had no effect on levels of enzymes27. Accordingly, MER-25 alone or MER-25 in combination with EB was given to gonadectomized female rats for 4 days. In the EB group, one day of pretreatment with MER-25 was given, and then 5/~g of EB was given for 3 days, preceded each day by MER-25 supplements. Results presented in Table VI show that EB given in combination with MER-25 does not lead to increased ChAc activity in the MPOA or CM-amygdala. This result can be compared to Table II where 5 ~g of EB alone led to 17-20 % increases in ChAc levels in these two areas.

TABLE VI EFFECT OF ESTRADIOL BENZOATE ( E B ) GIVEN IN COMBINATION WITH

MER-25

ON ENZYME LEVELS IN

OVARIECTOMIZED FEMALES

Activity is expressed as #moles/g protein/h. Numbers in parentheses indicate number of animals used. For treatment regimen, see Methods. Analysis by Student's t-test showed no significant differences between the MER-25 group and the MER-25 -b EB group. Enzyme

MER-25

Hypothalamic M A O Amygdaloid M A O Amygdaloid ChAc Pre-optic ChAc

121.0 119.0 27.2 26.3

zk 10 =k 12 :k 1 4- 2

M E R - 2 5 Jr- E B

(6) (7) (7) (7)

141.0 123.0 27.6 26.7

± 444-

6 (6) 8 (7) 1 (7) 2 (6)

300 MONOAMINE OXIDASE 30 - ( ~

lo

o

- -

-lo

HYPOTHALAMUS AMYGDALAPRE-OPTIC 0 -IO

g_ -20

-30

in

!

I

-40

-50

Fig. 2. Activity of monoamine oxidase in brain of gonadectomized male and female rats given estradiol benzoate and testosterone propionate. Activity is expressed as per cent change between gm~uadectomized and hormone treated-gonadectomized animals. Closed bars represent animals treated with 30 pg EB, open bars represent animals treated with 28 pg TP. Treatment was given for 7 days. Each bar represents the average of determinations in 6-9 animals per group. Differences between groups were tested by Dunnett t-test where *P < 0.05, **P < 0.01.

Likewise, M A O levels in the BM-hypothalamus and CM-amygdala were not significantly reduced by EB given in combination with MER-25 whereas EB alone led to significant depression of M A O activity in both areas (see Fig. 1).

Sex-dependent responses to gonadal hormones Distinct differences exist between male and female rats in their behavioral and neuroendocrine responses to gonadal hormones 24,29. Therefore, we have compared responses of M A O and ChAc in gonadectomized male and female rats to both EB and testosterone propionate (TP) to determine if hormone responsiveness of these enzymes may be altered by sexual differentiation of the brain. Fig. 2 shows the percentage changes in M A O activity in gonadectomized male and female rats after treatment with equimolar doses of EB or TP (30 #g EB/220 g body weight/day for 7 days; 28 #g TP/220 g body weight/day for 7 days). In the females, only EB led to significant depressions of M A O activity in the BM-hypothalamus and CM-amygdala. While TP administration did cause a depression of activities in these regions, the changes did not reach a statistically significant level. Gonadal hormone administration to males presents a strikingly different pattern of M A O responses. First, EB did not lead to significant changes in M A O levels in males while TP was effective. Second, the TP-dependent change in M A O activity, an increase, was localized in the M P O A of males in contrast to the female where El3-

301

~oic/.

CHOLINE

6O

ACET LY ASEI

50 40 30 ~ ao

o~HYPOTHALAMUS AMYGDALA PRE-OPTIC Fig. 3. Activity of choline:acetylase in brain of gonadeetomized male and female rats given estradiol benzoate and testosterone propionate. Activity is expressed as per cent change between gonadectomized and hormone treated-gonadectomized animals. Closed bars represent animals treated with 30 pg EB, open bars represent animals treated with 28/~g TP. Females were treated for 3 days while males were treated for 7 days. Each bar represents the average of determinations in 4-6 animals. Differences between groups were tested by Dunnett t-test where *P < 0.05, **P < 0.01.

dependent decreases in M A O activity were localized in the BM-hypothalamus and CM-amygdala. The response of ChAc to gonadal steroids is shown in Fig. 3. Equimolar doses of the hormones were given; however, males received treatment for 7 days while females were treated for 3 days. In the females, TP administration did not alter levels of ChAc in any brain region while EB administration led to elevations of activity in the M P O A and CM-amygdala. In the males, TP administration resulted in a 70 increase in ChAc in the M P O A and no changes in other areas. In males, EB administration had a tendency to increase levels of ChAc in the M P O A and CM-amygdala, but these changes were not statistically significant. DISCUSSION

This study has demonstrated the ability of exogenously administered gonadal hormones to affect the activity of two enzymes of neurotransmitter metabolism, choline acetyltransferase (ChAc) and monoamine oxidase (MAO) in those regions of brain, the corticomedial (CM)-amygdala, basomedial (BM)-hypothalamus and medial preoptic area (MPOA), where gonadal hormones are known to exert effects on sexual behavior and/or gonadotropin secretionS,24, 29 and where previous investigations have noted the presence of specific proteins which bind estrogen and lead to the association of estrogen with components of the cell nucleus 8JT,19,a4,43,52. Experimental evidence presented supports the conclusion that changes of M A O activity in the CM-amygdala and ChAc activity in the M P O A and CM-amygdala are the direct result of estrogen action in these tissues and the mechanism most likely depends on estrogen interaction

302 with cellular components other than the enzymes themselves. These conclusions were based on the observations that changes in levels of enzymes were directly related to the administered dose of estrogen, that they were obtainable in hypophysectomized animals (in which pituitary secretions are absent), and that they were readily blocked by the anti-estrogen, MER-25. Action of estrogen in the enzyme reaction itself was ruled out when estrogen was without effect when added directly to the enzyme incubation reagent with homogenates from brains of ovariectomized females. Indirect nutritional changes in the animals also could not account for changes in MAO activity after EB administration. Exactly the same pattern of experimental results were obtained previously on another group of enzymes which were studied in the same brain regions and pituitary of ovariectomized females receiving EB 26. Estrogen-dependent increases in activity of glucose-6-phosphate dehydrogenase (G6PDH), NADP +-dependent malate dehydrogenase (MDH), and isocitrate dehydrogenase (1CDH) were found in the BM-hypothalamus. The CM-amygdala showed increased activity of MDH and ICDH after EB while the pituitary showed elevated G6PDH, 6-phosphogluconate dehydrogenase and lactate dehydrogenase activity. The response of MAO to EB in the BM-hypothalamus stands in striking contrast compared to all other enzymes and brain regions studied. First, within a 200-fold range of EB doses, the percentage decrease in the activity of this enzyme remained constant. Second, EB administration to hypophysectomized females did not result in decreased MAO activity. These observations suggest that changes in MAO activity may be a result of factor(s) released from the pituitary, and therefore not a direct result of estrogen action. The feedback effects of estrogen on circulating levels of pituitary gonadotropins are well known, and this finding is similar to those of Anton-Tay e t a L 1 who reported that norepinephrine metabolism in the hypothalamus may be indirectly affected by estrogen through inhibition of FSH secretion. However, it must be considered whether hypophysectomy might have damaged nerve endings in the median eminence area where MAO is known to be localized and thereby altered the function of cellular mechanisms responsible for generating the response to EB. Clearly, further experiments are needed to distinguish among these possibilities. The effects of sexual differentiation of the brain on enzymatic responses to gonadal hormone were prominent in terms of the effectiveness of specific hormones, the location of hormonal effects, and the direction of enzymatic changes. EB administration to ovariectomized females resulted in depression of MAO levels in the BMhypothalamus and CM-amygdala, and an elevation of ChAc in the MPOA while TP was without significant effect in any of these areas. In males, TP was the active steroid, leading to increased levels of MAO and ChAc in the MPOA. In the females, results can be correlated with the presence in the cell nucleus in the hypothalamus, preoptic area, amygdala and pituitary of stereospecific putative estrogen receptorsS,17,19,34,4a,52. Evidence linking enzymatic changes with these putative estrogen receptors is as follows: (1) enzymatic changes were found where putative receptors are found and are absent in regions where they are not present; (2) putative estrogen receptors bind only 17fl-estradiol and diethylstilbestrol and not testosterone

303 or 17a-estradiol 3,32,52. In this study enzymatic changes in females were found only with EB and not TP. Also, a previous investigation of pituitary G 6 P D H showed increased enzyme activity after 17fl-estradiol or diethylstilbestrol but not after 17aestradiol or testosterone26; (3) estrogen-dependent changes were blocked by the antiestrogen, MER-25. Such anti-estrogens are known to occupy putative estrogen receptor sites in peripheral target tissues and in the brain 18,18,42. MER-25 also blocks estrogen-dependent sexual behavior 40 and interferes with ovulation 37. Enzymatic changes in castrate males after testosterone treatment can be accounted for by a number of alternative explanations. One possibility is mediation by nuclear binding sites for testosterone. Autoradiographic studies have strongly suggested accumulation of cell nuclear bound radioactivity injected as testosterone in males 33, 35,36, but conclusive evidence of such nuclear binding by cell fractionation techniques is lacking 30. Another alternative is based on the observations that testosterone can be metabolized to estrogen in the brain of males and females 31,49. Thus, some investigators have hypothesized that estrogen may be responsible for the neuroendocrine effects of testosterone in males. This possibility does not receive support from our data since testosterone itself was more effective than estrogen in males, and we also saw a distinct difference in the direction of the effects of estrogen and testosterone on MAO activity. EB decreased MAO activity while TP increased MAO activity. Finally, TP-dependent changes may be related to the conversion of testosterone to 5a-dihydrotestosterone by 5a-reductase 6. High levels of this enzyme are found in the hypothalamic-preoptic area 6, and dihydrotestosterone is known to exert androgenic effects in the periphery 5°. Clearly, further experiments in males are needed to distinguish between these possible mechanisms. Gonadal hormone-dependent changes in the activity of MAO may be important in regulation of behavioral and neuroendocrine aspects of the gonadal-hypothalamicpituitary axis. It is known that central amine levels and turnover rates are altered when circulating levels of gonadal and pituitary hormones are altered T M , and recent evidence supports the view that adrenergic synapses within the preoptic-hypothalamic-amygdaloid areas may be related to the control of gonadotropin and prolactin secretion14,39,46. The relationship of dopamine and serotonin to male sexual behavior have also been reported 11. The involvement of the cholinergic system in neuroendocrine events has received less study than the adrenergic system. However, modifications of gonadotropin secretion can be obtained by application of acetylcholine to the brain or pituitary 9. Moreover, Libertun e t al. 23 found that levels of ChAc were higher in the female than in the male preoptic-suprachiasmatic area and that this sex difference was dependent on the early hormonal environment. No differences were found between males and females in the arcuate-mammillary area of the hypothalamus or in cerebral cortex. In this study we found gonadal hormone effects also could be elicited in the adult preoptic area. In addition, another brain area which was not previously explored, the amygdala, also responded to EB with increased levels of ChAc. The amygdala is known to have putative estradiol receptors34, 52, and has been implicated in the regulation of gonadotropin secretiona3,44,45.

304 We also found changes in the level of ChAc in the hippocampus after a high dose of EB although nuclear uptake of estradiol in the hippocampus is 1C0-fold lower than in the hypothalamus a°. However, it has been shown that lesioning connections to the hippocampus from the medial septal area results in a 7 0 ~ decrease in the level of ChAc in the hippocampus 21. The increased ChAc activity in the hippocampus after EB may thus have been the result of EB action in the cell bodies in the medial septal area where estrogen-labeled cells are found a4 and where enzyme proteins would be synthesized and then transported along the neuron into the hippocampus. In this and previous papers 26,27 we have reported hormone-dependent changes in a number of enzymes in brain regions and pituitary of male and female rats where gonadal hormones are known to have effects on behavior and neuroendocrine function. Previous studies considered enzymes involved in intermediary metabolism 26,27. In this study we have considered two enzymes which are more intimately related to synaptic activity. In those areas of neural metabolism investigated thus far, we find that gonadal hormones may play an important regulatory role. We have also consistently found correlations between effects of gonadal hormones in peripheral target tissues and central nervous system target areas suggesting that the mechanisms underlying changes in both target areas may be similar. In the uterus, investigators have reported that M A O levels are depressed by exogenous EB administration 4 and that levels of M A O change during the estrus cycle la. We report here that brain levels in females are depressed by exogenous estrogen administration and other laboratories have found changes in activity in the hypothalamus and amygdala during the estrus cycle 1a,15,~°,,53. We thus feel that studies of gonadal hormone action on brain enzymes in males and females may help to delineate mechanisms responsible for hormone action in the brain. Moreover, studies of this nature may be useful in defining the neurochemical consequences of sexual differentiation of the brain. ACKNOWLEDGEMEN ]-S

The authors are grateful to Ms. Carew Magnus for technical assistance. This work was supported by research grant NS 07(380 from the National Institutes of Health, and by Rockefeller Foundation grant RF 713095 for research in reproductive biology. V N L is supported by a postdoctoral fellowship from the National Institutes of Health. R I K was supported by the International Research and Exchanges Board (|REX).

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