α1 adrenergic regulation of estrogen-induced increases in luteinizing hormone-releasing hormone mRNA levels and release

Molecular Brain Research, 17 (1993) 77-82

77

© 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00

BRESM 70546

a 1 Adrenergic regulation of estrogen-induced increases in luteinizing hormone-releasing hormone mRNA levels and release Gary D. Weesner a Lewis C. Krey b and Donald W. Pfaff

a

a Laboratory o f Neurobiology and Behavior, and b Laboratory o f Neuroendocrinology, The Rockefeller University, New York, N Y 10021 (USA)

(Accepted 25 August 1992)

Key words: LHRH; Surge; Prazosin; mRNA; Adrenergic; Estrogen

Prazosin, an a I adrenergic antagonist, was used to examine the relationship between adrenergic inputs and the stimulatory effects of estrogen on LHRH mRNA and release. Bilateral cannulae were implanted just dorsal to the preoptic area (POA). Estrous cycles were monitored daily by vaginal smears. On the morning of diestrus, each rat was ovariectomized and assigned to one of three treatment groups: Control - injected with sesame oil (n = 5); Surge - injected with estradiol benzoate (EB, 10 p.g) to produce an LH surge (n = 5); or, Surge + Prazosin - injected with EB and a prazosin-filled inner cannula was put into the POA (n = 6). Between 4-6 pm of the following day, rats were anesthetized, decapitated, trunk blood collected, and brains were stored in liquid nitrogen. In situ hybridization was performed using a 32p end-labelled 59-mer complementary to LHRH mRNA. Reduced silver grains, proportional to LHRH mRNA content, were quantified. Treatment with estrogen alone resulted in an LH surge and a 50% increase (P < 0.05) in numbers of cells expressing LHRH. This estrogen-induced increase and the LH surge were completely blocked (P < 0.01) by prazosin. Prazosin also decreased (P < 0.01) the median number of grains per cell from 81 (Surge) to 65 grains per cell (Surge + Prazosin). When the number of grains in LHRH-expressing neurons were totalled, EB increased (P < 0.05) LHRH gene expression by 53%, and local administration of prazosin completely blocked (P < 0.01) this increase. Since, coincident with estradiol stimulating the LH surge, it increased potential LHRH synthetic capacity (as measured by LHRH mRNA) and since administration of prazosin into POA blocked the ability of estrogen to do so, it appears that an endogenous a I ligand, probably norepinephrine, is a mediator of stimulatory effects of estrogen on LHRH biosynthesis as well as release.

INTRODUCTION T h e stimulatory effect of e s t r o g e n o n l u t e i n i z i n g h o r m o n e (LH) secretion is a well known, b u t n o t fully characterized physiological p h e n o m e n o n . I n female rats, e s t r o g e n stimulates the p r e o v u l a t o r y surge of L H R H , a n d thus LH, d u r i n g the e v e n i n g of proestrus 27. T h e stimulatory effects of e s t r o g e n are believed to occur at the p r e o p t i c - h y p o t h a l a m i c region of the b r a i n 5'21 w h e r e it facilitates the release of L H R H . W h i l e u n d e r some c o n d i t i o n s e s t r o g e n c a n be associated with d e c r e a s e d L H R H m R N A 36, w h e n estrogen is used in its 'positive f e e d b a c k ' mode, a schedule of h o r m o n e a d m i n i s t r a t i o n associated with L H surges, it c a u s e s i n c r e a s e d e x p r e s s i o n of the L H R H gene 17'23-25. This effect is likely indirect since previous studies have i n d i c a t e d that L H R H n e u r o n s themselves do not possess e s t r o g e n receptors 28.

Overall, c h a n g e s in L H R H m R N A levels are generally in the same direction as c h a n g e s in L H secretion. E s t r o g e n - t r e a t e d ovariectomized ( O V X ) rats, which have d e c r e a s e d s e r u m c o n c e n t r a t i o n s of L H ( c o m p a r e d to O V X ) except d u r i n g the daily a f t e r n o o n L H surge, also have d e c r e a s e d levels of L H R H m R N A 36. However, w h e n such rats are sacrificed in the a f t e r n o o n a r o u n d the time of the L H surge, i n c r e a s e d levels of L H R H m R N A are r e p o r t e d 17'21'23-25. I n addition, L H R H m R N A levels increase d u r i n g the a f t e r n o o n of p r o e s t r o u s 17'37 a n d following N M D A a d m i n i s t r a t i o n 2°. T h o u g h it is not k n o w n which, of the several possible 34, n e u r o c h e m i c a l s m e d i a t e the stimulatory signal from the e s t r o g e n - c o n c e n t r a t i n g n e u r o n s to the L H R H n e u r o n s , n o r e p i n e p h r i n e is o n e likely c a n d i d a t e . First, central n o r a d r e n e r g i c n e u r o n s have b e e n shown to c o n c e n t r a t e estradio126. Also, i n f u s i o n of n o r e p i n e p h r i n e or a 1 a d r e n e r g i c agonists stimulate the

Correspondence: G.D. Weesner, Laboratory of Neurobiology and Behavior, The Rockefeller University, 1230 York Avenue, New York, NY

10021, USA.

78 release of LHRH

( o r L H ) in r a t s 3'12 a n d m o n k e y s 3°.

A d m i n i s t r a t i o n o f c~t a d r e n e r g i c a n t a g o n i s t s s u p p r e s s e s the

release

of

LHRH

(or

LH)

o v a r l e c t o m t z e d 4'~'"~'L'1~'3" a n d in O V X - s t e r o i d

animal

m o d e l s 2.

norepinephrine

Additionally,

estrogen

in primed

influences

t u r n o v e r as w e l l as t h e n u m b e r

o f o~

a d r e n e r g i c r e c e p t o r s in r e l e v a n t b r a i n r e g i o n s s'33. W h i l e it s e e m s c l e a r t h a t N E a n d e s t r o g e n c a n b o t h a c t as p h y s i o l o g i c a l stimuli of LHRH mRNA l e v e l s 17'21"23-25'31 a n d r e l e a s e 27'3°, t h e r e l a t i o n s b e t w e e n t h e s e t w o a g e n t s a r e n o t fully u n d e r s t o o d . bility is t h a t t h e N E - c o n t a i n i n g

One possi-

neurons which concen-

t r a t e estradio126 m a k e e i t h e r d i r e c t o r i n d i r e c t c o n t a c t with LHRH

n e u r o n s 6'7'~'~4. T h u s , N E r e l e a s e w o u l d b e

required

somewhere

neurons

and

LHRH

between

estrogen-concentrating

neurons.

m e n t s to d e t e r m i n e if e n d o g e n o u s ing t h r o u g h

We

designed

a~ a d r e n e r g i c r e c e p t o r s , is a m e d i a t o r

actof

mRNA

levels. T h e s p e c i f i c o b j e c t i v e s o f t h i s s t u d y w e r e t w o fold. F i r s t , to c o n f i r m if a n e s t r o g e n - i n d u c e d is a c c o m p a n i e d

b y a n i n c r e a s e in L H R H

sion; and second, to determine

In situ hybridization

The procedure for ISH was similar to that described by McCabe and Pfaff 16. The probe was a 3Zp end-labelled oligonucleotide (59met) complementary to a portion of LHRH mRNA as reported by Rothfeld et al. 25. Most sections received saturating concentrations (approximately 300,000 cpm) of probe dissolved in 35 Ixl of hybridization buffer. The remaining sections were treated with an equivalently labelled sense probe to serve as a control. Hybridization proceeded at 37°C for 48 h in humidified boxes. Following hybridization, the sections were washed at 0.5 × SSC for 48 h at room temperature. All sections were then rinsed in gradations of 300 mM ammonium acetate : ethanol (7: 3, 1 : 1, 3 : 7), culminating in 100% ethanol. Slides were vacuum dried then dipped in Kodak autoradiographic emulsion (NTB3). Slides were then stored (4°C) in total darkness for 4 weeks. Finally, the sections were developed (Kodak, D-19), fixed (Kodak), lightly counterstained with Cresyl violet, and coverslipped.

experi-

norepinephrine,

the stimulatory effects of estrogen on LHRH

OVLT to the most caudal sections containing the anterior commissure. Most of these sections were treated with 32p-labelled hybridization probe while the remaining served as sense-strand controls.

LH surge

gene expres-

if t h e s t i m u l a t o r y ef-

f e c t s o f e s t r o g e n c a n b e b l o c k e d by t h e a~ a d r e n e r g i c receptor antagonist, prazosin. MATERIALS AND METHODS To evaluate the stimulatory effects of estrogen on LHRH, we utilized an animal model similar to that reported by Rosie et alfl 4. Mature, female Sprague-Dawley rats were maintained on a 14:10 h light-dark cycle (lights on from 05.00 to 19.00 h) under standard laboratory conditions. After an adequate adjustment period, a 22gauge, stainless-steel bilateral guide cannula was stereotaxically implanted I mm dorsal to the POA (0.1 mm caudal to Bregma, 7.6 mm ventral to top of skull, and 0.75 mm lateral to midlinel~). An internal dummy cannula remained inside the guide cannula to maintain the lumen. Following a recovery period of about 2 weeks, collection of daily vaginal smears began. Only rats showing at least two consecutive 4-day estrous cycles were further utilized in this study. On the morning of diestrus (09.00-11.00 h) rats were ovariectomized under flurothane anesthesia and divided into three treatment groups: (i) Control injected with sesame oil (100 ~1, s.c.), and no treatment through the POA cannula (n = 5); 2) Surge - injected with 10 /xg estradiol benzoate (EB, in 100 pA sesame oil, s.c.), and receiving an empty POA cannula (n = 5); 3) Surge + Prazosin - injected with l0 #g EB (as above) and a prazosin-filled (tamped crystals) inner cannula put into the POA (n = 6). Between 17.00 and 18.00 h on the following day (presumptive proestrus), final vaginal smears were collected and rats were quickly rendered unconscious with CO~ and then decapitated. We, and others, have found that this time window is optimal for seeing surge concentrations of LH. A sample of trunk blood was collected from each rat. Brains were immediately removed, blocked, and frozen without fixation. All tissue was stored in liquid nitrogen until further processing. Brains were sectioned (7/xm) at -25°C and mounted on acidwashed, diethylpyrocarbonate-treated, organosilane-subbed glass slides. Sectioned tissues were then fixed in a 4% paraformaldehyde solution in PBS, and processed as previously described 3t. For the in situ hybridization, approximately 30 brain sections per rat were chosen. Anatomically, these sections ranged from rostral to the

Quantification

of L H R H mRNA

Brain sections were examined under a light microscope. From each rat, brain sections from the rostral POA, the region with the largest number of LHRH cells, were initially selected for further evaluation. Across all treatment groups, anatomically similar brain sections (one per animal) were then chosen for more complete quantitative analysis. This selection process provides a stable quantitative measure which reliably reflects the quantitative aspects of LHRH-expressing cells. Additionally, cannula tips were present in all sections quantitated in this study. Reduced silver grains, proportional to LHRH mRNA, were counted and three parameters were evaluated per brain section. First, the number of positively labelled cells (which, in all cases, were unambiguously identifiable) were quantified. Second, the number of grains per cell was recorded. Finally, the number of grains in all labelled cells of the selected brain section was totalled, yielding an index theoretically reflective of total LHRH synthetic capacity. Radioimmunoassay o[" L H

Serum concentrations quantified by radioimmunoassay using monoclonal antiserum no. 518B7 which is generated against bovine LH but is reactive against LH from many species, including rat 15. The tracer was I251-rat LH (NIDDK). All serum samples were run in a single assay with an intra-assay coefficient of variation of 8.9%. Statistical analysis

The Wilcoxin-Mann-Whitney test was used to compare number of LHRH-expressing cells among treatment groups as well as for comparing total quantity of LHRH mRNA. The Kolmolgorov-Smirnov test was used to test for differences in the distributions of the number of grains per cell among the three experimental groups. For presentation purposes, the average of the Control group representing each parameter was assigned a value of 100% and other experimental groups are presented in relation to this value. All data analyses were performed prior to this normalization. The serum concentration of LH data were subjected to analysis of variance with means separated by Student-Newman-Keuls test. RESULTS Neurons

expressing

the

LHRH

gene

were

dis-

tributed throughout the region of the diagonal band of Broca, the organum

vasculosum of the lamina termi-

nalis (OVLT), and around the most rostral portions of t h e t h i r d v e n t r i c l e , in a m a n n e r

s i m i l a r t o p r e v i o u s in

s i t u h y b r i d i z a t i o n r e p o r t s 17'2°. L a b e l l e d cells a v e r a g e d

79

Fig. 1. Photomicrograph of a labelled LHRH-expressing neuron. Grains cover and surround the L H R H cell body consistent with the use of a 32p-oligomer for in situ hybridization.

78 g r a i n s / n e u r o n and were clearly distinguishable from unlabelled neurons. Even lightly labelled neurons, those possessing 20-40 grains, were unambiguously different than unlabelled neurons - typically possessing 0-3 grains per cell (Fig. 1). For all rats, the rostral POA was the brain region showing the maximum number of

L H R H expressing neurons, with fewer neurons found in both rostral and caudal directions. Serum L H concentrations In the control animals, serum LH concentrations at the time of sacrifice averaged 108.2 _+ 10.7 ng/ml. Ani-

Number of Cells Expressing LHRH mRNA

Serum LH Concentration 600

no/ml

200

Control I%...........................

500 160 400 100

300

200

50

100

0

Control

8urge

8urgec/~lz

Fig. 2. Serum concentrations of LH in the three experimental groups. * P < 0.01 compared to control. ** P < 0.05 compared to control and P < 0.01 compared to surge.

Control

Surge

Surge+Prazosln

Fig. 3. Number of cells expressing the L H R H gene as detected by in situ hybridization (mean_+ S.E.M.). The average of the control group was assigned a value of 100%. * P < 0.05 compared to control group. • * P < 0.01 compared to surge group.

80 mals treated with estrogen had significantly increased ( P < 0.01) serum LH concentrations, as expected, averaging 442.3 _+ 68.1 n g / m l . Treatment with prazosin in the P O A sharply attenuated estrogen's ability to induce an LH surge ( P < 0.01) in the estrogen + prazosin treated rats (Fig. 2). These animals had LH concentrations which averaged 186.1 + 28.1 n g / m l .

Total Grains (LHRH mRNA) 200

% Control

/r

160

100

Number of cells The number of positively identified LHRH-expressing cells was quantified across treatment groups. The average number of ceils in control group brain sections was 17.8 cells. As shown in Fig. 3, rats with an estrogen induced LH surge had a mean of 50% more ( P < 0.05) detectable L H R H neurons then those rats not receiving estrogen. The ability of estrogen to increase the number of L H R H cells was blocked ( P < 0.01) by the administration of prazosin into the preoptic area.

50

Control

Surge

8urge*Prazosin

Fig. 5. Total number of grains present in all L H R H neurons in representative sections from each rat (Mean_+ S.E.M.). The average of the Control group was assigned a value of 100%. * P < 0.05 compared to control group. ** P < 0.0l compared to surge group.

Grains per cell In L H R H cells from Control animals, the median cell possessed 78 grains (Fig. 4) with a smooth distribution above and below the median. L H R H neurons from rats displaying an estrogen induced LH surge had a median of 81 grains, with a distribution not significantly different than the control group. Treatment with prazosin significantly reduced ( P < 0.01) the median number of grains per cell to 65 grains. A leftward shift in the grain distribution readily apparent in Fig. 4.

measure of the L H R H synthetic capacity at the time of sacrifice. Rats showing an estrogen-induced LH surge had a 53% increase ( P < 0.05) in the number of grains per section when compared to the nonsurging controls. Rats primed with estrogen which also received prazosin in the preoptic area averaged 63% fewer grains ( P < 0.01) than the animals showing the LH surge (Fig. 5).

Total number of grains The total number of grains was determined by adding the number of grains in all positively labelled cells in a given section. This value is interpreted as one

In the current experiment, estrogen clearly induced an LH surge; and this surge was accompanied by an increase in the number of L H R H mRNA-expressing cells as well as a measure of total L H R H m R N A in the POA. These results are consistent with other reports that an estrogen-induced LH surge is accompanied by an increase in L H R H m R N A i n t h e P O A 17'21'24. We infer that the increase in L H R H m R N A , correlated with elevated LH release, is primarily a result of estrogen operating in a positive feedback mode. Although the estrogen-induced LH surge was sharply attenuated by local administration of prazosin into the POA, the estrogen + prazosin rats did have higher LH concentrations than did the non-estrogen treated rats. The fact that prazosin did not totally eliminate a rise in LH is likely due to one or more of the following factors: local administration of prazosin did not reach all the L H R H neurons stimulated by systemic estrogen; sensitivity of the pituitary changed such that the observed increase in LH was not reflecting a similar increase in L H R H release; or, estrogen stimulated a slight L H R H release via a non-a t adrenergic agent.

Percent of Cells

40~

* .....

30

20

10

0

20-40

40-60

60-80 80-100 lOO-120 12o-140 Number of Grains Per Cell

>14o

Fig. 4. Frequency distribution histogram showing numbers of grains per positively identified LHRH-expressing cell. * P < 0.05 between prazosin and surge groups. No difference found between control and surge groups.

DISCUSSION

81 It was interesting that, although treatment with estrogen increased the number of detectable LHRH-expressing neurons, the amount of LHRH mRNA in each cell did not appear to change (Figs. 3 and 4). Park et al. 17 likewise reported that estrogen increased the number of LHRH-expressing neurons in ovariectomized rats. In the current study, the stimulatory effects of estrogen on total L H R H mRNA were manifested primarily by increases in LHRH cell number. Though not observed in this study, other researchers have reported that estrogen can increase the cellular content of LHR H mRNA 21'23-a5. The quantity of L H R H mRNA fluctuates with the stage of the estrous cycle showing increased expression during the evening of proestrus - coincident with 17, or just after 37 the preovulatory LH surge. By comparison, the turnover of norepinephrine in the POA increases just prior to and during the LH surge 2. This sequence of .events is consistent with the theory that estrogen stimulates L H R H biosynthesis, at least in part, by activating a mechanism dependent on norepinephrine inputs into the POA. The stimulatory effects of norepinephrine on LHRH (or LH) release are well known 3't2'3°. Although it remains unresolved if norepinephrine neurons synapse directly onto LHRH neurons in rats, they are known to terminate in brain regions proximal to L H RH cell bodies 6'7'11'14. There is evidence that norepinephrine producing neurons possess estrogen receptors 26 and that the estrogen-induced increase in serum LH is preceded by an increase in norepinephrine turnover in the POA 1'35. Therefore, it appears that noradrenergic nerve terminals in the POA are well positioned for r~ediating the stimulatory effects of ovarian steroids on I~HRH neurons. Perhaps the most interesting result of the current study was the fact that both the estrogen-induced LH surge and the increase in L H R H mRNA were eliminated by local administration of prazosin in the POA. These results are consistent with the hypothesis of Akabori and Barraclough I that the stimulatory effects of estrogen on LH R H release and synthesis require a functional oq adrenergic receptor connection located somewhere between the steroid-concentrating neurons and the LHR H neurons. This formulation still allows the possibility that additional interneurons a n d / o r neurochemicals (e.g., GABA) play an important role in mediating the effects of estrogen on L H R H biosynthesis and release 34. The current results provide additional evidence that the endogenous noradrenergic system is positively coupled to LHR H gene expression. We recently reported 3t that preoptic administration of prazosin in OVX rats

resulted in a 32% decrease in LHRH mRNA leading to the idea that an endogenous a I ligand plays a role in maintaining levels of LHRH mRNA. The current study suggests further that an al ligand is required for mediating the stimulatory effects of estrogen on the LHRH system. We do not yet know whether estrogen stimulates the adrenergic system, which then stimulates the LHRH neurons; or, if the adrenergic system merely provides a permissive environment for the stimulatory effects of estrogen. In summary, since administration of an a~ adrenergic antagonist had a pronounced inhibitory effect on estrogen-induced LHRH gene expression and release, it seems clear that an endogenous a~ ligand, probably norepinephrine, is required for mediating the stimulatory effects of estrogen on L H RH neurons and subsequent LH release. Acknowledgements. This research was supported by NRSA fellowship HD07373 (G.D.W.) and by HD05751 (D.W.P.).

REFERENCES 1 Akabori, A. and Barraclough, C.A., Effects of morphine on luteinizing hormone secretion and catecholamine turnover in the hypothalamus of estrogen-treated rats, Brain Res., 362 (1986) 221-226. 2 Drouva, S.V., Laplante, E. and Kordon, C., Alpha-1 adrenergic receptor involvement in the LH surge in ovariectomized estrogen-primed rats, Eur. J. Pharmacol., 81 (1982) 341-344. 3 Gallo, R.V. and Drouva, S.V., Effect of intraventricular infusion of catecholamines on luteinizing hormone release in ovariectomized and ovariectomized, steroid-primed rats, Neuroendocrinology, 29 (1979) 149-162. 4 Gearing, M. and Terasawa, E., The alpha-l-adrenergic system is involved in the pulsatile release of luteinizing hormone-releasing hormone in the ovariectomized rhesus monkey, Neuroendocrinology, 53 (1991) 373-381. 5 Goodman, R.L., The site of the positive feedback action of estradiol in the rat, Endocrinology, 102 (1978) 151-159. 6 Hoffman, G.E., Wray, S. and Goldstein, M., Relationship of catecholamines and LHRH: light microscopic study, Brain Res. Bull., 9 (1982) 417-430. 7 Hoffman, G.E., Organization of LHRH cells: differential apposition of neurotensin, substance P and catecholamine axons, Peptides, 6 (1985) 439-461. 8 Honma, K. and Wunke, W., Norepinephrine and dopamine turnover rates in the medial preoptic area and the mediobasal hypothalamus of the rat brain after various endocrinological manipulations, Endocrinology, 106 (1980) 1848-1853. 9 Jackson, G.L., Effect of adrenergic blocking drugs on secretion of luteinizing hormone in the ovariectomized ewe, Biol. Reprod., 16 (1977) 543-548. 10 Jarry, H., Leonhardt, S. and Wuttke, W., A norepinephrine-dependent mechanism in the preoptic/anterior hypothalamic area but not in the mediobasal hypothalamus is involved in the regulation of the gonadotropin-releasing hormone pulse generator in ovariectomized rats, Neuroendocrinology, 51 (1990) 337-344. 11 Jennes, L., Beckman, W.C., Stumpf, W.E., and Grzanna, Anatomical relationships of serotoninergic and noradrenalinergic projections with the GnRH system in septum and hypothalamus, Exp. Brain Res., 46 (1982) 331-338. 12 Kalra, S.P. and Gallo, R.V., Effects of intraventricular administration of catecholamines in luteinizing hormone release in morphine-treated rats, Endocrinology, 113 (1983)23-28.

82 13 Kaufman, J.-M, Kesner, J.S., Wilson, R.C. and Knobil, E., Electrophysiological manifestation of luteinizing hormone-releasing hormone pulse generator activity in the rhesus monkey: influence of alpha-adrenergic and dopaminergic blocking agents, Endocrinology, 116 (1985) 1327-1333. 14 Leranth, C., MacLuskry, N.J., Shanabtough, M. and Naftolin, F., Catecholaminergic innervation of luteinizing hormone-releasing hormone and glutamic acid decarboxylase immunopositive neurons in the rat medial preoptic area, Neuroendocrinology, 48 (1988) 591-602. 15 Matteri, R.L., Roser, J.F., Baldmin, D.M., Liporetskv, V. and Papkoff, H., Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Dom. Anim. Endocrinol., 4 (1987) 157-165. 16 McCabe, J.T. and Pfaff, D.W., In situ hybridization: a methodological guide, Methods Neurosci., 1 (1989) 98-126. 17 Park, O.-K., Gugneja, S. and Mayo, K.E., Gonadotropin-releasing hormone gene expression during the rat estrous cycle: effects of pentobarbital and ovarian steroids, Endocrinology, 127 (1990) 365-372. 18 Pau, K.-Y F, Hess, D.L., Kaynard, A.H., Ji, W.-Z., Gliessman, P.M. and Spies, H.G., Suppression of mediobasal hypothalamic gonadotropin-releasing hormone and plasma luteinizing hormone pulsatile patterns by phentolamine in ovariectomized rhesus macaques, Endocrinology, 124 (1989) 891-898. 19 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2rid edn., Academic Press, Sydney, 1986. 20 Petersen, S.L., McCrone, S., Keller, M. and Gardner, E., Rapid increase in LHRH mRNA levels following NMDA, Endocrinology, 129 (1991) 1679-1681. 21 Petersen, S.L., Cheuk, C., Hartman, R.D. and Barraclough, C.A., Medial preoptic microimplants of the antiestrogen, Keoxifene, affect luteinizing hormone-releasing hormone mRNA levels, median eminence luteinizing hormone-releasing hormone concentrations and luteinizing hormone release in ovariectomized, estrogen-treated rats, J. Neuroendocrinol., 1 (1989) 279-283. 22 Rance, N., Wise, P.M., Selmanoff, M.K. and Barraclough, C.A., Catecholamine turnover rates in discrete hypothalamic areas and associated changes in median eminence luteinizing hormone-releasing hormone and serum gonadotropins on proestrus and diestrous day 1,, Endocrinology, 108 (1981) 1795-1802. 23 Roberts, J.L., Dutlow, C.M., Jakubowski, M., Blum, M. and Millar, R.P., Estradiol stimulates preoptic area-anterior hypothalamic proGnRH-GAP gene expression in ovariectomized rats, Mol. Brain Res., 6 (1989) 127-134. 24 Rosie, R., Thomson, E. and Fink, G., Oestrogen positive feedback stimulates the synthesis of LHRH mRNA in neurons of the rostral diencephalon of the rat, Z Endocrinol., 124 (1990) 285-289.

25 Rothfeld, J., Hejtmancik, J.F., Conn, P.M. and Pfaff, D.W., In situ hybridization for LHRt! mRNA following estrogen treatment, Mol. Brain Res., 6 (1989) 121-125. 26 Sar, M. and Stumpf, W.E., Central noradrenergic neurons concentrate 3H-oestradiol, Nature, 289 (1981) 500-502. 27 Sarkar, D.K. and Fink, G., Effects of gonadal steroids on output of luteinizing hormone releasing factor into pituitary stalk blood in the female rat, J. Endocrinol., 80 (1979) 303-313. 28 Shivers, B.D., Harlan, R.E., Morrell, J.I. and Pfaff, D.W., Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones, Nature, 304 (1983) 345-347. 29 Shivers, B.D., Harlan, R.E., Hejtmancik, J.F., Conn, P.M. and Pfaff, D.W., Localization of cells containing LHRH-like mRNA in rat forebrain using in situ hybridization, Endocrinology, 118 (1986) 883-885. 30 Terasawa, E., Krook, C., Hei, D.L., Gearing, M., Schultz, N.J. and Davis, G.A., Norepinephrine is a possible neurotransmitter stimulating pulsatile release of luteinizing hormone releasing hormone in the rhesus monkey, Endocrinology, 123 (1988) 181)81816. 31 Weesner, G.D., Bergen, tI.T. and Pfaff, D.W., Differential regulation of luteinizing hormone-releasing hormone and galanin mRNA levels by alpha-I adrenergic agents in the ovariectomized rat, J. Neuroendocrinol., 4 (1992) 331-336. 32 Weick, R.F., Acute effects of adrenergic blocking drugs and neuroleptic agents on pulsatile discharges of luteinizing hormone in the ovariectomized rat, Neuroendocrinology, 26 (1978) 108-117. 33 Weiland, N.G. and Wise, P.M.. Estrogen alters the diurnal rhythm of alpha-I adrenergic receptor densities in selected brain regions, Endocrinology, 121 (1987) 1751- 1758. 34 Weiner, R.I., Findell, P.R. and Kordon, C., Role of classic and peptide neuromediators in the neuroendocrine regulation of LH and prolactin. In E. Knobil, J. Neill et al. (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1235-1281. 35 Wise, P.M., Rance, N. and Barraclough, C.A., Effects of estradiol and progesterone on catecholamine turnover rates in discrete hypothalamic regions in ovariectomized rats, Endocrinology, 108 (1981) 2186-2193. 36 Zoeller, R.T., Seeburg, P.H. and Young Ill, W.S., In situ hybridization histochemistry for messenger ribonucleic acid (mRNA) encoding gonadotropin-releasing hormone (GnRH): effect of estrogen on cellular levels of GnRH mRNA in female rat brain, Endocrinology, 122 (1988) 2570-2577. 37 Zoeller, R.T. and Young Ill, W.S., Changes in cellular levels of messenger ribonucleic acid levels encoding gonadotropin-releasing hormone in the anterior hypothalamus of female rats during the estrous cycle, Endocrinology, 123 (1988) 1688-1689.