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Origin of noradrenergic projections to GnRH perikarya-containing areas in the medial septum-diagonal band and preoptic area
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Brain Research, 621 (1993) 272-278 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19168
Origin of noradrenergic projections to GnRH perikarya-containing areas in the medial septum-diagonal band and preoptic area D.E. Wright and L. Jennes Department of Anatomy and Neurobiology, University of Kentucky, College of Medicine, Lexington, KY 40536-0084 (USA) (Accepted 30 March 1993)
Key words: Gonadotropin-releasing hormone; Brainstem; Retrograde tracing; Dopamine-/3-hydroxylase; Rat
The purpose of the present study was to identify the sites of origin of the noradrenergic fibers that project to areas containing gonadotropin-releasing hormone (GnRH) perikarya since norepinephrine (NE) is known to influence the activity of GnRH neurons. Fluorescent retrograde tracers were used in combination with immunohistochemistry for dopamine-/3-hydroxylase (DBH) and GnRH. Small volumes of either Fluoro-gold (FG) or Fluoro-Ruby (FR) were pressure injected into areas that contain the largest number of GnRH cell bodies, i.e., the medial septum-diagonal band complex or preoptic area. Retrogradely labeled neurons were observed ipsilaterally in the following noradrenergic cell groups: A2 (in the nucleus tractus solitarii), A1 (in the ventrolateral medulla) and locus coeruleus. Approximately 8% of all DBH-positive neurons within the A2-cell group were retrogradely labeled, while 12% of DBH-ir neurons in the Al-group were double-labeled. Only a few retrogradely labeled DBH-ir neurons were observed in the locus coeruleus ( < 1%). Double-labeled neurons were not organized into discrete cell groups, but were dispersed among other NE-neurons within the A2- and Al-cell groups. The highest concentrations of double-labeled neurons were located in the central one-third of both the A2 and A1 cell groups. The results suggest that most noradrenergic terminals in the region of the GnRH perikarya in the medial septum-diagonal band/rostral preoptic area originate from ipsilateral neurons in areas A1 and A2. These data support the view that the medullary cell groups A1 and A2, and not the locus coeruleus, are the primary sites of origin of the noradrenergic component that is crucial for the direct regulation of GnRH perikarya.
INTRODUCTION Gonadotropin-releasing hormone ( G n R H ) is synthesized in the rat central nervous system (CNS) by neurons located in the medial septum-diagonal band complex, preoptic area, and anterior hypothalamus 33. These neurons extend axons to fenestrated capillaries in the median eminence, delivering the peptide to gonadotropes in the anterior pituitary where G n R H stimulates the synthesis and release of L H and FSH 38. During the female estrous cycle, gonadotropins stimulate the release of estrogens by the ovary which in turn provide both negative and positive feedback to the brain depending on the stage of the estrous cycle 6A9. The feedback action of estrogen does not affect G n R H neurons directly, since G n R H neurons lack steroid hormone receptors 3~. Thus, intermediary neurotransmitter systems are required to convey the feedback effects of estrogen to G n R H neurons.
Several neurotransmitter systems appear to be important for the propagation of the estrogen signal to G n R H neurons 3s. One such system is thought to be the noradrenergic system, since most brainstem noradrenergic neurons contain steroid receptors within their nuclei 9,m and norepinephrine (NE) neurons project to a variety of relevant forebrain regions 3'4'37. Several studies have shown that NE is critical for G n R H release during the preovulatory surge on proestrous ~'28. For instance, administration of alpha-adrenergic receptor blockers suppresses LH surges 5'2~, while inhibition of N E synthesis ~8'2°'21 or depletion of N E stores 35 result in a suppression of G n R H release on proestrus. Moreover, the turnover rate of NE in the preoptic area and mediobasal hypothalamus increases in synchrony with G n R H release during the preovulatory LH surgel3, 29,39.
The stimulatory effects of NE on G n R H neurons appear to be exerted at the level of the G n R H perikarya
Correspondence: L. Jennes, University of Kentucky, College of Medicine, Department of Anatomy and Neurobiology, Lexington, KY 40536, USA. Fax: (1) (606) 257-5946.
273 a n d at G n R H axons t e r m i n a l s . I m m u n o c y t o c h e m i c a l studies have d e m o n s t r a t e d n o r a d r e n e r g i c - i m m u n o r e a c tive (ir) nerve t e r m i n a l s closely a p p o s e d to G n R H - i r axon t e r m i n a l s in t h e m e d i a n e m i n e n c e a n d j u x t a p o s i t i o n e d to G n R H p e r i k a r y a in t h e m e d i a l s e p t u m - d i a g o nal b a n d o f B r o c a c o m p l e x ( M S - D B B ) a n d p r e o p t i c a r e a 11,12'14. F u r t h e r s u p p o r t for n o r a d r e n e r g i c i n n e r v a tion o f G n R H n e u r o n s is p r o v i d e d by r e c e n t e l e c t r o n m i c r o s c o p i c s t u d i e s in t h e m o u s e d e m o n s t r a t i n g G n R H - i r p e r i k a r y a a n d p r o c e s s e s c o n t a c t e d by D B H - i r synaptic t e r m i n a l s 24. T h e origin o f t h e n o r a d r e n e r g i c i n p u t to t h e r a t f o r e b r a i n arises f r o m several N E - c e l l g r o u p s l o c a t e d in t h e b r a i n s t e m 3'25. A x o n s f r o m t h e s e N E cell g r o u p s a s c e n d r o s t r a l l y via t h e p e r i v e n t r i c u l a r N E b u n d l e s (dorsal and ventral) and the ventral NE bundle. The ventral noradrenergic bundle supplies the majority of NE-fibers that innervate hypothalamic and ventral f o r e b r a i n a r e a s 26. Specifically, lesion a n d r e t r o g r a d e tracing studies have s u g g e s t e d t h a t t h e A 1 a n d A 2 cell groups are the predominant NE-groups that innervate h y p o t h a l a m i c areas, however, t h e r e m a i n i n g cell g r o u p s s u p p l y a m i n o r c o m p o n e n t as well 4'27. H o w e v e r , b e c a u s e t h e N E n e r v e fibers o f d i f f e r e n t sites o f origin a r e i n t e r m i x e d in t h e s e a s c e n d i n g N E - p r o j e c t i o n s , t h e p r e c i s e c o n t r i b u t i o n o f e a c h N E - c e l l g r o u p in t h e innervation of areas containing GnRH neurons remains unclear. In the present study we examined the anatomical relationship between brainstem noradrenergic neurons a n d a r e a s c o n t a i n i n g G n R H cell b o d i e s . T h e n e u roanatomical tracers Fluoro-Gold and Fluoro-Ruby w e r e u s e d in c o m b i n a t i o n with D B H i m m u n o c y t o c h e m i s t r y to d e t e r m i n e t h e l o c a t i o n o f t h e n o r a d r e n e r gic n e u r o n s t h a t m a y i n f l u e n c e G n R H n e u r o n a l activity at t h e level o f t h e G n R H p e r i k a r y a . M A T E R I A L S A N D METHODS Twenty-two 50-60 day old female Sprague-Dawley rats (150-175 g) were used for these experiments. Animals were anesthetized with with ketamine-acepromazine maleate (50 mg/kg and 5 mg/kg i.p., respectively) and placed into a stereotaxic apparatus. One to 200 nl of either Fluoro-Gold (2.5% in saline, Fluorochrome, Inc.) or Fluoro-Ruby (10% in saline, Molecular Probes) were pressure-injected through a 1 ttl Hamilton syringe into the MS-DBB and rostral preoptic area over a period of 15 min. Bregma was used as the zero point and the coordinates were: 0.1 mm lateral; 8.5 mm down from the surface of the skull; -0.1 mm posterior (MS-DBB) or -0.27 mm (rostral preoptic area). After an additional 10 rain the needle was slowly retracted and the skin closed with a wound clip. Control injections included pressure injection (200 nl) of FR and FG into the lateral ventricle adjacent to areas containing GnRH neurons in order to rule out that diffusion of the tracer into the cerebrospinal fluid was responsible for retrogradely labeling select NE neurons. Seven to 10 days following the injection, animals were perfused via cardiac puncture with 200 ml phosphate-buffered saline (PBS; 0.1
M, pH 7.4) followed by 500 ml of 4% paraformaldehyde in PBS. The brains were removed and postfixed overnight in the above fixative. Thirty micron coronal vibratome sections were taken from the brainstem, washed for 15 min in Tris-HCl buffer (0.05 M, pH 7.6) and incubated overnight at room temperature in guinea pig-anti DBH antiserum (1 : 2000, kindly provided by Dr. Grzanna7). The following day sections were washed for 15 min in Tris-HC1 buffer and incubated in goat anti-guinea pig IgG coupled to dichlorotriazinylaminofluorescein (DTAF, 1 : 70; Chemicon). After 1 hr, the sections were rinsed for 15 min in Tris-HCl buffer, mounted on gelatin coated slides and coverslipped with Vectashield mounting medium (Vector Labs). All antisera were diluted in Tris-HCl buffer containing 4% normal lamb serum, 0.2% Triton X-100 and 0.1% sodium azide. Specificity controls for the antisera included omission of the primary antibody and substitution of the antibody with normal guinea pig serum. The appropriateness of the injection sites was confirmed by GnRH immunocytochemistry as described above, except rabbit antiGnRH antiserum (635-5, 1 : 5000) was applied to sections at the level of the injection site. Only animals containing injections restricted to the MS-DBB or rostral preoptic area were used in the analysis. At least 30 sections were examined from each animal through regions containing DBH-ir cell groups. Cell counts of retrogradely labeled DBH-ir neurons in the brainstem were performed on 5 animals in which serial sections were obtained.
RESULTS The general identification of brainstem noradrenergic cell g r o u p s was b a s e d o n t h e study o f D a l h s t r o m a n d F u x e 3 a n d t h e specific analysis o f D B H - i r n e u r o n s within t h e m e d u l l a r y g r o u p s A 2 a n d A 1 was b a s e d o n t h e study o f K a l i a et al., (1985) 17. I n o r d e r to avoid inclusion o f a d r e n e r g i c n e u r o n s p r e s e n t in a d j a c e n t areas, only D B H - i r n e u r o n s in t h e m e d i a l a n d i n t e r m e d i a t e subdivision o f t h e n u c l e u s t r a c t u s solitarii w e r e c o u n t e d . A l l D B H - i r n e u r o n s in t h e v e n t r o l a t e r a l m e d u l l a w e r e a n a l y z e d since t h e A1 cell g r o u p consists a l m o s t exclusively o f n o r a d r e n e r g i c n e u r o n s 17. I n j e c t i o n with F R r e t r o g r a d e l y l a b e l e d n e u r o n s m o r e i n t e n s e l y t h a n F G injection; however, t h e d i s t r i b u t i o n a n d n u m b e r o f l a b e l e d n e u r o n s w e r e i d e n t i c a l for b o t h r e t r o g r a d e tracers. F o l l o w i n g u n i l a t e r a l p r e s s u r e injections o f e i t h e r F G o r F R into t h e M S - D B B o r r o s t r a l p r e o p t i c area, r e t r o g r a d e l y l a b e l e d n e u r o n s w e r e o b s e r v e d in t h r e e o f t h e seven n o r a d r e n e r g i c cell groups: A 2 r e g i o n in t h e n u c l e u s t r a c t u s solitarii (Figs. 1 a n d 4B, C), A 1 in t h e v e n t r o l a t e r a l m e d u l l a (Figs. 2 a n d 4B, C) a n d t h e locus c o e r u l e u s (A6; Figs. 3 a n d 4A). T h e r e m a i n i n g n o r a d r e n e r g i c g r o u p s (A3, A4, A5, a n d A 7 ) d i d n o t c o n t a i n r e t r o g r a d e l y l a b e l e d cells. T h e m a j o r i t y o f F G - o r F R - p o s i t i v e n e u r o n s w e r e i p s i l a t e r a l to t h e injection site; only a few c o n t r a l a t e r a l n e u r o n s w e r e o b s e r v e d ( < 2 % o f all d o u b l e - l a b e l e d n o r a d r e n e r g i c n e u r o n s ) . T h e A 2 cell g r o u p c o n t a i n e d t h e h i g h e s t n u m b e r o f d o u b l e - l a b e l e d n e u r o n s p e r a n i m a l (48 + 5.05, S.E.M.), while t h e A 1 r e g i o n a n d locus c o e r u l e u s c o n t a i n e d 29 + 1.60 a n d 2 + 0.052, r e s p e c t i v e l y (Fig. 5A). I n o r d e r to a c c o u n t for t h e overall lower n u m b e r
274
Figs. 1-3. Immunohistochemical stainings of brainstem dopamine-fl-hydroxylase-ir neurons (DBH, column a) that were retrogradely labeled with Fluoro-Ruby (FR) after unilatateral injection of the tracer into the medial septum-diagonal band of Broca (column b). Figs. la, b: A2 noradrenergic neurons in the medial subdivision of the nucleus of the tractus solitarii. Fig. 2a, b: A1 noradrenergic neurons in the ventrolateral medulla. Fig. 3a, b: Noradrenergic neurons in the locus coeruleus. Arrows point to double-labeled neurons, x 264.
275
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of NE neurons in the A1 region compared to the A2, the data are also expressed as percentage of doublelabeled cells of the total DBH-positive neurons ipsilateral to the injection site. Thus, the A1 cell group contained a higher percentage of retrogradely labeled DBH-positive neurons (11.8%) than the A2 cell group and locus coeruleus (7.6% and < 0.1%, respectively, Fig. 5B). The distribution a n d / o r number of retrogradely filled DBH-ir neurons did not differ between injections into the MS-DBB or into more caudal levels of the rostral preoptic area. Moreover, double-labeled cells were not organized into definable subgroups, but appeared as a loose band of diffuse NE neurons situated predominantly in the central one-third of the A2 and A1 cell groups. DISCUSSION
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Fig. 4. Schematic drawings of coronal sections of the rat brainstem illustrating the relative distribution of dopamine-/3-hydroxylase neurons (*) retrogradely labeled with Fluoro-Ruby (o). A: level of the locus coeruleus. B: A2 and A1 cell groups (rostral level). C: A2 and A1 cell groups (caudal level).
The present study identifies noradrenergic neurons of the caudal medulla that project to areas in the brain which contain the majority of GnRH perikarya. These findings suggest that some of the NE neurons in the areas A1 and A2 provide the major NE input to the region of the GnRH perikarya while the contribution of the neurons in the locus coeruleus appears to be minimal. The distribution of central noradrenergic systems has been well studied, however, the details of the noradrenergic innervation to brain areas that contain G nRH perikarya is less understood. In our study, injections into the MS-DBB or rostral preoptic area labeled DBH-ir neurons with the same distribution and number in the A2 and A1 cell group, suggesting that
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Fig. 5. Total number (A) and percent (B) of retrogradely labeled ipsilateral dopamine-/3-hydroxylase-ir neurons in the cell groups A2, A1, and locus coeruleus following unilateral injection of Fluoro-Ruby into the medial septum-diagonal band or preoptic area. Bars equals S.E.M.
276 NE-fibers ascending in the ventral N E bundle branch to innervate multiple hypothalamic and extrahypothalamic areas. At present it is not clear whether or not the N E fibers innervating the MS-DBB are collaterals of axon which terminate in the MPOA. However, the similar number and distribution of the N E perikarya that are retrogradely labeled from the MS-DBB andfrom the M P O A suggest that the same N E neurons in the central A1 and A2 innervate both the MS-DBB and the preoptic area. The relatively low number of retrogradely labeled DBH-ir neurons may have resulted from several factors. For instance, the injection sites were kept small ( < 200 nl injection volume) in order to prevent spread of the tracer into areas that may not be related to a direct innervation of G n R H neurons. In addition, since GnRH-containing neurons are dispersed throughout the MS-DBB and preoptic area, only a small fraction of the total number of G n R H neurons was covered by injection of the tracer. Immunocytochemical studies have shown that the number of DBH-ir fibers within the MS-DBB is relatively sparce 12A4 compared to adjacent areas such as the bed nucleus of the stria terminalis 37. Moreover, the overall number of synaptic inputs to G n R H perikarya and dendrites appears very lOW 16'22'32'34'40. These findings support the view that the number of N E neurons that innervate G n R H perikarya may be limited. Since G n R H neurons make synaptic contact with other G n R H neurons 22'34'4°, the activity of many G n R H neurons may be synchronized, and the restricted N E signal to a limited number of G n R H neurons may have profound influences on the entire G n R H neuronal population. The view that noradrenergic neurons convey the effects o f estrogen to G n R H neurons is supported by both anatomical and physiological studies. Thus, N E neurons in the A1 and A2 contain estrogen receptors as determined by a combination of [3H]estradiol autoradiography combined with formaldehyde induced fluorescence or immunohistochemistry for D B H 9'3°. Similar results were obtained with in situ hybridization which localized estrogen receptor m R N A to a large number of N E neurons in the A1 and A236. It appears that up to 5 0 - 7 0 % of caudal A1 NE-neurons contain estrogen receptors, while 7 5 - 8 5 % of A2 neurons contain 3H-estradiol9. It is unclear, however, whether N E neurons that project to G n R H perikarya-containing areas also contain estrogen receptors. Based on the high percentage of N E neurons which contain estrogen receptors 9 as well as on the overlap in the distribution of retrogradely labeled N E neurons with estrogen target cells, it is likely that at least some of the retrogradely labeled neurons identified in this study are
estrogen-sensitive. Since the number of estrogen target neurons in the A1 and A2 is much greater than the number of retrogradely labeled N E neurons, it is suggested that most of the estrogen-sensitive NE neurons are not involved in the propagation of the estrogen signal directly to the G n R H neurons, but instead, these neurons convey the estrogen signal to other as yet unidentified neurons in the CNS. Such neurons may be located in regions in the brainstem, hypothalamus, amygdala, or olfactory tubercle, all of which are areas in the CNS which receive N E innervation from the A1 or A2 N E cell groups and which have been shown to be involved in the regulation of reproductive behavior or G n R H release 23'25. The precise effects of estrogen on the neurons in the A1 and A2 are not clear. Three hours after estrogen administration to ovariectomized rats, a dramatic increase in los expression occurs in about 80% of the N E neurons in the A2, while long-term ovariectomy results in the disappearance of fos from A2 neurons 15. The presence of fos could be regarded as an indicator of the synthetic rate of tyrosine hydroxylase (TH) since an appropriate AP-1 sequence is present in the T H gene 2. It is therefor suggested that in the rat, estrogen can stimulate T H synthesis in the N E target neurons of the A2. Similarly, estrogen causes an increase in T H m R N A in the A1 region of the male rat. In this model, however, the T H levels in N E neurons in the A2 appear not to be affected by estrogen 8. In summary, the results of the present study show that the major N E input to the MS-DBB and preoptic area originates from brainstem N E neurons in the A1 and A2, and it is suggested that these N E neurons convey the estrogen signal to the G n R H neurons, thereby regulating G n R H release throughout the estrous cycle. Acknowledgements. Supported by NIH HD24697. ABBREVIATIONS a ab ap ce CSP cul cum DCT FLD FLM gc gr IAF LL LM lm
nucleus (n.) alpha n. ambiguus area postrema locus coeruleus tractus corticospinalis n. cuneatus lateralis n. cuneatus medialis decussatio corporis trapezoidei fasiculus longitudinalis dorsalis fasiculus longitudinalis medialis griseum centrale n. gracilis fibrae arcuatae internae lemniscus lateralis lemniscus medialis n. reticularis lateralis magnocellularis
277 lp old olm phi pbm PCM PCS pols rap rd rpa rob rpm rpo rtp SOL sol SPC SPCV STH tdo trl trm Vm Vmes VS Vs Vspc Xdm XII
n. reticularis lateralis parvocellularis n. olivaris accessorius dorsalis n. olivaris accessorius medialis n. parabrachialis lateralis n. parabrachialis medialis pedunculus cerebellaris medius pedunculus cerebellaris superior n. paraolivaris superior n. raphe pontis n. reticularis dorsalis medullae oblongata n. raphe pallidus n. raphe obscurus n. raphe paramedianus n. reticularis pontis oralis n. reticularis tegmenti pontis tractus solitarii n. tractus solitarii tractus spinocerebellaris tractus spinocerebellaris ventralis tractus spinothalamicus n. tegmentalis dorsalis n. trapezoides lateralis n. trapezoides medialis n. motorius nervi trigemini n. tractus mesencephali nervi trigemini tractus spinalis nervi trigemini n. sensibilis nervi trigemini n. caudalis tractus spinalis nervi trigemini n. dorsalis motorius nervi vagi n. nervi hypoglossi
REFERENCES 1 Barraclough, C.A., Wise, P.M. and Selmanoff, M.K., A role for hypothalamic catecholamines in the regulation of gonadotropin secretion, Recent Prog. Hormone Res., 40 (1984) 487-529. 2 Chambi, F., Fung, B. and Chikaraishi, D., 5' flanking DNA sequences direct cell-specific expression of rat tyrosine hydroxylase, J. Neurochem., 53 (1989) 1656-1659. 3 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 (1964) 1-55. 4 Day, T.A., Blessing, W. and Willoughby, J.O., Noradrenergic and dopaminergic projections to the medial preoptic area of the rat. A combined horseradish peroxidase/catecholamine fluorescence study, Brain Res., 193 (1980) 543-548. 5 Drouva, S.V., LaPlante, E. and Kordon, C., Alphavadrenergic receptor involvement in the LH surge in ovariectomized estrogen-primed rats, Eur. J. Pharmacol., 81 (1982) 341-344. 6 Freeman, M.E., The ovarian cycle of the rat. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1893-1928. 7 Grzanna, R. and Coyle, J.T., Rat adrenal dopamine beta hydroxylase: purification and immunologic characteristics, Z Neurochem., 27 (1976) 1091-1096. 8 Hartman, R.D. and Barraclough, C.A., Changes in tyrosine hydroxylase mRNA levels in medullary A1 and A2 neurons and locus coeruleus following castration and estrogen replacement in rats, Brain Res., 13 (1992) 231-238. 9 Heritage, A.S., Grant, L.D. and Stumpf, W.E., 3H-Estradiol in catecholamine neurons of rat brain stem: combined localization by autoradiography and formaldehyde-induced fluorescence, J. Comp. Neurol., 176 (1977) 607-630. 10 Heritage, A.S., Stumpf, W.E., Sar, M. and Grant, L.D., Brainstem catecholamine neurons are target sites for sex steroid hormones, Science, 207 (1980) 1377-1379. 11 Hoffman, G.E., Organization of LHRH cells: Differential apposition of neurotensin, substance P and catecholamine axons, Peptides, 6 (1985) 439-461.
12 Hoffman, G.E., Wray, S. and Goldstein, M., Relationship of catecholamines and LHRH: light microscopic study, Brain Res. Bull., 9 (1982) 417-430. 13 Honma, K. and Wuttke, 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. 14 Jennes, L., Beckman, W.C., Stumpf, W.E. and Grzanna, R., Anatomical relationships of serotoninergic and noradrenergic projections with the GnRH system in septum and hypothalamus, Exp. Brain Res., 46 (1982) 331-338. 15 Jennes, L., Jennes, M.E., Purvis, C. and Nees, M., C-fos expression in noradrenergic A2 neurons of the rat during the estrous cycle and after steroid hormone treatments, Brain Res., 586 (1992) 171-175. 16 Jennes, L., Stumpf, W.E. and Sheedy, M.E., UItrastructural characterization of gonadotropin-releasing hormone (GnRH)-producing neurons, J. Comp. Neurol., 232 (1985) 534-547. 17 Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. II. Dopaminergic, noradrenergic (A1 and A2) and adrenergic neurons, nerve fibers, and presumptive terminal processes, J. Comp. Neurol., 233 (1985) 308-332. 18 Kalra, S.P., Suppression of serum LH-RH and LH in rats by an inhibitor of norepinephrine synthesis, J. Reprod. Fert., 49 (1977) 371-373. 19 Kalra, P.S. and Kalra, S.P., Control of gonadotropin secretion. In H. Imura (Ed.), The Pituitary Gland, Raven, New York, 1985, pp. 189-220. 20 Kalra, P.S., Kalra, S.P., Krulich, L., Fawcett, C.P. and McCann, S.M., Involvement of norepinephrine in transmission" of the stimulatory influence of progesterone on gonadotropin release, Endocrinology, 90 (1972) 1168-1176. 21 Kalra, S.P. and McCann, S.P., Effects of drugs modifying catecholamine synthesis on plasma LH and ovulation in the rat, Neuroendocrinology, 15 (1974) 75-91. 22 L6r~nth, Cs., Segura, L.M.G., Palkovits, M., MacLusky, N.J., Shanabrough, M. and Naftolin, F., The LH-RH-containing neuronal network in the preoptic area of the rat: demonstration of LH-RH-containing nerve terminals in synaptic contact with LHRH neurons, Brain Res., 345 (1985) 332-336. 23 McKellar, S. and Loewy, A.D., Efferent projections of the A1 catecholamine cell group in the rat: an autoradiography study, Brain Res., 241 (1982) 11-29. 24 Miller, M.M. and Zhu, L., Immunocytochemical colocalization of dopamine beta-hydroxylase and luteinizing hormone releasing hormone neuronal elements by electron microscopy in the rostral forebrain of the female C57BL6J mouse, Soc. Neurosci. Abstr., (1992) 344.4. 25 Moore, R.Y. and Card, J.P., Noradrenaline-containing neuron systems. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Elsevier, Amsterdam, 1984, pp. 123-156. 26 Palkovits, M., Catecholamines in the hypothalamus: an anatomical review, Progress in Neuroendocrinology, 33 ( 1981) 123-128. 27 Palkovits, M., Zaborszky, L., Feminger, A., Mezey, E., Fekete, M.I.K., Herman, J.P., Kanyicska, B. and Szabo, D., Noradrenergic innervation of the rat hypothalamus: experimental, biochemical, and electron microscopic studies, Brain Res., 191 (1980) 161-171. 28 Ramirez, V.D., Feder, H.H. and Sawyer, C.H., The role of brain catecholamines in the regulation of LH secretion: a critical inquiry. In L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrinolgy, Vol. 8, Raven, New York, 1984, pp. 27-84. 29 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 diestrus day 1. Endocrinology, 108 (1981) 1795-1802. 30 Sat, M. and Stumpf, W.E., Distribution of androgen-concentrating neurons in rat brain, In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 120-133. 31 Shivers, B.D., Harlan, R.E., Morrell, J.I. and Pfaff, D.W., Ab-
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sence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones, Nature, 304 (1983) 345-347. Silverman, A.J., Luteinizing hormone releasing hormone containing synapses in the diagonal band and preoptic area of the guinea pig, J. Comp. Neurol., 227 (1984) 452-458. Silverman, A.J., The gonadotropin-releasing hormone (GnRH) neuronal systems: immunocytochemistry. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1283-1304. Silverman, A.J. and Witkin, J.W., Synaptic interactions of luteinizing hormone-releasing hormone (LHRH) neurons in the guinea pig preoptic area, J. Histochem. Cytochem., 33 (1985) 69-72. Simpkins, J.W., Advis, J.P., Hodson, C.A. and Meites, J., Blockade of steroid-induced luteinizing hormone release by selective depletion of anterior hypothalamic norepinephrine activity, Endocrinology, 104 (1979) 506-509. Simmerly, R.B., Chang, C., Muramatsu, M. and Swanson, L.W.,
37
38
39
40
Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study, Z Comp. Neurol., 294 (1990) 76-95. Swanson, L.W. and Hartman, B.K., The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-/3-hydroxylase as a marker, J. Comp. Neurol., 163 (1975) 467-506. 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 and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1235-1282. 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. Witkin, J.W. and Silverman, A.J., Synaptology of luteinizing hormone-releasing hormone neurons in rat preoptic area, Peptides, 6 (1985) 263-271.
Brain Research, 621 (1993) 272-278 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19168
Origin of noradrenergic projections to GnRH perikarya-containing areas in the medial septum-diagonal band and preoptic area D.E. Wright and L. Jennes Department of Anatomy and Neurobiology, University of Kentucky, College of Medicine, Lexington, KY 40536-0084 (USA) (Accepted 30 March 1993)
Key words: Gonadotropin-releasing hormone; Brainstem; Retrograde tracing; Dopamine-/3-hydroxylase; Rat
The purpose of the present study was to identify the sites of origin of the noradrenergic fibers that project to areas containing gonadotropin-releasing hormone (GnRH) perikarya since norepinephrine (NE) is known to influence the activity of GnRH neurons. Fluorescent retrograde tracers were used in combination with immunohistochemistry for dopamine-/3-hydroxylase (DBH) and GnRH. Small volumes of either Fluoro-gold (FG) or Fluoro-Ruby (FR) were pressure injected into areas that contain the largest number of GnRH cell bodies, i.e., the medial septum-diagonal band complex or preoptic area. Retrogradely labeled neurons were observed ipsilaterally in the following noradrenergic cell groups: A2 (in the nucleus tractus solitarii), A1 (in the ventrolateral medulla) and locus coeruleus. Approximately 8% of all DBH-positive neurons within the A2-cell group were retrogradely labeled, while 12% of DBH-ir neurons in the Al-group were double-labeled. Only a few retrogradely labeled DBH-ir neurons were observed in the locus coeruleus ( < 1%). Double-labeled neurons were not organized into discrete cell groups, but were dispersed among other NE-neurons within the A2- and Al-cell groups. The highest concentrations of double-labeled neurons were located in the central one-third of both the A2 and A1 cell groups. The results suggest that most noradrenergic terminals in the region of the GnRH perikarya in the medial septum-diagonal band/rostral preoptic area originate from ipsilateral neurons in areas A1 and A2. These data support the view that the medullary cell groups A1 and A2, and not the locus coeruleus, are the primary sites of origin of the noradrenergic component that is crucial for the direct regulation of GnRH perikarya.
INTRODUCTION Gonadotropin-releasing hormone ( G n R H ) is synthesized in the rat central nervous system (CNS) by neurons located in the medial septum-diagonal band complex, preoptic area, and anterior hypothalamus 33. These neurons extend axons to fenestrated capillaries in the median eminence, delivering the peptide to gonadotropes in the anterior pituitary where G n R H stimulates the synthesis and release of L H and FSH 38. During the female estrous cycle, gonadotropins stimulate the release of estrogens by the ovary which in turn provide both negative and positive feedback to the brain depending on the stage of the estrous cycle 6A9. The feedback action of estrogen does not affect G n R H neurons directly, since G n R H neurons lack steroid hormone receptors 3~. Thus, intermediary neurotransmitter systems are required to convey the feedback effects of estrogen to G n R H neurons.
Several neurotransmitter systems appear to be important for the propagation of the estrogen signal to G n R H neurons 3s. One such system is thought to be the noradrenergic system, since most brainstem noradrenergic neurons contain steroid receptors within their nuclei 9,m and norepinephrine (NE) neurons project to a variety of relevant forebrain regions 3'4'37. Several studies have shown that NE is critical for G n R H release during the preovulatory surge on proestrous ~'28. For instance, administration of alpha-adrenergic receptor blockers suppresses LH surges 5'2~, while inhibition of N E synthesis ~8'2°'21 or depletion of N E stores 35 result in a suppression of G n R H release on proestrus. Moreover, the turnover rate of NE in the preoptic area and mediobasal hypothalamus increases in synchrony with G n R H release during the preovulatory LH surgel3, 29,39.
The stimulatory effects of NE on G n R H neurons appear to be exerted at the level of the G n R H perikarya
Correspondence: L. Jennes, University of Kentucky, College of Medicine, Department of Anatomy and Neurobiology, Lexington, KY 40536, USA. Fax: (1) (606) 257-5946.
273 a n d at G n R H axons t e r m i n a l s . I m m u n o c y t o c h e m i c a l studies have d e m o n s t r a t e d n o r a d r e n e r g i c - i m m u n o r e a c tive (ir) nerve t e r m i n a l s closely a p p o s e d to G n R H - i r axon t e r m i n a l s in t h e m e d i a n e m i n e n c e a n d j u x t a p o s i t i o n e d to G n R H p e r i k a r y a in t h e m e d i a l s e p t u m - d i a g o nal b a n d o f B r o c a c o m p l e x ( M S - D B B ) a n d p r e o p t i c a r e a 11,12'14. F u r t h e r s u p p o r t for n o r a d r e n e r g i c i n n e r v a tion o f G n R H n e u r o n s is p r o v i d e d by r e c e n t e l e c t r o n m i c r o s c o p i c s t u d i e s in t h e m o u s e d e m o n s t r a t i n g G n R H - i r p e r i k a r y a a n d p r o c e s s e s c o n t a c t e d by D B H - i r synaptic t e r m i n a l s 24. T h e origin o f t h e n o r a d r e n e r g i c i n p u t to t h e r a t f o r e b r a i n arises f r o m several N E - c e l l g r o u p s l o c a t e d in t h e b r a i n s t e m 3'25. A x o n s f r o m t h e s e N E cell g r o u p s a s c e n d r o s t r a l l y via t h e p e r i v e n t r i c u l a r N E b u n d l e s (dorsal and ventral) and the ventral NE bundle. The ventral noradrenergic bundle supplies the majority of NE-fibers that innervate hypothalamic and ventral f o r e b r a i n a r e a s 26. Specifically, lesion a n d r e t r o g r a d e tracing studies have s u g g e s t e d t h a t t h e A 1 a n d A 2 cell groups are the predominant NE-groups that innervate h y p o t h a l a m i c areas, however, t h e r e m a i n i n g cell g r o u p s s u p p l y a m i n o r c o m p o n e n t as well 4'27. H o w e v e r , b e c a u s e t h e N E n e r v e fibers o f d i f f e r e n t sites o f origin a r e i n t e r m i x e d in t h e s e a s c e n d i n g N E - p r o j e c t i o n s , t h e p r e c i s e c o n t r i b u t i o n o f e a c h N E - c e l l g r o u p in t h e innervation of areas containing GnRH neurons remains unclear. In the present study we examined the anatomical relationship between brainstem noradrenergic neurons a n d a r e a s c o n t a i n i n g G n R H cell b o d i e s . T h e n e u roanatomical tracers Fluoro-Gold and Fluoro-Ruby w e r e u s e d in c o m b i n a t i o n with D B H i m m u n o c y t o c h e m i s t r y to d e t e r m i n e t h e l o c a t i o n o f t h e n o r a d r e n e r gic n e u r o n s t h a t m a y i n f l u e n c e G n R H n e u r o n a l activity at t h e level o f t h e G n R H p e r i k a r y a . M A T E R I A L S A N D METHODS Twenty-two 50-60 day old female Sprague-Dawley rats (150-175 g) were used for these experiments. Animals were anesthetized with with ketamine-acepromazine maleate (50 mg/kg and 5 mg/kg i.p., respectively) and placed into a stereotaxic apparatus. One to 200 nl of either Fluoro-Gold (2.5% in saline, Fluorochrome, Inc.) or Fluoro-Ruby (10% in saline, Molecular Probes) were pressure-injected through a 1 ttl Hamilton syringe into the MS-DBB and rostral preoptic area over a period of 15 min. Bregma was used as the zero point and the coordinates were: 0.1 mm lateral; 8.5 mm down from the surface of the skull; -0.1 mm posterior (MS-DBB) or -0.27 mm (rostral preoptic area). After an additional 10 rain the needle was slowly retracted and the skin closed with a wound clip. Control injections included pressure injection (200 nl) of FR and FG into the lateral ventricle adjacent to areas containing GnRH neurons in order to rule out that diffusion of the tracer into the cerebrospinal fluid was responsible for retrogradely labeling select NE neurons. Seven to 10 days following the injection, animals were perfused via cardiac puncture with 200 ml phosphate-buffered saline (PBS; 0.1
M, pH 7.4) followed by 500 ml of 4% paraformaldehyde in PBS. The brains were removed and postfixed overnight in the above fixative. Thirty micron coronal vibratome sections were taken from the brainstem, washed for 15 min in Tris-HCl buffer (0.05 M, pH 7.6) and incubated overnight at room temperature in guinea pig-anti DBH antiserum (1 : 2000, kindly provided by Dr. Grzanna7). The following day sections were washed for 15 min in Tris-HC1 buffer and incubated in goat anti-guinea pig IgG coupled to dichlorotriazinylaminofluorescein (DTAF, 1 : 70; Chemicon). After 1 hr, the sections were rinsed for 15 min in Tris-HCl buffer, mounted on gelatin coated slides and coverslipped with Vectashield mounting medium (Vector Labs). All antisera were diluted in Tris-HCl buffer containing 4% normal lamb serum, 0.2% Triton X-100 and 0.1% sodium azide. Specificity controls for the antisera included omission of the primary antibody and substitution of the antibody with normal guinea pig serum. The appropriateness of the injection sites was confirmed by GnRH immunocytochemistry as described above, except rabbit antiGnRH antiserum (635-5, 1 : 5000) was applied to sections at the level of the injection site. Only animals containing injections restricted to the MS-DBB or rostral preoptic area were used in the analysis. At least 30 sections were examined from each animal through regions containing DBH-ir cell groups. Cell counts of retrogradely labeled DBH-ir neurons in the brainstem were performed on 5 animals in which serial sections were obtained.
RESULTS The general identification of brainstem noradrenergic cell g r o u p s was b a s e d o n t h e study o f D a l h s t r o m a n d F u x e 3 a n d t h e specific analysis o f D B H - i r n e u r o n s within t h e m e d u l l a r y g r o u p s A 2 a n d A 1 was b a s e d o n t h e study o f K a l i a et al., (1985) 17. I n o r d e r to avoid inclusion o f a d r e n e r g i c n e u r o n s p r e s e n t in a d j a c e n t areas, only D B H - i r n e u r o n s in t h e m e d i a l a n d i n t e r m e d i a t e subdivision o f t h e n u c l e u s t r a c t u s solitarii w e r e c o u n t e d . A l l D B H - i r n e u r o n s in t h e v e n t r o l a t e r a l m e d u l l a w e r e a n a l y z e d since t h e A1 cell g r o u p consists a l m o s t exclusively o f n o r a d r e n e r g i c n e u r o n s 17. I n j e c t i o n with F R r e t r o g r a d e l y l a b e l e d n e u r o n s m o r e i n t e n s e l y t h a n F G injection; however, t h e d i s t r i b u t i o n a n d n u m b e r o f l a b e l e d n e u r o n s w e r e i d e n t i c a l for b o t h r e t r o g r a d e tracers. F o l l o w i n g u n i l a t e r a l p r e s s u r e injections o f e i t h e r F G o r F R into t h e M S - D B B o r r o s t r a l p r e o p t i c area, r e t r o g r a d e l y l a b e l e d n e u r o n s w e r e o b s e r v e d in t h r e e o f t h e seven n o r a d r e n e r g i c cell groups: A 2 r e g i o n in t h e n u c l e u s t r a c t u s solitarii (Figs. 1 a n d 4B, C), A 1 in t h e v e n t r o l a t e r a l m e d u l l a (Figs. 2 a n d 4B, C) a n d t h e locus c o e r u l e u s (A6; Figs. 3 a n d 4A). T h e r e m a i n i n g n o r a d r e n e r g i c g r o u p s (A3, A4, A5, a n d A 7 ) d i d n o t c o n t a i n r e t r o g r a d e l y l a b e l e d cells. T h e m a j o r i t y o f F G - o r F R - p o s i t i v e n e u r o n s w e r e i p s i l a t e r a l to t h e injection site; only a few c o n t r a l a t e r a l n e u r o n s w e r e o b s e r v e d ( < 2 % o f all d o u b l e - l a b e l e d n o r a d r e n e r g i c n e u r o n s ) . T h e A 2 cell g r o u p c o n t a i n e d t h e h i g h e s t n u m b e r o f d o u b l e - l a b e l e d n e u r o n s p e r a n i m a l (48 + 5.05, S.E.M.), while t h e A 1 r e g i o n a n d locus c o e r u l e u s c o n t a i n e d 29 + 1.60 a n d 2 + 0.052, r e s p e c t i v e l y (Fig. 5A). I n o r d e r to a c c o u n t for t h e overall lower n u m b e r
274
Figs. 1-3. Immunohistochemical stainings of brainstem dopamine-fl-hydroxylase-ir neurons (DBH, column a) that were retrogradely labeled with Fluoro-Ruby (FR) after unilatateral injection of the tracer into the medial septum-diagonal band of Broca (column b). Figs. la, b: A2 noradrenergic neurons in the medial subdivision of the nucleus of the tractus solitarii. Fig. 2a, b: A1 noradrenergic neurons in the ventrolateral medulla. Fig. 3a, b: Noradrenergic neurons in the locus coeruleus. Arrows point to double-labeled neurons, x 264.
275
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',
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of NE neurons in the A1 region compared to the A2, the data are also expressed as percentage of doublelabeled cells of the total DBH-positive neurons ipsilateral to the injection site. Thus, the A1 cell group contained a higher percentage of retrogradely labeled DBH-positive neurons (11.8%) than the A2 cell group and locus coeruleus (7.6% and < 0.1%, respectively, Fig. 5B). The distribution a n d / o r number of retrogradely filled DBH-ir neurons did not differ between injections into the MS-DBB or into more caudal levels of the rostral preoptic area. Moreover, double-labeled cells were not organized into definable subgroups, but appeared as a loose band of diffuse NE neurons situated predominantly in the central one-third of the A2 and A1 cell groups. DISCUSSION
C
Fig. 4. Schematic drawings of coronal sections of the rat brainstem illustrating the relative distribution of dopamine-/3-hydroxylase neurons (*) retrogradely labeled with Fluoro-Ruby (o). A: level of the locus coeruleus. B: A2 and A1 cell groups (rostral level). C: A2 and A1 cell groups (caudal level).
The present study identifies noradrenergic neurons of the caudal medulla that project to areas in the brain which contain the majority of GnRH perikarya. These findings suggest that some of the NE neurons in the areas A1 and A2 provide the major NE input to the region of the GnRH perikarya while the contribution of the neurons in the locus coeruleus appears to be minimal. The distribution of central noradrenergic systems has been well studied, however, the details of the noradrenergic innervation to brain areas that contain G nRH perikarya is less understood. In our study, injections into the MS-DBB or rostral preoptic area labeled DBH-ir neurons with the same distribution and number in the A2 and A1 cell group, suggesting that
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Fig. 5. Total number (A) and percent (B) of retrogradely labeled ipsilateral dopamine-/3-hydroxylase-ir neurons in the cell groups A2, A1, and locus coeruleus following unilateral injection of Fluoro-Ruby into the medial septum-diagonal band or preoptic area. Bars equals S.E.M.
276 NE-fibers ascending in the ventral N E bundle branch to innervate multiple hypothalamic and extrahypothalamic areas. At present it is not clear whether or not the N E fibers innervating the MS-DBB are collaterals of axon which terminate in the MPOA. However, the similar number and distribution of the N E perikarya that are retrogradely labeled from the MS-DBB andfrom the M P O A suggest that the same N E neurons in the central A1 and A2 innervate both the MS-DBB and the preoptic area. The relatively low number of retrogradely labeled DBH-ir neurons may have resulted from several factors. For instance, the injection sites were kept small ( < 200 nl injection volume) in order to prevent spread of the tracer into areas that may not be related to a direct innervation of G n R H neurons. In addition, since GnRH-containing neurons are dispersed throughout the MS-DBB and preoptic area, only a small fraction of the total number of G n R H neurons was covered by injection of the tracer. Immunocytochemical studies have shown that the number of DBH-ir fibers within the MS-DBB is relatively sparce 12A4 compared to adjacent areas such as the bed nucleus of the stria terminalis 37. Moreover, the overall number of synaptic inputs to G n R H perikarya and dendrites appears very lOW 16'22'32'34'40. These findings support the view that the number of N E neurons that innervate G n R H perikarya may be limited. Since G n R H neurons make synaptic contact with other G n R H neurons 22'34'4°, the activity of many G n R H neurons may be synchronized, and the restricted N E signal to a limited number of G n R H neurons may have profound influences on the entire G n R H neuronal population. The view that noradrenergic neurons convey the effects o f estrogen to G n R H neurons is supported by both anatomical and physiological studies. Thus, N E neurons in the A1 and A2 contain estrogen receptors as determined by a combination of [3H]estradiol autoradiography combined with formaldehyde induced fluorescence or immunohistochemistry for D B H 9'3°. Similar results were obtained with in situ hybridization which localized estrogen receptor m R N A to a large number of N E neurons in the A1 and A236. It appears that up to 5 0 - 7 0 % of caudal A1 NE-neurons contain estrogen receptors, while 7 5 - 8 5 % of A2 neurons contain 3H-estradiol9. It is unclear, however, whether N E neurons that project to G n R H perikarya-containing areas also contain estrogen receptors. Based on the high percentage of N E neurons which contain estrogen receptors 9 as well as on the overlap in the distribution of retrogradely labeled N E neurons with estrogen target cells, it is likely that at least some of the retrogradely labeled neurons identified in this study are
estrogen-sensitive. Since the number of estrogen target neurons in the A1 and A2 is much greater than the number of retrogradely labeled N E neurons, it is suggested that most of the estrogen-sensitive NE neurons are not involved in the propagation of the estrogen signal directly to the G n R H neurons, but instead, these neurons convey the estrogen signal to other as yet unidentified neurons in the CNS. Such neurons may be located in regions in the brainstem, hypothalamus, amygdala, or olfactory tubercle, all of which are areas in the CNS which receive N E innervation from the A1 or A2 N E cell groups and which have been shown to be involved in the regulation of reproductive behavior or G n R H release 23'25. The precise effects of estrogen on the neurons in the A1 and A2 are not clear. Three hours after estrogen administration to ovariectomized rats, a dramatic increase in los expression occurs in about 80% of the N E neurons in the A2, while long-term ovariectomy results in the disappearance of fos from A2 neurons 15. The presence of fos could be regarded as an indicator of the synthetic rate of tyrosine hydroxylase (TH) since an appropriate AP-1 sequence is present in the T H gene 2. It is therefor suggested that in the rat, estrogen can stimulate T H synthesis in the N E target neurons of the A2. Similarly, estrogen causes an increase in T H m R N A in the A1 region of the male rat. In this model, however, the T H levels in N E neurons in the A2 appear not to be affected by estrogen 8. In summary, the results of the present study show that the major N E input to the MS-DBB and preoptic area originates from brainstem N E neurons in the A1 and A2, and it is suggested that these N E neurons convey the estrogen signal to the G n R H neurons, thereby regulating G n R H release throughout the estrous cycle. Acknowledgements. Supported by NIH HD24697. ABBREVIATIONS a ab ap ce CSP cul cum DCT FLD FLM gc gr IAF LL LM lm
nucleus (n.) alpha n. ambiguus area postrema locus coeruleus tractus corticospinalis n. cuneatus lateralis n. cuneatus medialis decussatio corporis trapezoidei fasiculus longitudinalis dorsalis fasiculus longitudinalis medialis griseum centrale n. gracilis fibrae arcuatae internae lemniscus lateralis lemniscus medialis n. reticularis lateralis magnocellularis
277 lp old olm phi pbm PCM PCS pols rap rd rpa rob rpm rpo rtp SOL sol SPC SPCV STH tdo trl trm Vm Vmes VS Vs Vspc Xdm XII
n. reticularis lateralis parvocellularis n. olivaris accessorius dorsalis n. olivaris accessorius medialis n. parabrachialis lateralis n. parabrachialis medialis pedunculus cerebellaris medius pedunculus cerebellaris superior n. paraolivaris superior n. raphe pontis n. reticularis dorsalis medullae oblongata n. raphe pallidus n. raphe obscurus n. raphe paramedianus n. reticularis pontis oralis n. reticularis tegmenti pontis tractus solitarii n. tractus solitarii tractus spinocerebellaris tractus spinocerebellaris ventralis tractus spinothalamicus n. tegmentalis dorsalis n. trapezoides lateralis n. trapezoides medialis n. motorius nervi trigemini n. tractus mesencephali nervi trigemini tractus spinalis nervi trigemini n. sensibilis nervi trigemini n. caudalis tractus spinalis nervi trigemini n. dorsalis motorius nervi vagi n. nervi hypoglossi
REFERENCES 1 Barraclough, C.A., Wise, P.M. and Selmanoff, M.K., A role for hypothalamic catecholamines in the regulation of gonadotropin secretion, Recent Prog. Hormone Res., 40 (1984) 487-529. 2 Chambi, F., Fung, B. and Chikaraishi, D., 5' flanking DNA sequences direct cell-specific expression of rat tyrosine hydroxylase, J. Neurochem., 53 (1989) 1656-1659. 3 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 (1964) 1-55. 4 Day, T.A., Blessing, W. and Willoughby, J.O., Noradrenergic and dopaminergic projections to the medial preoptic area of the rat. A combined horseradish peroxidase/catecholamine fluorescence study, Brain Res., 193 (1980) 543-548. 5 Drouva, S.V., LaPlante, E. and Kordon, C., Alphavadrenergic receptor involvement in the LH surge in ovariectomized estrogen-primed rats, Eur. J. Pharmacol., 81 (1982) 341-344. 6 Freeman, M.E., The ovarian cycle of the rat. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1893-1928. 7 Grzanna, R. and Coyle, J.T., Rat adrenal dopamine beta hydroxylase: purification and immunologic characteristics, Z Neurochem., 27 (1976) 1091-1096. 8 Hartman, R.D. and Barraclough, C.A., Changes in tyrosine hydroxylase mRNA levels in medullary A1 and A2 neurons and locus coeruleus following castration and estrogen replacement in rats, Brain Res., 13 (1992) 231-238. 9 Heritage, A.S., Grant, L.D. and Stumpf, W.E., 3H-Estradiol in catecholamine neurons of rat brain stem: combined localization by autoradiography and formaldehyde-induced fluorescence, J. Comp. Neurol., 176 (1977) 607-630. 10 Heritage, A.S., Stumpf, W.E., Sar, M. and Grant, L.D., Brainstem catecholamine neurons are target sites for sex steroid hormones, Science, 207 (1980) 1377-1379. 11 Hoffman, G.E., Organization of LHRH cells: Differential apposition of neurotensin, substance P and catecholamine axons, Peptides, 6 (1985) 439-461.
12 Hoffman, G.E., Wray, S. and Goldstein, M., Relationship of catecholamines and LHRH: light microscopic study, Brain Res. Bull., 9 (1982) 417-430. 13 Honma, K. and Wuttke, 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. 14 Jennes, L., Beckman, W.C., Stumpf, W.E. and Grzanna, R., Anatomical relationships of serotoninergic and noradrenergic projections with the GnRH system in septum and hypothalamus, Exp. Brain Res., 46 (1982) 331-338. 15 Jennes, L., Jennes, M.E., Purvis, C. and Nees, M., C-fos expression in noradrenergic A2 neurons of the rat during the estrous cycle and after steroid hormone treatments, Brain Res., 586 (1992) 171-175. 16 Jennes, L., Stumpf, W.E. and Sheedy, M.E., UItrastructural characterization of gonadotropin-releasing hormone (GnRH)-producing neurons, J. Comp. Neurol., 232 (1985) 534-547. 17 Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. II. Dopaminergic, noradrenergic (A1 and A2) and adrenergic neurons, nerve fibers, and presumptive terminal processes, J. Comp. Neurol., 233 (1985) 308-332. 18 Kalra, S.P., Suppression of serum LH-RH and LH in rats by an inhibitor of norepinephrine synthesis, J. Reprod. Fert., 49 (1977) 371-373. 19 Kalra, P.S. and Kalra, S.P., Control of gonadotropin secretion. In H. Imura (Ed.), The Pituitary Gland, Raven, New York, 1985, pp. 189-220. 20 Kalra, P.S., Kalra, S.P., Krulich, L., Fawcett, C.P. and McCann, S.M., Involvement of norepinephrine in transmission" of the stimulatory influence of progesterone on gonadotropin release, Endocrinology, 90 (1972) 1168-1176. 21 Kalra, S.P. and McCann, S.P., Effects of drugs modifying catecholamine synthesis on plasma LH and ovulation in the rat, Neuroendocrinology, 15 (1974) 75-91. 22 L6r~nth, Cs., Segura, L.M.G., Palkovits, M., MacLusky, N.J., Shanabrough, M. and Naftolin, F., The LH-RH-containing neuronal network in the preoptic area of the rat: demonstration of LH-RH-containing nerve terminals in synaptic contact with LHRH neurons, Brain Res., 345 (1985) 332-336. 23 McKellar, S. and Loewy, A.D., Efferent projections of the A1 catecholamine cell group in the rat: an autoradiography study, Brain Res., 241 (1982) 11-29. 24 Miller, M.M. and Zhu, L., Immunocytochemical colocalization of dopamine beta-hydroxylase and luteinizing hormone releasing hormone neuronal elements by electron microscopy in the rostral forebrain of the female C57BL6J mouse, Soc. Neurosci. Abstr., (1992) 344.4. 25 Moore, R.Y. and Card, J.P., Noradrenaline-containing neuron systems. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Elsevier, Amsterdam, 1984, pp. 123-156. 26 Palkovits, M., Catecholamines in the hypothalamus: an anatomical review, Progress in Neuroendocrinology, 33 ( 1981) 123-128. 27 Palkovits, M., Zaborszky, L., Feminger, A., Mezey, E., Fekete, M.I.K., Herman, J.P., Kanyicska, B. and Szabo, D., Noradrenergic innervation of the rat hypothalamus: experimental, biochemical, and electron microscopic studies, Brain Res., 191 (1980) 161-171. 28 Ramirez, V.D., Feder, H.H. and Sawyer, C.H., The role of brain catecholamines in the regulation of LH secretion: a critical inquiry. In L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrinolgy, Vol. 8, Raven, New York, 1984, pp. 27-84. 29 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 diestrus day 1. Endocrinology, 108 (1981) 1795-1802. 30 Sat, M. and Stumpf, W.E., Distribution of androgen-concentrating neurons in rat brain, In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 120-133. 31 Shivers, B.D., Harlan, R.E., Morrell, J.I. and Pfaff, D.W., Ab-
278
32
33
34
35
36
sence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones, Nature, 304 (1983) 345-347. Silverman, A.J., Luteinizing hormone releasing hormone containing synapses in the diagonal band and preoptic area of the guinea pig, J. Comp. Neurol., 227 (1984) 452-458. Silverman, A.J., The gonadotropin-releasing hormone (GnRH) neuronal systems: immunocytochemistry. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1283-1304. Silverman, A.J. and Witkin, J.W., Synaptic interactions of luteinizing hormone-releasing hormone (LHRH) neurons in the guinea pig preoptic area, J. Histochem. Cytochem., 33 (1985) 69-72. Simpkins, J.W., Advis, J.P., Hodson, C.A. and Meites, J., Blockade of steroid-induced luteinizing hormone release by selective depletion of anterior hypothalamic norepinephrine activity, Endocrinology, 104 (1979) 506-509. Simmerly, R.B., Chang, C., Muramatsu, M. and Swanson, L.W.,
37
38
39
40
Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study, Z Comp. Neurol., 294 (1990) 76-95. Swanson, L.W. and Hartman, B.K., The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-/3-hydroxylase as a marker, J. Comp. Neurol., 163 (1975) 467-506. 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 and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 1235-1282. 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. Witkin, J.W. and Silverman, A.J., Synaptology of luteinizing hormone-releasing hormone neurons in rat preoptic area, Peptides, 6 (1985) 263-271.