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Endocrinology Vol. 140, No. 9 4335-4341
Copyright © 1999 by The Endocrine Society

ARTICLES

Estrogen Receptor Immunoreactivity Is Present in the Majority of Central Histaminergic Neurons: Evidence for a New Neuroendocrine Pathway Associated with Luteinizing Hormone-Releasing Hormone-Synthesizing Neurons in Rats and Humans1

Cs. Fekete, P. H. Strutton, F. R. A. Cagampang, E. Hrabovszky, I. Kalló, P. J. Shughrue, E. Dobó, E. Mihály, L. Baranyi, H. Okada, P. Panula, I. Merchenthaler, C. W. Coen and Zs. Liposits

Institute of Experimental Medicine (Cs.F., E.H., Zs.L.), Hungarian Academy of Sciences, Budapest, Hungary; Biomedical Sciences Division (P.H.S., F.R.A.C., C.W.C.), King’s College, London, United Kingdom; Department of Anatomy (I.K., E.D., E.M.), Albert Szent-Györgyi Medical University, Szeged, Hungary, The Women’s Health Research Institute (P.J.S., I.M.), Wyeth-Ayerst Research, Radnor, Pennsylvania; Department of Molecular Biology (H.O.), Nagoya City University School of Medicine, Nagoya, Japan; Department of Biology (P.P.), Abo Akademi University, Turku, Finland; Department of Membrane Biochemistry (L.B.), Walter Reed Army Institute for Research, Washington D.C.

Address all correspondence and requests for reprints to: Dr. Zsolt Liposits, Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Szigony u. 43, Hungary. E-mail: liposits{at}koki.hu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ER{alpha}-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge in ovariectomized estrogen-treated rats. These data are consistent with the hypothesis that the positive feedback effect of estrogen in the induction of the LH surge involves estrogen-receptive histamine-containing neurons in the tuberomammillary nucleus that relay the steroid signal to LHRH neurons via H1 receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
THE POSITIVE feedback effect of elevated plasma estradiol levels in proestrous animals initiates a surge of LH from the anterior pituitary gland, which is triggered by an increased discharge of LH-releasing hormone (LHRH) from nerve terminals in the median eminence into the hypophysial portal circulation (1). Although the LHRH secretion unequivocally depends on available estrogen levels, efforts to detect a significant uptake of estradiol (2) or estrogen receptor (ER) immunoreactivity (3, 4, 5) in LHRH neurons have been unsuccessful until very recently (see Note Added in Proof). Consequently, it has been assumed that the positive feedback effect of estrogen upon LHRH neurons is mediated by estrogen-sensitive interneurons. The neuronal circuits that relay information to LHRH neurons about the circulating levels of gonadal steroid hormones have been the subject of intensive investigation (1). Any candidate neurotransmitter system for mediating the feedback effects of estrogen on LHRH neurons must satisfy the criteria of (a) expressing ERs, (b) innervating LHRH neurons, and (c) exerting a regulatory influence upon LHRH neurons via specific neurotransmitter receptors.

In this report, we present data consistent with the hypothesis that the histaminergic neuronal system of the brain, the perikarya of which are confined to the tuberomammillary nuclear (TM) complex, provides an interneuron system capable of mediating the feedback effects of estrogen on LHRH neurons. This study was prompted by reports indicating (a) that administration of estrogen into the medium of perifused hypothalamic blocks stimulates the release of histamine (6), (b) that numerous histaminergic fibers project to the preoptico-septal area of the rat brain (7), the site at which most of the LHRH neurons are located in rats, (c) that histamine administered intracerebroventricularly stimulates ovulation in the rabbit (8), and (d) that an immortalized LHRH cell-line (GT1) expresses H1 receptors (9). The present studies demonstrate ER{alpha}-immunoreactivity in histamine-containing neurons, reveal the histaminergic pathway to LHRH neurons and provide in vivo pharmacological evidence concerning the histamine receptor subtype involved in regulating the LHRH surge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Tissue samples
Human brains. Diencephalic tissue samples were obtained from routine autopsies of five individuals (2 males and 3 females) whose clinical and pathological histories included neither neurological nor endocrine disturbances. The autopsy and tissue processing were carried out in accordance with the regulation and permission (No. 372) of the Ethics Board of the Albert Szent-Györgyi Medical University.

Rat brains. The animal experiments were performed on adult female Wistar rats that were ovariectomised bilaterally (day 0), treated with colchicine intracerebroventricularly (50 µg/100 g body wt.; day 14), and killed by transcardiac fixation (day 15) under Nembutal anesthesia (35 mg/kg). Each histological study detailed below comprised sections from 5 animals.

Immunocytochemical studies
Fixation
Human tissue.
The diencephalic blocks were fixed by immersion, first in buffered 4% 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDCDI; Sigma Chemical Co.) (8 days), then in 4% formaldehyde (4 days).

Rat tissue.
Following an initial flushing with 0.1 M PBS, the animals were perfused with 50 ml phosphate buffered 4% EDCDI. The brains were postfixed either in EDCDI (4 days) and then in 2% formaldehyde (1 day), or, for the purposes of estrogen receptor colocalization, in 4% formaldehyde (1 day).

Section preparation. Serial frozen sections were cut from the human and rat hypothalami at 30 µm and 20 µm thickness, respectively.

Immunocytochemical single-labeling. The detailed immunocytochemical protocol using the PAP technique has been published elsewhere (10).

Detection of histamine-containing neuronal elements.
Sections including the rat TM complex were incubated with a polyclonal antiserum raised against histamine (1:25,000) (11). Preabsorption of the primary antiserum with the histamine-ovalbumin conjugate that was used for immunization abolished all the immunoreactivity.

Localization of estrogen receptor-immunoreactivity.
ER-immunoreactive (IR) neurons of the TM were detected by three different polyclonal anti-ER{alpha} sera: AS409 rabbit antirat ER{alpha} (1:25,000) (12), 715 rabbit antirat ER{alpha} (1:1,000) (13) and ZS08–174 rabbit antihuman ER{alpha} (Zymed Laboratories, Inc., San Francisco, CA) (0.5 µg/ml). Nickel-3,3'-diaminobenzidine (Ni-DAB) was used as the chromogen in the peroxidase reaction; this was then silver-intensified (14). Preabsorption of ER{alpha} antibodies 715 and ZS08–174 with the corresponding synthetic ER peptides (1 µg/ml, overnight) resulted in loss of all immunoreactivity.

Immunocytochemical double-labeling.
For the simultaneous detection of two antigens, a previously reported double-labeling technique was used (15). This utilizes the color difference between the DAB (brown) and silver-intensified Ni-DAB (black) reaction products.

Simultaneous detection of histamine-containing axons and LHRH neurons in human and rat hypothalami.
At first, histamine immunoreactivity was detected by means of the PAP method with the silver-intensified Ni-DAB chromogen. Following incubation in monoclonal antibodies generated against LHRH (1:1,000) (16), the LHRH-IR neurons were visualized with the DAB reaction product. Some of the double-labeled sections from rats were embedded in Epon-resin for preparation of semithin sections.

Colocalization of ER and histamine in the tuberomammillary nucleus of the rat.
The immunostained ER{alpha}-IR nuclei were identified by the black silver-intensified Ni-DAB chromogen, whereas the histamine-IR perikarya were detected with the brown DAB alone. In addition to mapping the distribution of ER- and histamine-immunoreactive neurons in the subnuclei (E1–E5) of the TM complex (17), the ratio of signal coexpression was also assessed by counting single- and double-labeled histamine-IR neurons. This analysis included every sixth section from serial samples taken through the posterior hypothalamus of three rats (16 sections from each animal). The data are presented as the mean ± SE (SEM).

Effects of H1 and H2 receptor antagonists on the LH surge: in vivo studies
Animals. Adult female Wistar rats (250–320 g) were maintained under controlled conditions (lights on from 0600 to 1800 h, dim red light from 1800 to 0600 h; temperature 21 ± 1 C). Food and water were available ad libitum. All animals were bilaterally ovariectomized; 7 days later an icv cannula (C313G; Plastics One, Roanoke, VA) was implanted into the lateral cerebral ventricle. After a further 3–4 days an iv cannula was implanted into the right atrium of the heart via the external jugular vein. This cannula was directed sc and passed into a cranial attachment, which allowed for the Luer lock fitting of a protective flexible metal coil (Instech Laboratories, Plymouth Meeting, PA). On the following day, each animal was given a sc injection of oestradiol benzoate (50 µg/0.2 ml arachis oil) at 1200 h (day 1 of the experiment). These experiments were undertaken in accordance with the UK Animals (Scientific Procedures) Act, 1986, and associated guidelines.

Experimental protocol. At 1000 h on the day of sampling (day 4 of the experiment), an icv injection cannula (C313I; Plastics One) was attached to the central channel of a dual channel swivel (Instech Laboratories); this cannula was filled with the drug or the vehicle and inserted into the icv guide cannula. The iv cannula was attached to the second channel of the swivel. Blood sampling commenced 3 h later at 1300 h; an automated sampling system was used to withdraw two 25-µl blood samples within a period of 5 min every 30 min for 12 h (from 1300 to 0100 h). The samples were stored at -20 C before RIA for LH. Pyrilamine maleate (mepyramine; Research Biochemicals International, Natick, MA) or ranitidine (RBI) was dissolved in 0.9% sterile saline at 100 nmol/30 µl. After an initial sampling period of 1 h, mepyramine or ranitidine or vehicle was infused icv at a rate of 0.5 µl/min for 6 h using a 250 µl gas tight microsyringe driven by a syringe pump.

RIA and statistical analysis. The whole blood LH concentrations were measured in a single RIA as described previously (18). Within group comparisons were made using one-way repeated measures ANOVA followed by the Tukey multiple comparison test; between group comparisons were made using the unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Colocalization of estrogen receptor- and histamine-immunoreactivity in the tuberomammillary nucleus
Histamine-immunoreactive (IR) neurons appeared in all of the five subgroups (E1–E5) of the tuberomammillary complex (Figs. 1bGo and 2Go, a–d), corroborating the results of previous immunocytochemical studies (11, 19). The largest population of these neurons was found in the E2 subnucleus. Most histamine-IR neurons were multipolar; however, scattered, fusiform neurons were also observed. Neurons exhibiting ER{alpha}-IR nuclei were identified in all subgroups of the tuberomammillary complex, and they also occurred in other regions of the caudal hypothalamus, including the ventromedial, dorsomedial, arcuate, ventral premammillary, and lateral mammillary nuclei (Figs. 1aGo and 2Go, a–d). Immunostaining with three different ER{alpha} antibodies revealed a comparable distribution of ER{alpha}-IR nuclei. Using an immunocytochemical double-labeling method, we found that nuclear ER{alpha} immunoreactivity was present within the majority of histamine-IR perikarya (Figs. 1Go, c–d, and 2, a–d). In the double-labeled neurons, the cytoplasmic expression of histamine was clearly segregated from the ER{alpha}-immunoreactivity of the cell nuclei (Fig. 1Go, c and d). Analysis of the double-immunostained sections indicated ER{alpha}-immunoreactivity in 66–81% of the histamine-synthesizing neurons in the different subgroups of the tuberomammillary complex (Fig. 2eGo); the mean percentage of histaminergic neurons that were immunoreactive for ER{alpha} was 76 ± 3.2 (SEM).



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  		Figure 1. Localization of ER{alpha}-IR and histamine-IR neurons in the TM of the rat. a, Neurons of the E2-subnucleus possessing strong nuclear labeling (arrows) for ERs. b, Histamine-IR neurons clustered in the E2-subnucleus of the TM. c, Black ER{alpha}-IR nuclei located within brown histamine-IR neurons. d, High power picture of a histamine-IR neuron displaying an ER{alpha}-IR nucleus (arrow). Scale bar: a–b, 150 µm; c, 75 µm; d, 25 µm.

 


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  		Figure 2. Distribution of ER{alpha}-IR and histamine-IR neurons in the TM of the rat diencephalon. a–d, Schematic representation of coronal sections from the TM complex indicating the location of the five major histaminergic subnuclei (E1–E5). The left half of each figure depicts the distribution of ER{alpha}-IR neurons; the right half shows the pattern of colocalization for ER{alpha} and histamine. e, Bar diagram indicating the percentage of histamine-IR cells that coexpress ER{alpha} within the different subdivisions (E1–E5) of the TM. •, ER{alpha}-positive cells; {blacktriangleup}, 1 ER{alpha}- + histamine-IR neuron; {blacksquare}, 10 ER{alpha}- + histamine-IR neurons; {triangleup}; 1 ER{alpha}-negative, histamine-positive neuron; {square}; 5 ER{alpha}-negative, histamine-positive neurons. Arc, Arcuate nucleus; DM, dorsomedial nucleus; E1–E5, subnuclei of the tuberomammillary nucleus; MM, medial mammillary nucleus; MR, mammillary recess; PMD, dorsal premammillary nucleus; PMV, ventral premammillary nucleus; 3V: 3rd ventricle.

 
Histaminergic innervation of LHRH neurons in the rat
In accordance with previous reports (7), a dense plexus of histamine-IR fibers was detected in the bed nucleus of stria terminalis, in the vicinity of the organum vasculosum of the lamina terminalis (OVLT), and along the vertical and horizontal limbs of the diagonal band of Broca. Our immunocytochemical double-labeling studies of the preoptic region revealed an intimate relationship between histamine-IR axons and LHRH-IR neurons (Fig. 3Go, a–b). Histaminergic axons approached LHRH neurons and exhibited axo-somatic (Fig. 3aGo) and axo-dendritic (Fig. 3bGo) appositions; 40 ± 2.3% of the LHRH neurons were apposed by histamine-IR axons.



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  		Figure 3. Juxtapositions between the central histamine- and LHRH-immunoreactive (IR) systems of the rat (a, b) and human (c, d). a, Black histaminergic bouton (arrow) juxtaposed to a brown, LHRH-IR perikaryon (arrowheads) in the preoptic area; the inset shows a similar axo-somatic apposition (arrow) at higher power in a 1 µm thick specimen. b, Histamine-IR fiber (arrowheads) apposed (arrow) to the dendritic process of a fusiform LHRH neuron in the preoptic region. c, Histamine-IR axon forming multiple en passant-type appositions (arrows) with a multipolar LHRH cell (asterisk) located in the preoptic area of the human brain. d, A histamine-IR axon (arrowheads) making an axo-somatic apposition (arrow) with a fusiform LHRH neuron located in the human infundibular nucleus. Scale bar: a–d, 20 µm; inset, 10 µm.

 
Histaminergic innervation of LHRH neurons in the human
Immunocytochemical double-labeling techniques applied to human hypothalamic sections revealed LHRH-IR neurons embedded in a rich network of varicose histamine-IR axons in both the preoptic and the infundibular regions. Histamine-IR fibers were found to approach LHRH neurons and, in many instances, to be juxtaposed to their perikarya and dendrites (Fig. 3Go, c–d). Histaminergic axons winding around LHRH cells and exhibiting serial appositions (Fig. 3dGo) were also apparent. At least one juxtaposition with histamine-IR fibers was observed in association with 51 ± 3.0% of the LHRH neurons.

In vivo effects of H1- and H2-histaminergic receptor antagonists on the LH surge in rats
To elucidate the involvement of H1- and H2-histaminergic receptors in the regulation of the estrogen-induced LH surge in vivo, whole blood LH concentrations were monitored in ovariectomised estrogen-treated rats during intracerebroventricular (icv) infusion of an H1 or H2 receptor antagonist or the vehicle between 1400 and 2000 h. A significant rise in LH concentrations was observed in the animals (P < 0.05; Fig. 4Go, a–b) that received the vehicle. Infusion of the H1 antagonist, mepyramine, (100 nmol/h) prevented the occurrence of the estrogen-induced surge (Fig. 4aGo). In contrast, the surge remained unaffected (Fig. 4bGo) in the presence of the H2 antagonist, ranitidine (100 nmol/h). The treatments with mepyramine or ranitidine were not associated with any apparent changes in the behavior of the animals.



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  		Figure 4. Mean (±SEM) whole blood LH concentrations in ovariectomized rats at times indicated on day 4 following sc treatment with 50 µg estradiol benzoate at 1200 h on day 1. Animals were given an intracerebroventricular infusion between 14.00 and 20.00h of (a) the H1 receptor antagonist mepyramine (100 nmol/30 µl/h) or (b) the H2 receptor antagonist ranitidine (100 nmol/30 µl/h) or the vehicle (30 µl/h) in concurrently treated control groups. *, P < 0.05 with respect to the level at 1300 h within the same group. {dagger}, P < 0.05 with respect to the concurrent level in the vehicle-treated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
The results of the present studies are consistent with the hypothesis that one of the routes by which estrogen influences LHRH neurons involves the central histaminergic system and that this action, in the context of the LH surge, is restricted to H1 receptors. It has been previously demonstrated that the release of histamine in vitro from the perifused mediobasal hypothalamus can be stimulated by estradiol (6). The possibility that this steroid has direct actions on histamine-containing neurons is indicated by the present discovery of ER{alpha}-immunoreactivity in 76% of these cells. The pioneering work of Pfaff and Keiner (20) demonstrated estradiol uptake in the lateral mammillary region with a distribution that is comparable to the immunocytochemical map of ER{alpha}-IR cells in the E2 and E3 subgroups of the TM presented here. The estrogen receptor antisera used in our work have been widely used for the visualization of the classical estrogen receptor ER{alpha}. Recently, a novel type of estrogen receptor, ERß has been cloned (21); the messenger RNA (mRNA) for this receptor has been detected in various regions of the rat brain including the TM (22, 23). Consequently, the role of ERß in mediating estrogenic effects within the TM merits attention in further studies. Whether ERß is present in the histaminergic neurons remains to be determined.

Histamine was first implicated in the regulation of gonadotropin secretion with the discovery that it was capable of inducing ovulation when injected intracerebroventricularly into pentobarbital anaesthetised rabbits (8). It was subsequently shown that this amine stimulates LHRH and LH secretion from an in vitro preparation containing the medial basal hypothalamus and pituitary of female rats (24); this stimulatory effect can also be achieved using an H1 but not an H2 agonist and can be blocked by an H1 antagonist (24). In contrast, in vitro studies on tissues taken from male rats have reported that histamine is without effect not only on LH release when the pituitary is perifused alone (24) but also on LHRH release from the mediobasal hypothalamus (25). A permissive role for estrogen in the stimulatory action of histamine on LH is suggested by the discovery that the central administration of this amine stimulates LH release in rats on the day of proestrus; no such effect was observed on other days of the estrous cycle or in male rats (26). Other studies have shown that intracerebroventricular histamine stimulates LH release in ovariectomized rats treated with a relatively high dose of estrogen and progesterone (27, 28) but not in orchidectomized rats following the same steroid treatment (28); only a weak stimulatory effect has been observed in the presence of a lower dose of estrogen (29). Our present observation of histaminergic fibers apposed to the perikarya and dendrites of LHRH neurons in both the rat and the human suggests that the effects of histamine on LH secretion may include direct actions on the LHRH neurons. This does not, however, exclude additional sites of interaction; an axo-axonic-type regulation might also occur at the level of the median eminence where scattered histaminergic fibers are found (30).

It should be noted that the method of postfixation used in this study was developed in our laboratory to optimize the detection of histamine-IR axons while retaining immunoreactivity for the other products examined. By using this procedure, we were able to demonstrate for the first time the relationship between histaminergic axons and an immunocytochemically characterized population of neurons (i.e. LHRH neurons in the rat and human brain). The requirements of our double-label immunohistochemistry were satisfied by postfixing the tissues in EDCDI over 4 or 8 days (for rat and human tissue, respectively) before the paraformaldehyde treatment; because this procedure provided poor membrane preservation, it was not appropriate to investigate the material at the electron microscopic level. Alternative methods will be required to establish whether the appositions identified in this study involve synaptic specializations or, alternatively, whether locally released histamine can affect the LHRH neurons via extrasynaptically located receptors.

The in vivo pharmacological data presented here demonstrate that central treatment with an antagonist against H1 but not H2 receptors blocks the estrogen-induced LH surge in rats. This study was designed to assess the involvement of these receptors in the spontaneous surge while minimizing the nonspecific disturbances that can affect its timing, amplitude, and occurrence. The drug- and vehicle-treated groups were sampled concurrently and received the intracerebroventricular infusion via a syringe pump located outside the cage; furthermore, the use of an automated blood sampling system permitted the frequent withdrawal of small blood samples (25 µl) with minimal stress to the animals. The discovery that the LH surge can be suppressed by mepyramine suggests that the histaminergic fibers that exhibit multiple appositions onto LHRH neurons may exert their effects via H1 receptors. This notion is supported by recent evidence (9) showing that H1 receptors are expressed in GT-1 cells, a cell line derived from LHRH-producing neurons (31). Furthermore, it has been found that the stimulation by estrogen of LHRH release from the hypothalamus in vitro can be blocked by an H1 but not an H2 antagonist (24).

The positive feedback actions of estrogen upon LHRH neurons are likely to operate via more than a single estrogen-sensitive neuronal system. Considerable evidence indicates that estrogen has potent regulatory effects on GABA transmission in the medial preoptic area and that changes in GABA-ergic tone in this region contribute to the induction of the LH surge (32, 33, 34). Within the context of the present study the evidence that all histaminergic neurons also contain GABA (35) may be highly significant; nevertheless, the region of the preoptic area in which the LHRH cells are located is also densely populated with GABA-ergic neurons (34). Additional neurotransmitter systems that have been implicated in the positive feedback action of estrogen include the central noradrenergic and adrenergic systems (36, 37, 38, 39). Other systems that might mediate the effects of estrogen on LHRH neurons include those employing neuropeptide-Y and substance P; both have been shown to innervate LHRH neurons and to express estrogen receptors (40, 41, 42, 43). In contrast to the various neuronal systems that are already recognized as potential sites for the action of estrogen in the context of LHRH regulation, the histaminergic neurons are not only concentrated in a particularly circumscribed part of the brain but also show a very high incidence (76%) of ER{alpha}-immunoreactivity.

Our understanding of the mechanisms underlying the positive feedback actions of estrogen in the human brain is limited. As in the case of several other species, morphological data indicate that human LHRH neurons do not express estrogen receptors (44). Among the neurotransmitters/modulators that might regulate human LHRH neurons via afferent connections neuropeptide Y (45), catecholamines (46), and substance P (47) have been implicated by double-label immunocytochemistry. The present study has revealed that histamine-IR fibres form close appositions with human LHRH neurons. Our current understanding of the role of histamine in the regulation of LH release in humans is restricted to a series of studies that predominantly involved H2 antagonists administered peripherally (48, 49, 50, 51, 52, 53, 54, 55); no H2 receptor-specific effects on circulating levels of LH have been demonstrated. In contrast, the reported effects of H1 antagonists include the suppression of LH in women and its elevation in men (50); paradoxically, comparable sex-dependent effects were achieved with peripherally administered histamine (50). Nevertheless, the H1 antagonist employed in another study (49) was without effect on LH levels in either sex. It should be noted that research designed to assess histamine involvement in the regulation of either the LH surge or LH pulses in humans remains to be undertaken.

In summary, the morphological and functional data presented here demonstrate that (a) the majority of histamine-IR neurons within the tuberomammillary nuclear complex exhibit ER{alpha} immunoreactivity in their cell nucleus, (b) histamine-IR neurons of the TM exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both rats and humans; and (c) intracerebroventricular administration of the H1 receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge induced by estrogen in ovariectomized rats. These data indicate that the positive feedback effect of estradiol on the preovulatory LH surge may involve estrogen-receptive histamine-containing neurons within the TM that relay their steroid-influenced signal to LHRH neurons via H1 receptors.


   Note Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
During the editorial processing of this paper, a report was published showing that 17% of the LHRH neurons are immunoreactive for ER-{alpha} in the rat. (Butler J, Sjöberg M, Coen CW 1999 Evidence for estrogen receptor {alpha} immunoreactivity in gonadotropin-releasing hormone expressing neurons. J Neuroendocrinol 11:331–335).


   Acknowledgments
 
The authors would like to express their appreciation to Dr. S. Hayashi for the generous gift of the ER antibody (AS409), and to Dr. H. F. Urbanski for the kind donation of the monoclonal LHRH antibodies. We also thank A. Kobolák for her valuable technical assistance.


   Footnotes
 
1 This study was supported by grants from National Science Foundation of Hungary (OTKA T0–16354 and F-22711), the BBSRC, the Wellcome Trust, the Royal Society, and NATO. Back

Received January 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

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