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Department of Pharmacology and Experimental Therapeutics (B.D.), Loyola University Chicago, Strich School of Medicine, Maywood, Illinois 60153, and Women’s Health Research Institute (I.M.), Wyeth-Ayerst Research, Collegeville, Pennsylvania 19426
Abstract
Catecholamines have been shown to modulate gonadal functions via interactions with hypothalamic LH-releasing hormone (LHRH)-synthesizing neurons. To reveal the morphological background of this phenomenon, the distribution of LHRH neurons and tyrosine hydroxylase (TH)-immunoreactive (IR), catecholaminergic structures were mapped in the human diencephalon. First, the location of LHRH and TH-IR neuronal elements was analyzed, and then the relationship between the two different systems was examined. The LHRH-IR cell bodies were mainly present in the medial preoptic and infundibular areas. The TH-IR perikarya were located in the periventricular, paraventricular, and supraoptic hypothalamic nuclei and also in the median eminence. The TH-IR fibers were numerous in septal, infundibular, periventricular, and lateral hypothalamic regions. The brown, diaminobenzidine-labeled LHRH-containing perikarya were found to receive black, silver-intensified, TH-positive axon terminals in the infundibular and medial preoptic areas. However, in the preoptic and caudal parts of the diencephalon, only a few juxtapositions were noted. The present results indicate that hormone released from diencephalic LHRH-IR neurons in humans may be influenced by the central catecholaminergic system via direct synaptic mechanisms.
IT IS WELL established that the catecholaminergic system plays a pivotal role in the regulation of gonadal functions in numerous mammalian species, including human. Acute and chronic stress as well as autonomic failure cause selective defect in the gonadal physiology (1, 2, 3, 4). Central catecholaminergic deficiency is present with altered LH release in polycystic ovarian syndrome (5, 6, 7). Moreover, catecholamines are known to be involved in the preovulatory hormonal changes (8, 9) and are crucial for the initiation of ovulation (10, 11, 12).
The mechanism(s) involved in this regulation are not entirely
understood, but several studies suggest that it is based on the control
of gonadotropin secretion by the hypothalamo-hypophyseal axis.
Catecholamines have been implicated in the modulation of LH release,
but reports of their effects have been contradictory. For example,
norepinephrine (NE) actively inhibits LH secretion and pulse frequency
in rat and ewe (13, 14), and in isolated pig pituitary
cells (15). In human, baboon, and rat, chronic stress also
inhibits the amount of circulating LH (1, 2, 3). Dopamine and
epinephrine administration presented similar blocking effect in human,
ewe, and pig pituitary cell culture (13, 15). In contrast
to these findings, an increase in LH release is evoked by NE in rat,
possibly through the 
-1 and -2 adrenergic receptors (8, 11, 16, 17, 18, 19, 20, 21). Le et al. (22) reported that
-1, but not -2, antagonists blocked LH secretion and reduced Fos
expression in LH-releasing hormone (LHRH) neurons at the time of LH
surge in the rat. Administration of an -adrenergic antagonist blocks
LH secretion in ovariectomized rhesus monkeys (23),
and electric stimulation of the locus ceruleus and medullary
NE-containing cell groups increases LH release in rat
(24). Moreover, the stimulatory and inhibitory effects of
NE and dopamine appear to be changed during sexual maturation, with
inhibitory effects predominating in the prepubertal rat (25, 26).
The presence of tyrosine hydroxylase (TH)-immunoreactive (IR) elements in the pituitary gland of several species (27, 28) raises the possibility of direct catecholaminergic control of LH release; however, the transmitter-synthesizing role of TH in the pituitary is not completely known. Although not directly demonstrated, there is a general consensus that catecholamines influence LH secretion through the hypothalamic LHRH system (13, 17, 22, 23, 29, 30). This could occur by a direct morphological connection between catecholaminergic and LHRH-IR elements. Some studies found that these systems not only colocalize in certain brain areas (31), but their perikarya comigrate during embryonic development (32). Earlier studies also revealed a close opposition between the LHRH cell bodies and catecholaminergic structures in rat (33) and mice (34) and suggested that most of the noradrenergic terminals in the region of the LHRH perikarya in the rat medial septum/rostral preoptic area originate from ipsilateral neurons in areas A1 and A2 (35). In contrast to these findings, dopaminergic neurotoxins administered in the rat preoptic area caused degeneration of the catecholamine terminals on LHRH neurons, suggesting that the terminals do arise locally and are dopaminergic (36). Although the morphology and distribution of LHRH-IR (37, 38, 39, 40, 41, 42) and catecholaminergic elements (43, 44, 45, 46) have been mapped in human, the morphological relationship between these systems has not been studied. Juxtapositions of these systems in particular areas would provide for the possibility of direct catecholamine-LHRH interaction and could be the morphological basis for catecholaminergic control on LH release in man.
In this study, antisera against both TH, the rate-limiting enzyme of catecholaminergic biosynthesis, and LHRH were used to visualize and systematically map TH-IR and LHRH-IR neuronal structures in the human hypothalamus. To reveal the location of putative interactions, the maps of these systems were superimposed. Finally, putative synapses between catecholaminergic and LHRH immunoreactive elements were identified and quantitated in the overlapping areas using double-label immunohistochemistry. Because the human tissue used in our studies had 24–48 h post mortem time, the justification of the synaptic interactions between the TH-IR and LHRH-IR elements using electron microscopy is severely limited. Thus, studying the juxtapositions, we decided to use confocal microscopy, which provides reliable results on tissues with longer post mortem time as well.
Materials and Methods
Brain samples
Brain samples of four adult women and three adult men (20–78 yr of age) were obtained from autopsies at 24–48 h post mortem. Review of medical records indicated specimens were obtained from individuals with no known neurological or neuroendocrinological disease. The brain samples were taken in accordance with the regulation and permission of the Ethics Board of the Szent-Györgyi Albert Medical University.
Fixation and section preparation
Hypothalami were dissected from the brains and fixed by
immersion in 0.1 M phosphate-buffered (PB; pH 7.4) 4%
formaldehyde at 4 C for 2–24 weeks. Each block contained half of the
hypothalamus split along the midsagittal line. The samples were
cryoprotected in 30% sucrose in phosphate buffer containing 0.9%
sodium chloride (PBS) and 0.1% sodium azide, and then frozen on dry
ice. Using a Reichert freezing microtome, 30-µm sections were
collected in the coronal plane. The sections (
450–500 from each
block) were collected in four series of wells into PBS containing 0.2%
sodium azide and stored at 4 C until processing. Adjacent sections were
processed for 1) Nissl staining using cresyl violet, 2) detection of
LHRH-IR, 3) detection of TH-IR, and 4) double-label
immunohistochemistry for TH and LHRH.
Immunohistochemistry
Single labeling was carried out using either peroxidase-antiperoxidase or streptavidin-biotin methods combined with silver intensification as described by Gallyas and co-workers (47). The samples were pretreated sequentially with 10% thioglycolic acid for 30 min to suppress the endogenous tissue argentophilia, 0.2% Triton X-100 for 20 min, and 10% normal horse serum in PBS for 1 h at room temperature to block nonspecific staining. Thereafter, the sections were incubated in a primary antisera solution composed of 10% normal horse serum, PBS, and 0.1% sodium azide for 24 h. The LHRH-containing structures were identified using a monoclonal mouse anti-LHRH antiserum (Chemicon, Temecula, CA) at a dilution of 1:20,000. The TH-IR structures were visualized by rabbit anti-TH serum (gift from Prof. Nagatsu, Fujita Health University, Toyoake Aichi, Japan; dilution, 1:50,000). Then, the sections were incubated for 2 h in a secondary antiserum containing unlabeled goat antirabbit IgG (Jackson ImmunoResearch, West Grove, PA; dilution, 1:50) for TH, or biotin-labeled horse antimouse IgG (Vector Laboratories, Inc., Burlingame, CA; dilution, 1:500) to detect LHRH. In each case, the cross-reactions of the secondary antibodies with the endogenous human IgGs were eliminated by the addition of 2% normal human serum to these reagents. Finally, the sections were incubated for 1 h in rabbit peroxidase-antiperoxidase (Jackson ImmunoResearch; dilution, 1:2,000) for the detection of TH, or peroxidase-labeled streptavidin (Jackson ImmunoResearch; dilution, 1:500) for the detection of LHRH. The chromogen solution was composed of 0.05% diaminobenzidine (DAB), 0.125% nickel-ammonium-sulfate, and 0.005% hydrogen peroxide in 0.1 M Tris-HCl (pH 7.6). The resulting DAB polymer was silver-intensified in a solution containing 0.1% silver nitrate, 0.1% ammonium nitrate, 1% silicotungstic acid, and 0.2% formaldehyde for 3–4 min (47).
Simultaneous detection of TH-IR and LHRH-IR structures was performed using double-label immunohistochemistry. First, TH immunohistochemistry was carried out, and then the LHRH-containing neuronal structures were immunolabeled as described above using DAB chromogen without nickel ions (48). The anti-LHRH serum was used in 1:8000 dilution.
For fluorescent immunohistochemistry, tetramethylrhodamine isothiocyanate was used to label LHRH-IR elements, and fluorescein isothiocyanate (FITC) was used to detect TH-IR fibers. Fluorescent secondary antibodies were purchased from DAKO Corp. (Carpinteria, CA). The anti-LHRH serum was used in 1:8000 dilution.
In control sections, the primary antibodies were omitted or replaced by nonimmune rabbit serum at the dilution of 1:50,000 (TH) or 1:20,000 (LHRH), respectively. In all cases, immunoreaction was not observed.
Computer-assisted mapping
The hypothalamic sections were systematically scanned with the
aid of a plain-scanner (Hewlett-Packard Co., Palo Alto,
CA). The outline of the sections was traced using the CorelTrace
software 4.0 (Corel Corp., Ottawa, Ontario, Canada). Computer-generated
superimposition of eight, consecutive sections was summarized in each
of the slides shown in Fig. 1
, A–P. The neurons and fibers have been marked on
these figures using a Carl Zeiss (Thornwood, NY) Axiophot
microscope with camera lucida and Adobe (San Jose, CA) Photoshop
software, version 3.0.
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The fluorescent double-labeled sections were examined with a computerized confocal laser microscope (LSM 410 Invert, Carl Zeiss) with an Ar-Kr laser 488/568/647 using the x63/1.4 oil Plan-Apochromat objective. Simultaneous dual excitation was applied at 488/568 nm with FITC and rhodamine filters.
Terminology
The terminology of the diencephalic structures was adapted from Braak and Braak (49), Saper (50), and Silverman et al. (41).
Results
LHRH system
The distribution of the LHRH-IR neuronal structures was similar to our previous immunohistochemical study [Dudas et al. (42)]. LHRH-IR cell bodies formed a loose network throughout the hypothalamus and were located mainly in the medial preoptic, infundibular areas and median eminence. In addition, a few cells were found along the diagonal band of Broca. The perikarya were concentrated in the ventral part of the periventricular zone; few immunoreactive cells were present in the paraventricular and supraoptic nuclei. The majority of the neurons were fusiform, although a few multipolar cells (less than 5% of the total population) were found in the medial preoptic area. Immunoreactive fibers were located in the diagonal band of Broca and in the infundibular and periventricular areas. No sex- and age-related differences were observed in the number and/or pattern of IR perikarya and processes, and the different post mortem periods did not influence the number of IR structures.
Catecholaminergic system
In contrast to the LHRH-IR elements, the TH-IR perikarya were
often arranged into clusters in the diencephalon. The majority of the
cell bodies were located in the ventral part of the preoptic and
tuberal areas, whereas few were present in the posterior hypothalamus.
The septal area contained a small number of immunoreactive cells that
were concentrated on the medial-dorsal region (Fig. 1
, A and B), and no
perikarya were observed along the diagonal band of Broca and the lamina
terminalis (Fig. 1, B–D). In the preoptic region, the TH-IR neurons
were arranged periventricularly with few cells located at the bottom of
the optic recess sitting on the dorsal surface of the optic chiasm
(Fig. 1, D–F). The TH-IR cell bodies were densely packed in the
paraventricular and supraoptic nuclei (Fig. 1, G–I). In the tuberal
region, a large number of cells were present in the median eminence and
in the periventricular area (Fig. 1, K and L), whereas the infundibular
zone contained relatively small numbers of TH-IR perikarya (Fig. 1, I
and J). Dorsal to the median eminence, a cluster of cell bodies were
located between the fornix and the optic tract and in the dorsal part
of the diencephalon, close to the midline (Fig. 1, K and L). In the
dorsal hypothalamus, the cells were arranged periventricularly, with
few labeled neurons found around the mamillary body (Fig. 1, M–P).
Morphologically, the TH-IR neurons were mainly fusiform, with thin cell
bodies and two processes emanating from their opposite poles. Smaller
numbers of multipolar cells were also detected mainly in the
paraventricular and supraoptic nuclei.
TH-IR fibers were seen as a loose network in the periventricular and infundibular areas. Labeled axon varicosities were observed in the lateral hypothalamus and also in the septal area. Interestingly, the TH-IR processes formed well defined terminal fields in the paraventricular and supraoptic nuclei. Cresyl violet staining revealed that these fiber baskets contained large multipolar cells that were obviously not immunoreactive for either TH or LHRH.
Associations between TH-IR and LHRH-IR neurons
Superimposition of the maps of LHRH-IR and TH-IR elements revealed
regions of overlap in the medial preoptic area and median eminence.
Partial overlap was detected in the periventricular zone of the tuberal
part of the hypothalamus and in the paraventricular and supraoptic
nuclei. High magnification examination of the overlapping areas
revealed black TH-IR fibers abutted on brown LHRH-IR neurons in the
medial preoptic and infundibular areas and in the median eminence (Fig. 2). Here the TH-IR varicose fibers
targeted mainly fusiform LHRH-IR neurons and formed intimate contact
with the cell bodies and the stem dendrites. These juxtapositions
between the TH and LHRH systems were not observed in the
paraventricular and supraoptic nuclei or in the caudal part of the
periventricular area. All hypothalami showed a similar distribution of
LHRH-TH contacts irrespective of the sex or age.
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As described in our previous study [Dudas et al. (42)], all hypothalami contained contacting LHRH-IR elements in the infundibular and periventricular areas (data not shown). In the majority of these cases, LHRH-IR fibers with many varicosities passed along the LHRH-IR neurons forming multiple juxtapositions. In contrast to these findings, LHRH-IR axon varicosities juxtaposing with TH-IR fibers or perikarya were not found in the human diencephalon.
Examination of the juxtapositions with oil-immersion revealed no
synaptic cleft between the associating TH-LHRH neural elements. Also,
these interneuronal connections proved to be close axosomatic and
axodendritic contacts when we investigated the double-labeled
fluorescent preparations with confocal microscopy (Fig. 3).
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Experimental evidence shows that catecholamines can influence gonadotropin secretion either by a direct effect on LH release (15) or by modulating hypothalamic LHRH production (13, 17, 22, 23, 29, 30). The mechanism of this phenomenon is not known, however it was hypothesized that catecholamines influence gonadal function via the control of LHRH secretion (13, 17, 22, 23, 29, 30). In our studies, we found morphological evidence of direct association between catecholaminergic and LHRH-IR neural elements in human, which may be the basis of catecholaminergic control of LHRH release. To reveal the location of these juxtapositions, we also mapped the catecholaminergic and LHRH-IR structures in the human diencephalon.
The location and morphology of LHRH-IR elements agreed with our previous findings (42). The cell bodies were mainly located along the diagonal band of Broca, in the medial preoptic and infundibular areas, and in the median eminence, suggesting that the catecholaminergic control of LHRH release possibly takes place in these regions.
The distribution of TH-IR elements also corresponded to previous findings (43, 44, 45, 46); however, the present map of the TH-IR system provides better resolution in the entire hypothalamus and septal area. Immunostaining of the TH-IR elements revealed that the most dense cell populations were in the supraoptic and paraventricular nuclei, and numerous cells were packed together in the periventricular region and in the median eminence. These cell bodies may be the source of the loose network of fibers running periventricularly or at the base of the hypothalamus toward the infundibulum. Examination of the areas populated by TH-IR fibers revealed definite terminal fields formed by ramifying axon varicosities in the paraventricular and supraoptic nuclei. Although these fiber baskets did not contain LHRH-IR or TH-IR perikarya, cresyl violet staining revealed that the varicosities abut around large multipolar neurons. These findings suggest that the catecholaminergic system may influence other diencephalic functions as well.
Because the most intensive overlap between the TH and LHRH systems was in the medial preoptic and infundibular areas and in the median eminence, we expected the most numerous associations in these regions. Interestingly, the infundibular nucleus contained only small numbers of TH-IR perikarya but a dense fiber network, whereas in the other two regions, TH cell bodies dominated. Earlier studies reported that the cell bodies projecting to the medial preoptic area are located in areas A1 and A2 (35) or the projecting fibers do arise locally (36) in the preoptic area. In our studies, the distribution of TH-IR perikarya and fibers suggests that TH-IR axon varicosities juxtaposing LHRH-IR perikarya in the medial preoptic area and in the median eminence may originate locally, whereas LHRH-IR cell bodies in the infundibular region may receive TH-IR fibers from distant hypothalamic or extrahypothalamic regions. The difference between the pattern of the TH-IR structures in these areas suggests that LHRH cell subpopulations may receive catecholaminergic innervation from different sources.
Previous findings of Hoffman (33) showed that the majority of the juxtapositions between LHRH-IR perikarya and TH-IR fibers were located in the medial preoptic area in rat. In contrast to these data, 78% of these LHRH-TH-IR associations were present in the infundibular region and median eminence in our studies. These findings indicate that there may be significant difference between the pattern of catecholaminergic innervation of LHRH system in different species. Because the distribution of the juxtapositions between the LHRH-IR perikarya and neuropeptide Y system is similar to that of the LHRH and TH-IR elements (42), there is a possibility that the catecholaminergic terminals interacting with LHRH neurons might arise from the same neurons that provide neuropeptide Y innervation to LHRH cells.
Close examination of the associations between LHRH-IR perikarya and TH-IR axon varicosities revealed no synaptic cleft between the juxtaposing neuronal elements, suggesting that these structures may be functioning synapses. This finding was verified using confocal microscopy combined with double-label fluorescence immunohistochemistry, demonstrating that the TH-IR structures are in close proximity to the LHRH-IR perikarya without a sign of a gap.
In conclusion, catecholaminergic fibers terminating on the surface of LHRH-IR neurons may form the morphological basis for interactions between these two systems in the medial preoptic and infundibular regions of the human diencephalon. According to the present findings, the central catecholaminergic system may also regulate the gonadal function centrally via the LHRH neuronal network in the human diencephalon. Moreover, defined TH-IR terminal fields around large multipolar neurons lacking either LHRH or TH immunoreactivity suggest that catecholamines also modulate other diencephalic functions.
Acknowledgments
We thank Professor Toshiharu Nagatsu for providing the antibodies, Professor Janos Szabad (Department of Biology, University of Sciences, Szeged, Hungary) for access to the confocal microscope, Professor Zsolt Liposits (Hungarian Academy of Sciences, Budapest, Hungary) for his critical evaluation of the data, and Dr. Tammy Dellovade (Wyeth-Ayerst Research, Collegeville, PA) for her critical evaluation of this manuscript. We thank Mr. Csaba Bohata for the computer work and Ms. Gabriella Kovács for expert technical assistance.
Footnotes
Address all correspondence and requests for reprints to: Dr. István Merchenthaler, Women’s Health Research Institute, Wyeth-Ayerst Research, RN 3164, 500 Arcola Rd., Collegeville, Pennsylvania 19426.
Abbreviations: DAB, Diaminobenzidine; FITC, fluorescein isothiocyanate; IR, immunoreactive; LHRH, LH-releasing hormone; NE, norepinephrine; TH, tyrosine hydroxylase.
Received January 30, 2001.
Accepted August 14, 2001.
References
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