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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1842-1848
Copyright © 2003 by The Endocrine Society

Topography and Associations of Leu-Enkephalin and Luteinizing Hormone-Releasing Hormone Neuronal Systems in the Human Diencephalon

Bertalan Dudás and István Merchenthaler

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 Research, Collegeville, Pennsylvania 19426

Address all correspondence and requests for reprints to: Bertalan Dudas, M.D., Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, Illinois 60153. E-mail: bdudas{at}lumc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although several studies indicated that leu-enkephalin controls gonadal function, the morphological substrate of this modulation is unknown. To reveal potential interaction sites between leu-enkephalin and LH-releasing hormone (LHRH) in the hypothalamus, the distribution and connections of leu-enkephalin-immunoreactive (IR) and LHRH-IR systems were examined in the human diencephalon using double-label immunohistochemistry. First the leu-enkephalin-IR and LHRH-IR neural elements were mapped, then the maps of the two different neurotransmitter systems were superimposed unveiling the overlapping areas. The putative juxtapositions between leu-enkephalin-IR and LHRH-IR structures were revealed with double label immunocytochemistry. Close contacts were detected in the medial preoptic area and in the infundibulum/median eminence. In these areas, diaminobenzidine-silver-intensified, black leu-enkephalin-IR fibers abutted fusiform, brown, diaminobenzidine-labeled LHRH neurons often forming multiple contacts. Examination of semithin sections of these close associations with the aid of oil immersion revealed no cleft between the contacting LHRH-IR and leu-enkephalin-IR elements.

Our findings indicate that the juxtapositions between LHRH-IR and leu enkephalin-IR neurons may be functional synapses forming the morphological substrate of the leu-enkephalin-modulated LHRH secretion in the human diencephalon. Moreover, the wide distribution of leu-enkephalin-IR elements suggests leu-enkephalin control of other diencephalic functions as well.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEU-ENKEPHALIN IS a pentapeptide serving as an endogenous agonist for the receptors that are stimulated by opiate alkaloids. Leu-enkephalin has multiple effects on the central nervous system (1) including the neuroendocrine hypothalamus (2). Among other anterior pituitary hormones, leu-enkephalin also controls the gonadal function by regulating LH secretion (3, 4).

Because LH-releasing hormone (LHRH) receptor antagonists block the naloxone-induced LH release (5), and neither basal nor LHRH-stimulated LH secretion is blocked by opiates in hemipituitary preparations in vitro (6), there is a general consensus that opiates influence the pituitary LH secretion via regulating the hypothalamic LHRH release. However, the reported effects of opiate agonists and antagonists on LHRH secretion are contradictory. Opioid agonists were reported to decrease the LHRH controlled release of LH from the anterior pituitary gland in several species including rats (7, 8), monkeys (9), and humans (10, 11). Morphine administration inhibits the ovulation in rats by abolishing the preovulatory LH surge in proestrus (12). The release of LHRH from medial basal hypothalamic fragments can be inhibited by submicromolar concentrations of leu-enkephalin (13). Conversely, opiate antagonists stimulate LH secretion in rats (6, 14, 15), sheep (16), primates (17, 18, 19), and humans (11, 20, 21). In contrast to these findings, administration of either leu-enkephalin or met-enkephalin has been reported to increase LH in male and female rats (3, 4), and leu-enkephalin stimulates LH secretion in primary culture of anterior pituitary cells and enhances the stimulatory effect of LHRH on LH release (22). Moreover, naloxone induces a significant decrease in LH secretion of newborn pigs (23).

Although several studies reported that other neurotransmitter systems may be involved in the leu-enkephalin-controlled LHRH secretion (24, 25, 26), it has been suggested that the leu-enkephalin system may also directly innervate the LHRH neurons. Previous studies revealed that enkephalinergic axons innervate areas containing LHRH perikarya and fibers in the rat hypothalamus (27, 28, 29). Moreover, microinjection of naloxone to the medial basal hypothalamus and to medial preoptic area, which are the major locations of LHRH-IR elements, increases LH release from the pituitary gland (30). However, there is no available evidence whether LHRH neurons are innervated directly by leu-enkephalin-IR elements in rats or humans.

The distribution of LHRH-IR elements is well known in the human diencephalon (31, 32, 33, 34, 35, 36). In contrast to these data, studies regarding the pattern and morphology of enkephalin system were restricted to the rat brain (27, 28, 29). Because the juxtapositions between the leu-enkephalin-IR and LHRH-IR elements may be the morphological substrate of the leu-enkephalin-controlled LHRH release, it is crucial to reveal the pattern and morphology of the putative juxtapositions between the leu-enkephalin and LHRH systems in the human brain.

In these studies, we simultaneously mapped the LHRH and leu-enkephalin-IR elements in the human diencephalon using double-label immunohistochemistry. After superimposition of the LHRH and leu-enkephalin maps, the overlapping areas were closely examined to identify the putative juxtapositions between the LHRH-IR and leu-enkephalin-IR structures. The pattern and morphology of these LHRH-leu-enkephalin close associations were then characterized and quantitated. Because electron microscopy is not applicable in case of human samples with long postmortem periods, the ultrastructure of the juxtapositions between the LHRH-IR and leu-enkephalin-IR elements was studied using semithin plastic sections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples

Brain samples of two adult women and two adult men (31–72 yr of age) were obtained from autopsies at 24–48 h postmortem. The clinical and pathological records of the individuals did not reveal any neurological and neuroendocrinological diseases. The brain samples were taken in accordance with the regulation and permission of the Ethics Board of the Szent-Györgyi Albert Medical University.

Tissue preparation

Following the dissection of the hypothalami from the brains, the tissue blocks were fixed by immersion in 0.1 M phosphate-buffered (pH 7.4) 4% formaldehyde at 4 C for 2–24 wk. Each block contained half of the hypothalamus split at the midsagittal line. The samples were cryoprotected in 30% sucrose in phosphate buffer containing 0.9% sodium chloride (PBS) supplemented with 0.1% sodium-azide. Coronal sections (30 µm) were cut on a freezing microtome (Reichert) and collected in four series of wells of plastic 24-compartment plates with PBS containing 0.2% sodium-azide, and stored at 4 C until processing. The adjacent sections were processed as follows: 1) Nissl staining with cresyl-violet; 2) immunohistochemical detection of LHRH; 3) immunohistochemical detection of leu-enkephalin; and 4) double-label immunohistochemistry of leu-enkephalin and LHRH.

Immunohistochemistry

For single labeling, the streptavidin-biotin method was used in combination with silver intensification introduced by Gallyas and co-workers (37, 38). The samples were pretreated first with 10% thioglycolic acid for 30 min to suppress the endogeneous tissue argentophilia, then with 0.2% Triton X-100 for 20 min followed by with 10% normal horse serum in PBS for 1 h at room temperature to block nonspecific staining. Thereafter, the sections were incubated in a primary antiserum 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 leu-enkephalin-IR structures were visualized by monoclonal mouse anti-leu-enkephalin serum (1:8000; NOC1, Axell, Westbury, NY). The sections were then incubated in a secondary antiserum containing biotin-labeled horse antimouse IgG (Vector Laboratories, Burlingame, CA; 1:500) for 2 h. In each case, the cross-reactions of the secondary antibodies with the endogeneous human IgGs were eliminated by the addition of 2% normal human serum to these reagents. Finally, the sections were incubated for 2 h in peroxidase-labeled streptavidin (Jackson ImmunoResearch, West Grove, PA; at a dilution of 1:500) for the detection of LHRH and leu-enkephalin. 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 (37, 38).

Simultaneous detection of leu-enkephalin-IR and LHRH-IR structures was performed using double label immunohistochemistry. First, leu-enkephalin immunohistochemistry was carried out; then the LHRH-containing neuronal structures were immunolabeled as described above with a slight difference, using DAB chromogen without nickel ions. The anti-LHRH serum was used in 1:8000 dilution.

Some of the double-labeled sections were embedded in plastic to obtain thin (1 µm) sections. The large frozen sections were cut in smaller pieces, processed through graded ethanol series and propylene oxide, and then flat embedded in Durcupan. Semithin sections were cut with an ultramicrotome (Reichert, Vienna, Austria).

In control sections, the primary antibodies were omitted or replaced by nonimmune rabbit serum at the dilution of 1:8000 (leu-enkephalin) or 1:20000 (LHRH), respectively. Immunoreaction was not observed.

Computer-assisted mapping

The hypothalamic sections were systematically scanned using a plain-scanner (Hewlett-Packard Co., Palo Alto, CA), and the outlines of the sections were traced using CorelTrace software 4.0 (Corel Corp., Ottawa, Ontario, Canada). The computer-generated superimposition of eight, consecutive sections was summarized in each of the slides of Fig. 1Go denoted by letters from A to 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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  		Figure 1. Distribution of the leu-enkephalin-IR elements (circles: perikarya; lines: fibers) and LHRH-IR elements (dots: perikarya) in the human diencephalon. Each slide (A–P) is the superimposition of eight, 30 µm thick sections. Ac, Anterior commissure; Fx, fornix; Och, optic chiasm; Ot, optic tract; Pvn, paraventricular nucleus; Mb, mamillary body; Son, supraoptic nucleus. Scale bar, 5000 µm.

 
Terminology

The terminology of the diencephalic structures was adapted from Braak and Braak (39), Saper (40), and Silverman et al. (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LHRH system

The distribution of LHRH-IR elements were in good agreement with our previous studies (31, 32, 33, 34, 35, 36). LHRH-IR perikarya were located mainly in the medial preoptic area and in the infundibulum/median eminence (Fig. 1Go, H–L). Several cell bodies were detected periventricularly in the tuberal region (Fig. 1Go, H–L) and posterior hypothalamus (Fig. 1Go, M and N), along the diagonal band of Broca (Fig. 1BGo), and basally to the mamillary bodies (Fig. 1Go, M and N). Few cells were detected in the paraventricular and supraoptic nuclei (Fig. 1Go, F–I). The majority of the LHRH-IR cells were fusiform in shape, only few (less than 5%) multipolar neurons were observed. Dense LHRH-IR fiber network was located in the infundibulum and median eminence around the portal vessels. LHRH-IR fibers were also detected in the medial preoptic area, and along the diagonal band of Broca. Few fibers were found in the periventricular area of the tuberal region and posterior hypothalamus.

Leu-enkephalin system

Leu-enkephalin-IR cell bodies were concentrated in three different regions. 1) The vast majority of leu-enkephalin-IR perikarya (82%) were observed in the periventricular area of the tuberal region (Fig. 1JGo); 2) Few cells were found in the infundibulum in the close proximity of the portal vessels (Fig. 1Go, J–L); and 3) Periventricularly arranged in the medial preoptic area (Fig. 1Go, D and F). The leu-enkephalin-IR neurons were mainly fusiform in shape with processes emanating from the opposite poles of the cell body (Fig. 2AGo). The leu-enkephalin-IR cells were characteristically oriented with the axis of the fusiform cell bodies running parallel to the surface of the third ventricle, and the processes pointing dorsally and ventrally along the axis of the cells.



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  		Figure 2. Leu-enkephalin-IR elements in the human diencephalon. A, Fusiform leu-enkephalin-IR neuron receiving le-enkephalin-IR axon variosities (arrowheads) in the periventricular area of the tuberal region. B, Leu-enkephalin-IR fibers forming fiber baskets around fusiform cells in the dorsal part of the tuberal periventricular region and (C) in the medial preoptic area. The perikarya are marked by asterisks. D, Leu enkephalin-IR fibers are in close proximity of the portal vessels in the median eminence. The lumen of the vessel is marked by asterisk. Scale bar: 10 µm (A–C); 50 µm (D).

 
Leu-enkephalin-IR fibers were detected periventricularly, running along the medial surface of the hypothalamus (Fig. 1Go, F–L). An additional fiber network projected from the infundibulum toward the lateral hypothalamic area (Fig. 1Go, G–J). Dense leu-enkephalin-IR axonal network was revealed around the fornix and along the lateral part of the anterior commissure (Fig. 1Go, A–D). Leu-enkephalin-IR fibers were also found in the dorsal part of the lateral hypothalamic area (Fig. 1Go, E–I) and around the portal vessels (Fig. 1Go, H–L, and 2D). No leu-enkephalin-IR elements were detected in the supraoptic nucleus.

Leu-enkephalin-IR fiber varicosities were observed to form fiber baskets in the periventricular area of the preoptic and tuberal region (Fig. 2Go, B and C). Here, leu-enkephalin-IR axons abutted on fusiform neurons, that did not show LHRH-immunoreactivity. The fibers were in intimate contact with the unidentified neurons, and often covered the majority of their surface.

Associations between the LHRH and leu-enkephalin systems

Leu-enkephalin-IR and LHRH-IR elements showed overlap in the infundibulum/median eminence and the medial preoptic area after superimposing the maps of these two systems (Fig. 1Go, D–L). Partial overlap was detected in the periventricular zone of the tuberal region (Fig. 1Go, H–K), and at the ventral zone of the posterior hypothalamus, around the mamillary body (Fig. 1Go, M and N). Close examination of the overlapping areas revealed that black, silver-intensified, leu-enkephalin-IR axons abutted on brown, DAB-labeled, LHRH-IR cell bodies and axonal processes in these regions (Fig. 3Go). These juxtapositions between leu-enkephalin-IR fibers and LHRH-IR neurons were detected mainly in the infundibulum and median eminence (78%; Fig. 3Go, B, C, E, and G). Here, the 21% of the observed LHRH-IR perikarya were heavily innervated with more than three intimate leu-enkephalin-IR contacts. Numerous LHRH-IR neurons (23%) were lightly innervated in these regions (1–3 contacts) and 56% of the LHRH-IR cells were not closely associated with leu-enkephalin-IR axon varicosities. Close LHRH-leu-enkephalin-IR associations were also observed in the medial preoptic area and in the periventricular area of the tuberal region (18%; Fig. 3AGo). Few juxtapositions between the leu-enkephalin and LHRH systems were revealed in other areas including the anteroventral periventricular nucleus and the tuberomamillary nucleus (4%; Fig. 3Go, D and F). No juxtapositions were found rostrally to the lamina terminalis. Examination of the juxtapositions with oil immersion and analysis of the semithin plastic sections revealed no gap between the leu-enkephalin-IR and LHRH-IR elements (Fig. 3GGo). The pattern and morphology of the juxtapositions did not show any significant differences regarding the sex and age of the examined individuals.



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  		Figure 3. Juxtapositions between leu-enkephalin (black) and LHRH (brown) immunoreactive elements in the human diencephalon. The position of the demonstrated LHRH-IR neurons is shown by asterisks in the upper corners of the micrographs. The schematic drawings correspond to the slices denoted by the same letters in Fig. 1Go. The thickness of the sections is 30 µm (A–F), and that of the plastic section is 1 µm (G). Arrowheads point to juxtapositions between the LHRH-IR and leu-enkephalin-IR elements. Scale bar, 20 µm.

 
Associations between leu-enkephalin neurons

Fusiform leu-enkephalin-IR perikarya often received leu-enkephalin-IR fibers in the periventricular region (Fig. 2AGo). Few juxtapositions were detected in the infundibulum. The morphology of these juxtapositions were similar to those between the LHRH-IR and leu-enkephalin-IR elements.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although recent studies revealed that leu-enkephalin influences gonadal functions possibly via hypothalamic LHRH release, the morphological substrate of this modulation was unknown. The intimate juxtapositions between the leu-enkephalin-IR and LHRH-IR neurons in the human brain reported in the present studies may serve as functioning synapses and can be the morphological base of leu-enkephalin-controlled LHRH release.

The pattern and morphology of the LHRH-IR system was in good agreement with our previous findings (33, 34, 35, 36). LHRH perikarya were located in the infundibular and periventricular areas and along the diagonal band of Broca, whereas LHRH-IR fibers were detected periventricularly, and in the infundibular region and in the median eminence. The similar pattern of the LHRH-IR elements in individuals of different sex and age indicate that the sex- and age-related differences of the gonadal functions are not related to the hypothalamic LHRH system.

Previous studies described the morphology of the leu-enkephalin system in rat (27, 28, 29). However, the distribution and the morphology of the leu-enkephalin-IR elements were almost completely unknown in human. The present study is the first that provides a high resolution map of the leu-enkephalin-system in the human diencephalon and describes the morphology of the leu-enkephalin-IR elements.

In our study, a few leu-enkephalin-IR perikarya were detected mainly in the periventricular area of the hypothalamus and the infundibulum. In contrast to these findings, the leu-enkephalin-IR fibers formed a delicate network throughout the entire hypothalamus without any well-distinguishable pathways. Although the periventricularly located leu-enkephalin fibers seem to originate from the periventricularly located leu-enkephalin-IR cell bodies, the vast majority of leu-enkephalin-IR axon varicosities appear to be of extrahypothalamic origin.

Previous data indicated that leu-enkephalin influences hypothalamic LHRH release indirectly through other neurotransmitter systems (24, 25, 26). Our studies revealed intimate juxtapositions between the leu-enkephalin and LHRH systems. Close examination of these leu-enkephalin-LHRH-associations in semithin plastic sections did not reveal any gap between the contacting leu-enkephalin and LHRH-IR elements, strongly suggesting direct synaptic control of LHRH by leu-enkephalin-IR axon varicosities. These findings along with the previously published data suggest that the leu-enkephalin system may modulate pituitary LH release both directly at the level of anterior pituitary and indirectly by influencing hypothalamic LHRH secretion.

Leu-enkephalin microinjection into the medial preoptic area (MPOA) had no significant effect on circulating levels of LH in the rat (41). These findings suggest that the subset of LHRH neurons located in the MPOA may not participate significantly in leu-enkephalin-controlled LHRH release. Indeed, our studies revealed that the majority of the leu-enkephalin-LHRH juxtapositions (78%) were detected in the infundibulum, whereas in the MPOA only a few LHRH cells were in close association with leu-enkephalin-IR axonal varicosities. These data indicate that the modulation of LHRH by leu-enkephalin may be localized to the infundibular area and the median eminence. It has been reported that leu-enkephalin-IR axons form presynaptic nerve terminals on LHRH-IR nerve terminals in the frog median eminence (42). The tuberoinfundibular dopaminergic (TIDA) neurons of hyperprolactinemic female rats (pregnant, lactating, aged or pharmacologically hyperprolactinemic) coexpress tyrosine hydroxylase, the rate limiting enzyme for dopamine synthesis, and leu-enkephalin (43, 44, 45). Moreover, it has been shown that tyrosine hydroxylase-IR axons form presynaptic contacts with LHRH nerve terminals in the rat (46, 47) and the ewe (48). Therefore, under hyperprolactinemic conditions, when TIDA neurons coexpress leu-enkephalin, these TIDA/leu-enkephalin-IR axon terminals, via presynaptic contacts with LHRH-IR nerve terminals, may regulate LHRH release in the median eminence. The close juxtapositions between leu-enkephalin and LHRH processes in the human infundibulum reported in the present studies and the presence of opioid receptors in the infundibulum and median eminence (49) suggest that a similar mechanism may also exist in the human. Therefore, also still speculative, leu-enkephalin, colocalized with dopamine, the physiological prolactin (PRL) release-inhibiting factor, may either increase PRL synthesis/secretion from the anterior pituitary and counterbalance the inhibitory action of dopamine and/or it may modulate the pulsatile pattern of LHRH release into the hypophysial portal circulation. The lack of ovulation during pregnancy and aging and the lower frequency of ovulations during lactation, may be due, in part, to the elevated PRL secretion that leads to expression of leu-enkephalin in the TIDA neurons. This newly synthesized leu-enkephalin, then could either modulate the pulsatile pattern of LHRH release or maintain elevated PRL synthesis, both of which would block ovulation.

In the present studies, we noticed that the subset of LHRH-IR neurons in front of the lamina terminalis did not have contacts from leu-enkephalin-IR axon varicosities. Our previous studies also demonstrated that these LHRH-IR cells do not form juxtapositions with neuropeptide Y (33), substance P (35), corticotropin-releasing factor (36), and catecholaminergic systems (34) in the human diencephalon. Because this subset of LHRH-IR neurons in the rostral diencephalon do not appear to receive any significant input from any of the examined neurotransmitter systems, they may have an entirely different regulatory mechanism and function than the rest of the LHRH-IR perikarya. Moreover, the widespread distribution of leu-enkephalin-IR axon varicosities indicates leu-enkephalin control of other diencephalic functions as well. Because leu-enkephalin-IR fibers are in close proximity of blood vessels in the infundibulum/median eminence and in the paraventricular nucleus, leu-enkephalin may also regulate different extrahypothalamic functions via the blood stream.

The leu-enkephalin-IR neurons often received leu-enkephalin-IR axon varicosities that formed multiple contacts with the perikarya. These leu-enkephalin-leu-enkephalin juxtapositions were almost entirely restricted to the periventricular area of the preoptic area and tuberal region and may be functional synapses, raising the possibility of a direct leu-enkephalin-controlled leu-enkephalin secretion in the human diencephalon.

In conclusion, leu-enkephalin-IR axon varicosities form intimate contact with LHRH-IR neurons. These juxtapositions may be functional synapses, and may represent the morphological substrate of leu-enkephalin-controlled LH secretion via the modulation of hypothalamic LHRH release. Although not explored in the present studies, leu-enkephalin may modulate LHRH release and block ovulation via preterminal contacts with LHRH-IR nerve terminals. The close associations between leu-enkephalin-IR elements in the human diencephalon indicate direct leu-enkephalin influence on leu-enkephalin secretion. Moreover, the widespread pattern of leu-enkephalin-IR axon varicosities suggests leu-enkephalin-control of other diencephalic functions as well.


   Acknowledgments
 
We thank Professor Thackery S. Gray (Department of Cell Biology, Neurobiology and Anatomy, Loyola University, Chicago, IL) for access to the microscope, Csaba Bohata for the computer work, and Gabriella Kovács for expert technical assistance (Albert Szent-Gyorgyi University, Szeged, Hungary).


   Footnotes
 
Abbreviations: DAB, Diaminobenzidine; IR, immunoreactive; LHRH, LH-releasing hormone; MPOA, medial preoptic area; PRL, prolactin; TIDA, tuberoinfundibular dopaminergic.

Received September 9, 2002.

Accepted January 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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F. J P Ebling
The neuroendocrine timing of puberty
Reproduction, June 1, 2005; 129(6): 675 - 683.
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