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Department of Pharmacology and Experimental Therapeutics (B.D.), Loyola University Chicago, Stritch 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: Istvan Merchenthaler, M.D., D.Sc., Women’s Health Research Institute, Wyeth Research, RN 3164, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: . merchei{at}wyeth.com
Abstract
LHRH release is induced by substance P (SP) in the rat hypothalamus. Recent immunocytochemical studies indicate that SP-immunoreactive axons synapse on LHRH neurons in the diencephalon of the rat, but this phenomenon has not yet been demonstrated in human. Therefore, in the present study we visualized the SP- and LHRH-immunoreactive (IR) elements in the human diencephalon and evaluated the close juxtapositions between them. The distribution of LHRH- and SP-IR sites were investigated in diencephalic sections of six, postmortem human brains by means of double-labeling immunocytochemistry. The LHRH-containing perikarya were located in the diagonal band of Broca, lamina terminalis cinerea, preopticoseptal, medial preoptic, and infundibular areas of the brain. The SP-IR fibers formed a network in the periventricular zone in the infundibular region, median eminence, and corpus striatum. The SP-IR cell bodies were located mainly in the infundibular region, median eminence, basal part of the periventricular area, dorsomedial subdivision of the ventromedial nucleus, and basal perifornical area of the tuberal region. The juxtapositions between LHRH-IR cell bodies and SP-IR varicosities were detected in the infundibular and periventricular regions. In these sites black, silver-intensified, SP-IR fiber varicosities abutted on brown, DAB-labeled, LHRH-IR cell bodies. Similar structures were detected between the SP-IR fibers and SP-IR perikarya. These findings suggest that the juxtapositions between the SP and LHRH systems may be the morphological basis of SP-controlled LHRH release in the human diencephalon. Moreover, the intimate contacts between SP-IR fiber varicosities and SP-IR cell bodies or axons indicate direct control of SP on the diencephalic SP release.
SUBSTANCE P (SP) is an undecapeptide with multiple effects on the cardiovascular (1, 2, 3), gastrointestinal (4, 5), and urinary systems (6, 7) as well as complex central nervous system functions such as learning (8) and memory (9). Previous studies also revealed that SP modulates gonadal functions in several species including human (10, 11, 12, 13, 14, 15, 16, 17). Although the exact mechanism of this modulation is not known, there is a general consensus that this phenomenon is based on the influence of SP on hypophyseal LH release. Because LH secretion from the pituitary gland is under the control of hypothalamic LHRH release, SP can alter gonadal function via direct influence on pituitary LH secretion and/or by regulation of the hypothalamic LHRH system.
For example, SP stimulates LH release from rat (18) and pig (19) pituitary cells in vitro. Moreover, rat anterior pituitary cells both in vivo and in vitro (20, 21, 22) release SP, and SP mRNA has also been detected in the pituitary of humans (23). In addition, the pituitary gland contains SP receptors in both humans (12) and rats (11) suggesting the possibility of direct control by SP on LH secretion. Supporting this type of action, the presence of SP-immunoreactive (IR) fibers in the hypophysial stalk and median eminence has been reported in humans (24) and primates (25). However, there are also contrasting data showing that in dispersed anterior pituitary cells, harvested from estrogen-treated ovariectomized rats, SP did not affect gonadotropin release in vitro (26).
An indirect effect of SP on gonadotropin secretion could be mediated via the LHRH neuronal system. Subcutaneously administered SP inhibits LHRH-induced LH release via NK1 receptors (27, 28), and microinjection of the peptide into the medial preoptic area reduces LH and FSH levels in the plasma (29). A synaptic connection between SP-IR fibers and LHRH-IR cell bodies in the rat has been described by Tsuruo et al. (30), supporting a direct connection between these two neuropeptidergic systems. In vitro, SP inhibits LHRH-stimulated gonadotropin release from rat pituitary cells (28) but potentiates the response in prepubertal porcine cells (19). In contrast to these findings, SP antagonist or antiserum injected into the lateral or third ventricles inhibits LH release (20, 31).
The effect of SP on gonadotropin secretion also depends on the hormonal environment (32, 33). Although iv administration of SP antiserum had no effect on LH or FSH levels of ovariectomized rats, it stimulated LH (but not FSH) secretion in ovariectomized rats with estrogen priming (33). Moreover, iv SP administration directly increases LHRH concentrations in the pituitary portal blood of male rats (34).
Although the LHRH system has already been mapped in the human diencephalon (35), little is known about the pattern of the SP-IR elements. The information on the distribution of SP-IR structures in the brain is based on immunocytochemical studies in rats (36, 37) and humans (24) and in situ hybridization experiments in human tissue samples (38). Because the direct influence of the SP-IR elements on the hypothalamic LHRH system could be the morphological basis for SP-controlled gonadal function, the analysis of the putative juxtapositions between the LHRH- and SP-IR elements might reveal the site of interaction.
In the present study, we mapped LHRH-IR and SP-IR neuronal structures in the human hypothalamus. In an attempt to reveal the location of the putative juxtapositions and to confirm the observations that SP-IR nerve terminals form synaptic contacts on LHRH-IR perikarya in rats (30), the maps of LHRH- and SP-IR images obtained from human brains were superimposed. Finally, the putative synapses between the SP and LHRH immunoreactive elements were identified and quantitated in the overlapping areas using double-label immunohistochemistry.
Materials and Methods
Tissue
Brain samples of three adult women and three adult men (25–72 yr of age) were obtained from autopsies between 24 and 48 h postmortem. Review of the medical records indicated specimens were obtained from individuals with no neurological or neuroendocrinological disease. The brain samples were taken in accordance with the regulations of and with permission from the Ethics Board of the Szent-Györgyi Albert Medical University.
Tissue preparation
Hypothalami were dissected from the brains and immersion fixed in 4% paraformaldehyde/0.1 M phosphate-buffered (pH 7.4) at 4 C for 2–24 wk. Each tissue block contained half of the hypothalamus that was split along 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 and then cut on a Reichert freezing microtome at 30-µm intervals in coronal planes. The sections (
450–500 from each block) were collected in a series of four wells into PBS containing 0.2% sodium azide and stored at 4 C until processing. The adjacent sections were processed for Nissl staining with cresyl-violet, immunohistochemical detection of LHRH, immunohistochemical detection of SP, and double-label immunohistochemistry of SP and LHRH.
Immunohistochemistry
Single-labeling immunohistochemistry was carried out using either peroxidase-antiperoxidase (PAP) or streptavidin-biotin methods combined with silver intensification as introduced by Gallyas et al. (39). In brief, samples were pretreated with 10% thioglycolic acid for 30 min and then with 0.2% Triton X-100 for 20 min followed by incubation in 10% normal horse serum in PBS for 1 h at room temperature. 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 at 4 C. The LHRH-IR structures were identified using a monoclonal mouse anti-LHRH antiserum (Chemicon, Temecula, CA) at a dilution of 1:20,000. The SP-IR structures were visualized with a rabbit anti-SP serum (7451, Peninsula Laboratories, Inc., San Carlo, CA) at a dilution of 1:50,000. This antiserum was raised against the N-terminal portion of SP, and it shows 100% cross-reactivity with SP and the N-terminus sequence of SP (2–11 but not 7–11). It does not recognize neurokinin A, neurokinin B, neuropeptide K, or physelamin in Western blot analysis. The sections were then incubated for 2 h in a secondary antiserum containing either unlabeled goat antirabbit IgG (Jackson Immunocytochemicals, West Grove, PA) at a dilution of 1:50 for SP, or biotin-labeled horse antimouse IgG (Vector Laboratories, Inc., Burlingame, CA; 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 2 h in rabbit peroxidase-antiperoxidase (Jackson Immunocytochemicals) at a dilution of 1:2,000 for the detection of SP or peroxidase-labeled streptavidin (Jackson Immunocytochemicals) at a dilution of 1:500 for the detection of LHRH. The chromogen solution was composed of 0.05% diamino-benzidine (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 (39).
Simultaneous detection of SP- and LHRH-IR structures was performed using double-label immunohistochemistry. First, SP immunohistochemistry was carried out as described above using DAB/nickel chromogen, and then the LHRH-IR neuronal structures were immunolabeled with DAB alone as described above (39, 40). For dual labeling the anti-LHRH serum was used in a 1:8000 dilution.
Because it was not practical to perform electron microscopic analysis on tissue obtained after a 24- to 48-h postmortem period, some of the double-labeled sections were embedded in plastic to obtain thin (0.5 µm) sections. Thus, the large frozen sections were cut in smaller pieces, processed through graded ethanol series and propylene oxide, and then flat embedded in Durcupan (Sigma, St. Louis, MO). Semithin sections were cut with an ultramicrotome (Reichert).
In control sections, the primary antibodies were omitted or replaced by nonimmune rabbit serum at the dilution of 1:50,000 (SP) or 1:20,000 (LHRH), respectively. In both cases, immunoreaction was not observed. In addition, the SP antiserum used in these studies was preabsorbed with SP (Peninsula Laboratories, Inc.). Preabsorption with as low as 10 µg/ml of the working dilution of the primary antiserum completely eliminated the staining. According to the distributor (Peninsula Laboratories, Inc.), the antiserum is specific for SP and does not cross-react with other members of the tachykinin family, including neurokinin A, neurokinin B, or physelamin in Western blot analysis.
Data analysis
The hypothalamic sections were systematically scanned with the aid of a plain scanner (Hewlett-Packard Co., Palo Alto, CA). 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 were summarized in each of the slides shown in Fig. 1
. The neurons and fibers were marked on these figures using an Axiophot microscope with camera lucida (Carl Zeiss, Thornwood, NY) and Adobe Photoshop software, version 3.0.
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The terminology of the diencephalic structures was adapted from Braak and Braak (41), Saper (42), and Silverman et al. (35).
Results
LHRH-IR system
The pattern of the LHRH-IR neuronal elements was similar to our previous immunohistochemical study (43, 44). LHRH-IR cell bodies were concentrated in the ventral and caudal part of the periventricular zone, mainly in the medial preoptic, infundibular areas, and the median eminence. Few cells were found along the diagonal band of Broca and in either the paraventricular or supraoptic nuclei. The majority of LHRH-IR 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 periventricular and infundibular areas. No differences related to sex, age, and/or postmortem time were observed in the number and/or pattern of IR perikarya and processes.
SP-IR system
The majority (94%) of the SP-IR perikarya were located in the tuberal region. Only a few cell bodies were seen scattered in the periventricular zone of the preoptic area and basal part of the posterior hypothalamus. In the tuberal region, the neurons were arranged in several subclasses. Most of the perikarya were found in the infundibular region (Figs. 1
and 2, H–J) and the median eminence (Fig. 2, K and L). The basal part of the periventricular area and the dorsomedial subdivision of the ventromedial nucleus contained an additional cell population (Figs. 1 and 2, H–J). Neither the paraventricular nor supraoptic nuclei contained any SP-IR perikarya (Figs. 1 and 2, F–I). Finally, a well-defined cell group was distinguished in the basal perifornical area of the tuberal region (Figs. 1 and 2, H–J).
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). The multipolar cells, with triangle-shaped or rounded cell bodies, were usually situated in the dorsomedial subdivision of the ventromedial nucleus (Fig. 3A).
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, A and B) and around the anterior comissure (Fig. 1, C and D). In the preoptic area, SP-IR fibers were mainly periventricularly arranged, and a delicate fiber network appeared around the fornix (Fig. 1, E and F). In the lateral zone of the preoptic area, SP-IR processes were located in the corpus striatum and on the basal part of the diencephalon running parallel with the surface of the brain (Fig. 1, E and F). The supraoptic nuclei did not contain significant amounts of SP-IR fibers (Fig. 1, F–H). Most of the fiber varicosities were periventricularly arranged in the tuberal region of the diencephalon. Dense network of fibers was located around the portal vessels in the infundibulum/pituitary stalk (Fig. 3C). Few axons were detected in the paraventricular nucleus (Figs. 1 and 2, G–I). An additional SP-IR fiber population was distinguished running to the infundibulum and the median eminence, passing over the optic tract and located laterally on the basal region of the diencephalon (Figs. 1 and 2, H–L). Numerous SP-IR processes were located around the fornix in the tuberal region (Figs. 1 and 2, I–L). In the posterior hypothalamus, the axon varicosities were found at the base of the third ventricle and in the periventricular area (Fig. 2, M–P). On the lateral part of the posterior hypothalamus, SP-IR fibers were observed at the region of the corpus striatum (Fig. 2, M–P). No sex- and age-related differences were observed in the number and/or pattern of SP-IR perikarya and processes, and the different postmortem periods did not influence the number of IR structures. Juxtapositions between the LHRH and SP elements
Superimposing the map of the LHRH- and SP-IR neural elements revealed that the two systems were overlapped in the medial preoptic area, in the infundibulum and median eminence (Figs. 1 and 2, F–L). Partial overlap was detected in the periventricular zone of the tuberal region and in the paraventricular nucleus (Figs. 1 and 2, G–K). Dual-labeling immunohistochemistry in these areas revealed that LHRH-IR cell bodies were contacted by SP-IR fiber varicosities. Closer examination with high magnification revealed black, silver-intensified SP-IR fiber varicosities abutted on brown, diaminobenzidine-labeled LHRH-IR cell bodies in the medial preoptic area, basal part of the periventricular zone in the tuberal region, infundibulum, and median eminence (Fig. 4). None of these juxtapositions were detected in the paraventricular nucleus. All hypothalami contained a similar distribution of LHRH-SP contacts irrespective of sex or age.
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). Juxtapositions between the SP elements
In addition to the SP-LHRH connections, several SP-IR axons contacted SP-IR cell bodies or axons in the infundibular nucleus (Fig. 3B). The structure of these juxtapositions was similar to those between the LHRH-IR and SP-IR elements.
Discussion
Previous studies revealed that SP regulates gonadal functions by influencing LH release from the pituitary gland (13, 14, 20, 28, 29, 31, 32). Although the mechanism of this phenomenon is not entirely known, it is generally believed that SP might regulate LH secretion by affecting hypothalamic LHRH synthesis and/or release. In the present study, we mapped the LHRH and SP systems and reported evidences of juxtapositions between LHRH and SP-IR elements. These intimate contacts between the two systems may be synapses as suggested by others (30) that provide the morphological basis for SP regulation of gonadal function.
The pattern and morphology of LHRH-IR elements in the present study agrees with our previous findings (43, 44). LHRH-IR perikarya were present mainly along the diagonal band of Broca and in the medial preoptic area, infundibulum, and median eminence. In addition, the distribution of LHRH-IR neuronal elements was not affected by sex or age. Earlier studies reported SP-IR structures in the diencephalon of the rat (24, 25); however, little is known about the distribution and morphology of SP-IR structures in the human brain. Although the distribution of SP mRNA-expressing neurons in the human brain is similar to the distribution of SP-IR cell bodies, probably because of the sensitivity of the in situ hybridization technique, the number of SP mRNA-expressing cells is higher (38). The present study provides a high-resolution map of the SP-IR elements in the entire human hypothalamus. Interestingly, the location of SP-IR cell bodies was almost entirely restricted to the tuberal region of the diencephalon, whereas SP-IR fiber varicosities were widely distributed in the hypothalamus. These data suggest that a significant amount of SP-IR axons may have an extrahypothalamic origin, and they may play role in the regulation of several diencephalic functions.
In the tuberal region, SP-IR perikarya were found mainly in the infundibular area and median eminence, surrounded by a dense network of SP-IR fiber varicosities. This finding corresponds to earlier studies reporting SP in the median eminence of the rat (24, 25). Moreover, Wormald et al. (12) reported SP receptors in the human pituitary and found that SP acting at the level of the pituitary inhibits LHRH-induced LH secretion in man. Similar to the studies of Hökfelt et al. (25) and Mai et al. (24), in our studies also, a close relationship between the SP-IR structures and the portal vessels was detected, suggesting direct control of SP on the release of pituitary hormones in human.
Additional subgroups of SP-IR perikarya were located in the basal part of the periventricular zone, dorsomedial subdivision of the ventromedial nucleus, and in the basal perifornical area of the tuberal region. Although these cell bodies seemed to project toward the median eminence, their location and the wide distribution of SP-IR fibers in the diencephalon indicate that certain SP-IR structures may not have direct contact with the portal vessels. These data support the theory that SP may control other diencephalic functions as well.
Previous studies reported that microinjection of SP into the medial preoptic area, or SP antagonist or antiserum injected into the lateral and third ventricles, influence plasma LH and/or FSH levels in rats (20, 30, 32). These effects of SP are believed to be mediated by regulation of hypothalamic LHRH secretion, suggesting that the subpopulation of LHRH neurons close to the ventricles may be involved in SP-controlled LH release. This theory was supported by superimposing the maps of LHRH and SP-IR systems in humans. These superimposed images showed that the LHRH and SP-IR elements overlap mainly in the periventricular area of the tuberal region, infundibular region, and median eminence. Here LHRH cell bodies are surrounded by dense SP-IR fiber varicosities. Close examination of dual-labeled sections found that LHRH-IR perikarya in these areas revealed that individual LHRH-IR elements were in contact with the axonal swellings of two or more SP-IR fibers. Analysis of 0.5-µm-thick plastic sections did not reveal any gap between the contacting elements. These data suggest direct SP control on hypothalamic LHRH release in these regions and suggest that the juxtapositions between the LHRH-IR and SP-IR elements are functional synapses. Similar intimate contacts were detected between SP-IR fiber varicosities and SP-IR cell bodies or axons, indicating direct control of SP-IR elements on the diencephalic SP release.
Our earlier studies reported close contacts between the LHRH-IR and NPY-IR (43) or catecholaminergic (44) elements in the medial preoptic region, infundibular area, and median eminence. The similar morphology and pattern of LHRH and NPY/catecholaminergic juxtapositions and those between LHRH-IR and SP-IR elements suggest that some of the SP-IR axon varicosities might contain NPY or catecholamines as well. Therefore, these systems may interact at the diencephalic level and regulate complex functions. The putative interaction of the NPY, catecholaminergic, and SP systems with LHRH-IR neurons in the medial preoptic and infundibular area/median eminence raises the possibility that other neuromodulator systems may influence hypothalamic LHRH release at the same sites. Thus, LHRH secretion is the result of an interaction with several neural inputs on the LHRH-IR cells. Because LHRH-IR perikarya located in the septal area, lamina terminalis cinerea, lateral preoptic area, paraventricular, supraoptic, and lateral part of the posterior hypothalamus do not seem to receive inputs from NPY/catecholaminergic/SP systems, the function of LHRH neurons in these subgroups of nuclei and the control of the LHRH release may be entirely different in these sites. Alternatively, it is possible that the release of LHRH by NPY/catecholaminergic/SP systems also takes place in the median eminence.
In conclusion, the juxtapositions reported here between the LHRH- and SP-IR elements may be the morphological substrate for the SP-controlled regulation of gonadal functions in human. A direct interaction between SP and LHRH systems, as first suggested by Tsuruo et al. (30) based on electron microscopic observations in rats, may take place at the level of LHRH perikarya and dendrites in the medial preoptic area and infundibular stalk/median eminence regions. Intimate contacts between the SP-IR elements indicate that SP release may be directly controlled by juxtaposing SP-IR structures. Moreover, the wide distribution of SP-IR fiber varicosities in the hypothalamus suggests that SP controls other diencephalic functions as well.
Acknowledgments
We thank Prof. Zsolt Liposits (Hungarian Academy of Sciences, Budapest, Hungary) and Dr. Tammy Dellovade (Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, PA) for critical evaluation of the data and Gabriella Kovács for expert technical assistance.
Footnotes
Abbreviations: DAB, Diamino-benzidine; IR, immunoreactive; SP, substance P.
Received August 23, 2001.
Accepted February 26, 2002.
References
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