Neuroscience 297 (2015) 205–210
JUXTAPOSITIONS BETWEEN THE SOMATOSTATINERGIC AND GROWTH HORMONE-RELEASING HORMONE (GHRH) NEURONS IN THE HUMAN HYPOTHALAMUS N. PROUDAN, a M. PEROSKI, a G. GRIGNOL, a I. MERCHENTHALER b,c AND B. DUDAS a*
secretion via direct contacts. The rare GHRH to somatostatin juxtapositions indicate that the negative feedback effect of GH targets the somatostatinergic system directly and not via the GHRH system. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
a Laboratory of Neuroendocrine Organization (NEO), Lake Erie College of Osteopathic Medicine, Erie, PA, United States b Department of Epidemiology & Public Health, University of Maryland, School of Medicine, Baltimore, MD, United States c Department of Anatomy & Neurobiology, University of Maryland, School of Medicine, Baltimore, MD, United States
Key words: hypothalamus, somatostatin, GHRH, growth.
Abstract—Somatostatin is a 14–28 amino acid peptide that is located not only in the gastrointestinal system but also in multiple sites of the human brain. The inhibitory effect of somatostatin on the growth hormone (GH) secretion of the pituitary gland is a well-established phenomenon. There is a general consensus that somatostatin is released into the hypophysial portal blood and modulates GH secretion by hormonal action. In the present study, we explored the possibility that in addition to the hormonal route, somatostatin may also influence GH secretion via influencing the growth hormone-releasing hormone (GHRH) secretion by direct contacts that may be functional synapses. Since the verification of these putative synapses by electron microscopy is virtually impossible in humans due to the long post mortem time, in order to reveal the putative somatostatinergic-GHRH juxtapositions, light microscopic double-label immunohistochemistry was utilized. By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya received contacting somatostatinergic axonal varicosities in the arcuate nucleus. In contrast, GHRH axonal varicosities rarely contacted somatostatinergic perikarya. The morphology and the abundance of somatostatin to GHRH juxtapositions indicate that these associations are functional synapses, and they represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary by regulating the hypothalamic GHRH
INTRODUCTION The central control of growth hormone (GH) secretion from the anterior pituitary is under the control of two hypothalamic neurohormones: growth hormonereleasing hormone (GHRH) and growth hormone release-inhibiting hormone (GH-RIH) or somatostatin. The activity of these hypophysiotropic hormonesynthesizing neurons is regulated by a number of neuropeptides, neurotransmitters, and hormones. Interestingly, however, GHRH and somatostatinergic systems also communicate with each other directly to maintain a delicate control over GH secretion. Ultrastructural findings indicate a bidirectional synaptic contacts between GHRH and somatostatinimmunoreactive (IR) hypophysiotropic neurons in the rat (Daikoku et al., 1988; Liposits et al., 1988; Horvath et al., 1989) and somatostatin receptors have been shown in the majority of GHRH neurons of the ventrolateral portion of the arcuate nucleus (Bertherat et al., 1992, 1995; McCarthy et al., 1992; Muller et al., 1999). Moreover, selective membrane somatostatin receptors sst1 or sst2 have also been localized in a subpopulation of GHRH neurons (Tannenbaum et al., 1998). Contrary to these finding in rats, no studies have been conducted in humans to elucidate the connections between GHRH and somatostatin. We have previously described that the GHRH system receives input from other neurotransmitter systems (neuropeptide Y [NPY], endogenous opiate and catecholaminergic systems) in the human hypothalamus (Deltondo et al., 2008; Rotoli et al., 2011; Dudas and Peroski, 2013; Dudas and Merchenthaler, 2014; Olsen et al., 2014). Thus, in the present study we utilized light microscopic double-label immunohistochemistry in order to reveal the putative juxtapositions between the somatostatinergic and GHRH elements in the human diencephalon.
*Corresponding author. Address: Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16509, United States. Tel: +1-814-866-8142; fax: +1-814-866-8411. E-mail address: [email protected] (B. Dudas). Abbreviations: GH, growth hormone; GHRH, growth hormonereleasing hormone; GH-RIH, growth hormone release-inhibiting hormone; IGF, insulin-like growth factor; IR, immunoreactive; NPY, neuropeptide Y; PBS, phosphate buffer containing 0.9% sodium chloride. http://dx.doi.org/10.1016/j.neuroscience.2015.03.054 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 205
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EXPERIMENTAL PROCEDURES Tissue samples Tissue blocks containing half of the hypothalamus split by the midsagittal line (one man, 89 years of age [cause of death: chronic obstructive pulmonary disease] and two adult women, 82 [cause of death: pneumonia, hypertension] and 86 [cause of death: sudden cardiac death, pulmonary hypertension] years of age) were harvested from autopsies at less than 24 h post mortem period, in accordance with the regulations of the Institutional Review Board of Lake Erie College of Osteopathic Medicine (LECOM). The clinical records of the individuals did not indicate any neurological or neuroendocrinological disorders, no Alzheimer’s or Parkinson diseases or stroke have been recorded.
created by denoting the neurons on these figures using Olympus BX45 microscope with camera lucida, and Adobe Photoshop software, version 7.0. The 3D model of the hypothalamic somatostatinergic and GHRH systems were created by the computer-generated superimposition of the consecutive sections using VoxBlast NT/9x Version 3.0 Light (Vaytek, Image Analysis Facility, University of Iowa, Iowa City, IA, USA). Our criteria for somatostatinergic-GHRH associations were twofold; (1) the somatostatinergic axonal varicosities must contact the GHRH perikarya and (2) the fibers must follow the GHRH cell bodies and/or the emanating axons for a significant length.
RESULTS Somatostatinergic system
Tissue preparation The harvested hypothalami were fixed by immersion in 0.1 M phosphate-buffered (pH 7.4; PB) 4% formaldehyde at 4 °C for 2–8 weeks. Each block contained half of the hypothalamus divided in the midsagittal line. The samples were cryoprotected with 30% sucrose in phosphate buffer containing 0.9% sodium chloride (PBS) supplemented with 0.15% sodium-azide and then sectioned on a freezing microtome at 30 lm intervals in coronal planes. The sections were collected in three series of wells of plastic 24-compartment plates with PBS containing 0.2% sodium-azide, and stored at 4 °C until processing. Immunohistochemistry Immunohistochemistry was carried out on representative sections containing the infundibulum/median eminence using streptavidin–biotin (ABC) methods described previously (Dudas and Merchenthaler, 2006). Simultaneous detection of the somatostatinergic and GHRH-IR structures was performed using double-label immunohistochemistry. The somatostatinergic structures were visualized with rabbit anti-somatostatin serum (Chemicon, Temecula, CA, USA; dilution 1:5000) and the GHRH-containing structures were identified using a rabbit anti-GHRH serum (Chemicon, Temecula, CA) at a dilution of 1:8000. The first signal of the immunohistochemistry was visualized by using the black Vector SG chromogen (Vector Laboratories, Burlingame, CA, USA), and then the second signal was revealed with NovaRed (Vector Laboratories, Burlingame, CA). In control sections, the primary antibodies were omitted or replaced by non-immune rabbit serum at the dilution of the used primary antibodies. No immunoreaction was observed in these control sections. Computer assisted mapping and microscopic analysis Following mounting and coverslipping, the hypothalamic sections were scanned and the contours of the sections were traced by CorelTrace software 4.0. The map depicting the somatostatinergic and GHRH neurons was
The vast majority of the somatostatin-IR neuronal cell bodies are located in the periventricular area of the preoptic and infundibular regions and in the infundibulum/ median eminence (arcuate nucleus; Figs. 1 and 2A). Numerous somatostatin-IR perikarya can be observed in the nucleus of the diagonal band of Broca and in the suprachiasmatic and ventromedial nuclei. Paraventricular nuclei contain several somatostatinergic perikarya (Fig. 2B), while only negligible amounts of somatostatinergic axonal varicosities, without perikarya can be detected in the supraoptic nucleus. Mammillary nuclei and the supramammillary nuclei as well as the perifornical area of the tuberal region also contain cell bodies (Fig. 2C, D). Somatostatinergic cell bodies are scattered in the lateral hypothalamus, particularly at the infundibular and posterior hypothalamic regions. The lamina terminalis contains small numbers of perikarya along with fiber varicosities. Somatostatin-IR axonal varicosities form a relatively dense network in the infundibulum and periventricular area of the preoptic and infundibular regions, while the medial hypothalamic regions contain only few somatostatin-IR fibers. Occasionally, well-defined terminal fields can be observed in the periventricular region where somatostatinergic fibers form baskets around neurons that are obviously not IR for somatostatin (Fig. 2E). GHRH system The morphology and distribution of the GHRH-IR elements in human have been described in our previous studies (Dudas and Merchenthaler, 2006; Deltondo et al., 2008). Briefly, GHRH-IR perikarya are confined to the basal hypothalamus (Figs. 1 and 3). At the basal part of the infundibular region perikarya form four well-defined subdivisions (Fig. 3): (1) The majority of the GHRH-IR cell bodies can be observed in the infundibulum/median eminence and (2) in the basal part of the periventricular zone. (3) The dorsomedial subdivision of the ventromedial nucleus as well as (4) the basal perifornical area of the tuberal region also contains a group of neurons. GHRH neurons are generally fusiform in shape with processes emanating from the opposite ends of the cells. Only few
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Fig. 1. Stereoscopic images of the human diencephalon illustrating somatostatinergic perikarya (blue dots) and GHRH-IR perikarya (red dots). The overlapping sites between these systems are primarily located in the basal part of the infundibulum. Stereoscopic images can be seen using U or parallel vision. The eyes are relaxed to look into the distance until the pair of the images fuse, and then refocused by the brain. With this technique a 3D hypothalamus can be seen on the figure, appearing to float in front of the paper, with the immunolabeled perikarya (marked by black and red dots) discernible at different depth. If the separation is too great for the eyes (double vision), move the picture away and the required viewing angle will decrease. Stereoscopic magnifier is suggested to readers unfamiliar with U or parallel vision. Abbreviations: INF, infundibulum; MB, mammillary body; OCh, optic chiasm; PVN, paraventricular nucleus.
Fig. 2. Somatostatinergic neural elements in the human hypothalamus. (A) Infundibulum contains large numbers of somatostatin-IR perikarya. The pial surface is marked by asterisk. (B) Somatostatinergic perikarya are common in the paraventricular nucleus where their processes are often in close proximity of vessels. Asterisk denotes the ependyma lining the 3rd ventricle. (C) Fusiform somatostatin-IR neurons surround the fornix (asterisk). Somatostatin-IR cells are also located around the mammillary body (D). Occasionally, well-defined terminal fields can be observed in the periventricular region around neurons that are not immunoreactive for somatostatin (E). Magnification: (A–B): 200; (C–D): 400.
GHRH-IR perikarya can be detected in the medial preoptic area and in the posterior hypothalamus and none in the lateral hypothalamus. In the basal part of the infundibulum, GHRH-IR axonal varicosities form a dense network. GHRH-IR fibers can also be detected in the basal periventricular area and in the basal part of the medial hypothalamus, oriented parallel with the pial surface. Fibers can also be found in the perifornical area at the preoptic and tuberal regions. Few labeled axon varicosities can be detected in the lamina terminalis, in medial and lateral zones of the paraventricular nucleus, in the basal zone of the lateral hypothalamus and around the medial part of the mammillary body while no GHRH-IR fibers can be observed in the supraoptic nucleus. Juxtapositions between the somatostatinergic and GHRH systems By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya
received contacting somatostatinergic axonal varicosities in the arcuate nucleus (Fig. 4A–I). Here, the somatostatinergic axonal varicosities abutted GHRH perikarya in en passant fashion, often forming multiple contacts while passing by following the contours of the GHRH cell bodies and often the emanating axons as well. In contrast, GHRH fibers commonly avoided the close vicinity of the somatostatinergic perikarya (Fig. 4J) and abutted somatostatinergic neurons only rarely, forming only few contacts (Fig. 4K). These uncommon GHRH-somatostatinergic juxtapositions were restricted primarily to the basal part of the periventricular area.
DISCUSSION The release of GHRH from the hypothalamic infundibular nucleus (arcuate nucleus in rodents) regulates the secretion of GH from the anterior pituitary. The release of GHRH is antagonized by somatostatin synthesized in the anterior periventricular
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Fig. 3. Frontal section of the infundibular region of the human hypothalamus with Nissl staining, illustrating the location of the groups of GHRH neurons. The vast majority of the GHRH-IR perikarya are located in the (1) infundibulum/median eminence and in the (2) basal part of the periventricular zone. GHRH-IR cell bodies can also be found in the (3) ventromedial nucleus and in the (4) the basal perifornical area of the tuberal region. Abbreviations: Am, accessory magnocellular neurons; AN, arcuate nucleus; DM, dorsomedial nucleus; Fx, fornix; LHA, lateral hypothalamic area; OT, optic tract; PVNd, paraventricular nucleus, dorsal part; PVNm, paraventricular nucleus, magnocellular part; SON, supraoptic nucleus; VMd, ventromedial nucleus, dorsomedial subdivision; VMv, ventromedial nucleus, ventrolateral subdivision.
nucleus which project to the hypophyseal portal capillaries in the median eminence. While GHRH stimulates membrane depolarization and opens sodium channels leading to GH secretion, somatostatin activates and opens potassium channels leading to hyperpolarization and inhibition of GH secretion (Tsaneva-Atanasova et al., 2007). The released GH has direct effect on target cells or stimulates the production of insulin-like growth factor (IGF)-I. In addition to the antagonistic actions of GHRH and somatostatinergic systems on GH secretion, somatostatin regulates GH release by inhibiting GHRH secretion (Plotsky and Vale, 1985; Katakami et al., 1988). Indeed, in the present study, we have observed juxtapositions between the somatostinergic and GHRH-IR neuronal elements. The light microscopic analysis of these associations is not able to determine whether these contacts are functional synapses or not; however, the relatively long post mortem time of the cadavers does not make ultrastructural studies possible. Due to the rarity of the suitable samples, we were able to utilize a limited number of hypothalami. However, the abundance of the
juxtapositions we have observed between the GHRH and somatostatinergic elements suggests that these neurotransmitter systems communicate at the hypothalamic level directly by synaptic contacts. The secretion of GH from the pituitary is tightly regulated not only by GHRH and somatostatin in the hypothalamus but via negative feedback by GH and IGF-I at the level of the pituitary and hypothalamus. The negative feedback of GH and IGF-I at the level of the hypothalamus is predominantly acting to increase somatostatin secretion [see (Murray and Clayton, 2013) for a recent review] which is in turn decreases GHRH release. Indeed, the current morphological observations in the human hypothalamus support this view by describing primarily somatostatin to GHRH neuronal connections in the infundibulum. In contrast, the present study revealed that the GHRH to somatostatin connections are rare, indicating that GHRH does not influence the somatostinergic system significantly, and therefore, the negative feedback effect of GH and IGF-I targets the somatostatinergic system directly at the hypothalamic level and not via the GHRH system. These data are in consensus with previous studies describing more abundant somatostatin to GHRH juxtapositions than GHRH to somatostatin associations in rats (Willoughby et al., 1989). However, it is possible that GHRH may regulate somatostatin release indirectly by either hormonal routes or via other neurotransmitter systems and not via direct synaptic contacts. This negative feedback action of GH centered on increasing the somatostatin release may occur indirectly, via other neurotransmitter systems. The activity of GHRH and somatostatin is regulated by neurotransmitters, neuropeptides, and hormones. NPY, a potent orexigenic peptide, has been also recognized as an inhibitor of GH secretion. NPY stimulates hypophysiotropic somatostatin release and thereby inhibits GH secretion (Rettori et al., 1990). The indirect inhibitory role of NPY is also supported by the demonstration of synaptic connections between NPY-positive axon terminals and somatostatin-IR neurons in the periventricular nucleus (Hisano et al., 1990). GH is thought to exert a short-loop feedback action on the hypothalamic somatostatin- and GHRH-containing neurons. Indeed, GH receptors are found on somatostatin neurons in the periventricular nucleus, on NPY neurons in the arcuate nucleus, but not on GHRH neurons (Burton et al., 1992; Chan et al., 1996) which contradicts the claim that almost all NPY neurons also contain GHRH (Ciofi et al., 1987). We have shown that in the human hypothalamus GHRH-IR neurons have close contacts with NPY (Dudas and Merchenthaler, 2006), and therefore, NPY may play similar roles on GH secretion in humans and rodents. Another type of regulation of neuronal activity is paracrine or autocrine regulation is based on colocalization of multiple chemical messengers. GHRH and somatostatin neurons colocalize a variety of neuropeptides and neurotransmitters (Meister and Hokfelt, 1988; Sawchenko and Swanson, 1990; Fodor et al., 2006) but not each other. Colocalization of different
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Fig. 4. Somatostatinergic axonal varicosities (black) form intimate juxtapositions with the GHRH-IR perikarya (brown) in the human hypothalamus (A–I). The inserts J–K depict somatostatinergic perikarya in brown color and GHRH fibers in black. The majority of the somatostatinergic–GHRH associations are located in the infundibulum/median eminence where GHRH neurons receive contacting somatostatinergic axons in en passant fashion. Only few somatostatinergic neurons receive juxtaposing GHRH axons, mainly in the periventricular area (K); in the most frequent cases GHRH fibers do not contact somatostatinergic perikarya in the median eminence (J). The positions of the illustrated GHRH and somatostatinergic neurons are denoted by asterisks on the hypothalamic frontal sections at the corner of the inserts. Abbreviations: Fx, fornix; Ot, optic tract. Scale bar = 10 lm.
neurotransmitters and neuropeptides in neurohormonecontaining cells is functionally relevant, because it facilitates differential regulation of distinct subsets of cell populations and contributes to the fined-tuned regulation of their activities.
CONCLUSION The morphology and the abundance of the somatostatinGHRH juxtapositions indicate that these contacts between the somatostatinergic fibers and GHRH
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perikarya may be functional synapses, and may represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion in humans. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary in humans by regulating the hypothalamic GHRH secretion via direct synaptic contacts. In contrast, the relatively small numbers of GHRH-somatostatinergic juxtapositions we have detected indicate that GHRH does not have significant direct influence on the hypothalamic somatostatin secretion and may influence the somatostatinergic system mainly indirectly, via other neurotransmitter systems. The widespread distribution of the somatostatinergic elements in the human hypothalamus as well as the well-defined somatostatinergic terminal fields suggest that somatostatin, apart from its endocrine function to regulate GH secretion, may play a pivotal role in the regulation of other hypothalamic functions acting as a neurotransmitter/neuromodulator.
REFERENCES Bertherat J, Bluet-Pajot MT, Epelbaum J (1995) Neuroendocrine regulation of growth hormone. Eur J Endocrinol 132:12–24. Bertherat J, Dournaud P, Berod A, Normand E, Bloch B, Rostene W, Kordon C, Epelbaum J (1992) Growth hormone-releasing hormone-synthesizing neurons are a subpopulation of somatostatin receptor-labelled cells in the rat arcuate nucleus: a combined in situ hybridization and receptor light-microscopic radioautographic study. Neuroendocrinology 56:25–31. Burton KA, Kabigting EB, Clifton DK, Steiner RA (1992) Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat brain and its colocalization in hypothalamic somatostatin neurons. Endocrinology 131:958–963. Chan YY, Steiner RA, Clifton DK (1996) Regulation of hypothalamic neuropeptide-Y neurons by growth hormone in the rat. Endocrinology 137:1319–1325. Ciofi P, Croix D, Tramu G (1987) Coexistence of hGHRF and NPY immunoreactivities in neurons of the arcuate nucleus of the rat. Neuroendocrinology 45:425–428. Daikoku S, Hisano S, Kawano H, Chikamori-Aoyama M, Kagotani Y, Zhang RJ, Chihara K (1988) Ultrastructural evidence for neuronal regulation of growth hormone secretion. Neuroendocrinology 47:405–415. Deltondo J, Por I, Hu W, Merchenthaler I, Semeniken K, Jojart J, Dudas B (2008) Associations between the human growth hormone-releasing hormoneand neuropeptide-Yimmunoreactive systems in the human diencephalon: a possible morphological substrate of the impact of stress on growth. Neuroscience 153:1146–1152. Dudas B, Merchenthaler I (2006) Three-dimensional representation of the neurotransmitter systems of the human hypothalamus: inputs of the gonadotrophin hormone-releasing hormone neuronal system. J Neuroendocrinol 18:79–95. Dudas B, Merchenthaler I (2014) Central catecholaminergic regulation of growth. In: Dudas B, editor. The central catecholaminergic system: anatomy, functions and disorders. New York: NOVA. p. 85–100. Dudas B, Peroski M (2013) Hypothalamic regulation of growth. In: Dudas B, editor. The human hypothalamus: anatomy, functions and disorders. New York: NOVA. p. 173–190.
Fodor M, Kordon C, Epelbaum J (2006) Anatomy of the hypophysiotropic somatostatinergic and growth hormonereleasing hormone system minireview. Neurochem Res 31:137–143. Hisano S, Tsuruo Y, Kagotani Y, Daikoku S, Chihara K (1990) Immunohistochemical evidence for synaptic connections between neuropeptide Y-containing axons and periventricular somatostatin neurons in the anterior hypothalamus in rats. Brain Res 520:170–177. Horvath S, Palkovits M, Gorcs T, Arimura A (1989) Electron microscopic immunocytochemical evidence for the existence of bidirectional synaptic connections between growth hormonereleasing hormone- and somatostatin-containing neurons in the hypothalamus of the rat. Brain Res 481:8–15. Katakami H, Downs TR, Frohman LA (1988) Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormone-releasing factor in the rat. Endocrinology 123:1103–1109. Liposits Z, Merchenthaler I, Paull WK, Flerko B (1988) Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat. Histochemistry 89:247–252. McCarthy GF, Beaudet A, Tannenbaum GS (1992) Colocalization of somatostatin receptors and growth hormone-releasing factor immunoreactivity in neurons of the rat arcuate nucleus. Neuroendocrinology 56:18–24. Meister B, Hokfelt T (1988) Peptide- and transmitter-containing neurons in the mediobasal hypothalamus and their relation to GABAergic systems: possible roles in control of prolactin and growth hormone secretion. Synapse 2:585–605. Muller EE, Locatelli V, Cocchi D (1999) Neuroendocrine control of growth hormone secretion. Physiol Rev 79:511–607. Murray PG, Clayton PE (2013) Endocrine control of growth. Am J Med Genet C Semin Med Genet 163C:76–85. Olsen J, Peroski M, Kiczek M, Grignol G, Merchenthaler I, Dudas B (2014) Intimate associations between the endogenous opiate systems and the growth hormone-releasing hormone system in the human hypothalamus. Neuroscience 258:238–245. Plotsky PM, Vale W (1985) Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial-portal circulation of the rat. Science 230:461–463. Rettori V, Milenkovic L, Aguila MC, McCann SM (1990) Physiologically significant effect of neuropeptide Y to suppress growth hormone release by stimulating somatostatin discharge. Endocrinology 126:2296–2301. Rotoli G, Grignol G, Hu W, Merchenthaler I, Dudas B (2011) Catecholaminergic axonal varicosities appear to innervate growth hormone-releasing hormone-immunoreactive neurons in the human hypothalamus: the possible morphological substrate of the stress-suppressed growth. J Clin Endocrinol Metab 96:E1606–E1611. Sawchenko PE, Swanson LW (1990) Growth hormone releasing hormone. In: Bjorklund A, Hokfelt T, Kuhar MJ, editors. Handbook of chemical neuroanatomy. Amsterdam: Elsevier. p. 131–163. Tannenbaum GS, Zhang WH, Lapointe M, Zeitler P, Beaudet A (1998) Growth hormone-releasing hormone neurons in the arcuate nucleus express both Sst1 and Sst2 somatostatin receptor genes. Endocrinology 139:1450–1453. Tsaneva-Atanasova K, Sherman A, van GF, Stojilkovic SS (2007) Mechanism of spontaneous and receptor-controlled electrical activity in pituitary somatotrophs: experiments and theory. J Neurophysiol 98:131–144. Willoughby JO, Brogan M, Kapoor R (1989) Hypothalamic interconnections of somatostatin and growth hormone releasing factor neurons. Neuroendocrinology 50:584–591.
(Accepted 24 March 2015) (Available online 1 April 2015)
JUXTAPOSITIONS BETWEEN THE SOMATOSTATINERGIC AND GROWTH HORMONE-RELEASING HORMONE (GHRH) NEURONS IN THE HUMAN HYPOTHALAMUS N. PROUDAN, a M. PEROSKI, a G. GRIGNOL, a I. MERCHENTHALER b,c AND B. DUDAS a*
secretion via direct contacts. The rare GHRH to somatostatin juxtapositions indicate that the negative feedback effect of GH targets the somatostatinergic system directly and not via the GHRH system. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
a Laboratory of Neuroendocrine Organization (NEO), Lake Erie College of Osteopathic Medicine, Erie, PA, United States b Department of Epidemiology & Public Health, University of Maryland, School of Medicine, Baltimore, MD, United States c Department of Anatomy & Neurobiology, University of Maryland, School of Medicine, Baltimore, MD, United States
Key words: hypothalamus, somatostatin, GHRH, growth.
Abstract—Somatostatin is a 14–28 amino acid peptide that is located not only in the gastrointestinal system but also in multiple sites of the human brain. The inhibitory effect of somatostatin on the growth hormone (GH) secretion of the pituitary gland is a well-established phenomenon. There is a general consensus that somatostatin is released into the hypophysial portal blood and modulates GH secretion by hormonal action. In the present study, we explored the possibility that in addition to the hormonal route, somatostatin may also influence GH secretion via influencing the growth hormone-releasing hormone (GHRH) secretion by direct contacts that may be functional synapses. Since the verification of these putative synapses by electron microscopy is virtually impossible in humans due to the long post mortem time, in order to reveal the putative somatostatinergic-GHRH juxtapositions, light microscopic double-label immunohistochemistry was utilized. By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya received contacting somatostatinergic axonal varicosities in the arcuate nucleus. In contrast, GHRH axonal varicosities rarely contacted somatostatinergic perikarya. The morphology and the abundance of somatostatin to GHRH juxtapositions indicate that these associations are functional synapses, and they represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary by regulating the hypothalamic GHRH
INTRODUCTION The central control of growth hormone (GH) secretion from the anterior pituitary is under the control of two hypothalamic neurohormones: growth hormonereleasing hormone (GHRH) and growth hormone release-inhibiting hormone (GH-RIH) or somatostatin. The activity of these hypophysiotropic hormonesynthesizing neurons is regulated by a number of neuropeptides, neurotransmitters, and hormones. Interestingly, however, GHRH and somatostatinergic systems also communicate with each other directly to maintain a delicate control over GH secretion. Ultrastructural findings indicate a bidirectional synaptic contacts between GHRH and somatostatinimmunoreactive (IR) hypophysiotropic neurons in the rat (Daikoku et al., 1988; Liposits et al., 1988; Horvath et al., 1989) and somatostatin receptors have been shown in the majority of GHRH neurons of the ventrolateral portion of the arcuate nucleus (Bertherat et al., 1992, 1995; McCarthy et al., 1992; Muller et al., 1999). Moreover, selective membrane somatostatin receptors sst1 or sst2 have also been localized in a subpopulation of GHRH neurons (Tannenbaum et al., 1998). Contrary to these finding in rats, no studies have been conducted in humans to elucidate the connections between GHRH and somatostatin. We have previously described that the GHRH system receives input from other neurotransmitter systems (neuropeptide Y [NPY], endogenous opiate and catecholaminergic systems) in the human hypothalamus (Deltondo et al., 2008; Rotoli et al., 2011; Dudas and Peroski, 2013; Dudas and Merchenthaler, 2014; Olsen et al., 2014). Thus, in the present study we utilized light microscopic double-label immunohistochemistry in order to reveal the putative juxtapositions between the somatostatinergic and GHRH elements in the human diencephalon.
*Corresponding author. Address: Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16509, United States. Tel: +1-814-866-8142; fax: +1-814-866-8411. E-mail address: [email protected] (B. Dudas). Abbreviations: GH, growth hormone; GHRH, growth hormonereleasing hormone; GH-RIH, growth hormone release-inhibiting hormone; IGF, insulin-like growth factor; IR, immunoreactive; NPY, neuropeptide Y; PBS, phosphate buffer containing 0.9% sodium chloride. http://dx.doi.org/10.1016/j.neuroscience.2015.03.054 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 205
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EXPERIMENTAL PROCEDURES Tissue samples Tissue blocks containing half of the hypothalamus split by the midsagittal line (one man, 89 years of age [cause of death: chronic obstructive pulmonary disease] and two adult women, 82 [cause of death: pneumonia, hypertension] and 86 [cause of death: sudden cardiac death, pulmonary hypertension] years of age) were harvested from autopsies at less than 24 h post mortem period, in accordance with the regulations of the Institutional Review Board of Lake Erie College of Osteopathic Medicine (LECOM). The clinical records of the individuals did not indicate any neurological or neuroendocrinological disorders, no Alzheimer’s or Parkinson diseases or stroke have been recorded.
created by denoting the neurons on these figures using Olympus BX45 microscope with camera lucida, and Adobe Photoshop software, version 7.0. The 3D model of the hypothalamic somatostatinergic and GHRH systems were created by the computer-generated superimposition of the consecutive sections using VoxBlast NT/9x Version 3.0 Light (Vaytek, Image Analysis Facility, University of Iowa, Iowa City, IA, USA). Our criteria for somatostatinergic-GHRH associations were twofold; (1) the somatostatinergic axonal varicosities must contact the GHRH perikarya and (2) the fibers must follow the GHRH cell bodies and/or the emanating axons for a significant length.
RESULTS Somatostatinergic system
Tissue preparation The harvested hypothalami were fixed by immersion in 0.1 M phosphate-buffered (pH 7.4; PB) 4% formaldehyde at 4 °C for 2–8 weeks. Each block contained half of the hypothalamus divided in the midsagittal line. The samples were cryoprotected with 30% sucrose in phosphate buffer containing 0.9% sodium chloride (PBS) supplemented with 0.15% sodium-azide and then sectioned on a freezing microtome at 30 lm intervals in coronal planes. The sections were collected in three series of wells of plastic 24-compartment plates with PBS containing 0.2% sodium-azide, and stored at 4 °C until processing. Immunohistochemistry Immunohistochemistry was carried out on representative sections containing the infundibulum/median eminence using streptavidin–biotin (ABC) methods described previously (Dudas and Merchenthaler, 2006). Simultaneous detection of the somatostatinergic and GHRH-IR structures was performed using double-label immunohistochemistry. The somatostatinergic structures were visualized with rabbit anti-somatostatin serum (Chemicon, Temecula, CA, USA; dilution 1:5000) and the GHRH-containing structures were identified using a rabbit anti-GHRH serum (Chemicon, Temecula, CA) at a dilution of 1:8000. The first signal of the immunohistochemistry was visualized by using the black Vector SG chromogen (Vector Laboratories, Burlingame, CA, USA), and then the second signal was revealed with NovaRed (Vector Laboratories, Burlingame, CA). In control sections, the primary antibodies were omitted or replaced by non-immune rabbit serum at the dilution of the used primary antibodies. No immunoreaction was observed in these control sections. Computer assisted mapping and microscopic analysis Following mounting and coverslipping, the hypothalamic sections were scanned and the contours of the sections were traced by CorelTrace software 4.0. The map depicting the somatostatinergic and GHRH neurons was
The vast majority of the somatostatin-IR neuronal cell bodies are located in the periventricular area of the preoptic and infundibular regions and in the infundibulum/ median eminence (arcuate nucleus; Figs. 1 and 2A). Numerous somatostatin-IR perikarya can be observed in the nucleus of the diagonal band of Broca and in the suprachiasmatic and ventromedial nuclei. Paraventricular nuclei contain several somatostatinergic perikarya (Fig. 2B), while only negligible amounts of somatostatinergic axonal varicosities, without perikarya can be detected in the supraoptic nucleus. Mammillary nuclei and the supramammillary nuclei as well as the perifornical area of the tuberal region also contain cell bodies (Fig. 2C, D). Somatostatinergic cell bodies are scattered in the lateral hypothalamus, particularly at the infundibular and posterior hypothalamic regions. The lamina terminalis contains small numbers of perikarya along with fiber varicosities. Somatostatin-IR axonal varicosities form a relatively dense network in the infundibulum and periventricular area of the preoptic and infundibular regions, while the medial hypothalamic regions contain only few somatostatin-IR fibers. Occasionally, well-defined terminal fields can be observed in the periventricular region where somatostatinergic fibers form baskets around neurons that are obviously not IR for somatostatin (Fig. 2E). GHRH system The morphology and distribution of the GHRH-IR elements in human have been described in our previous studies (Dudas and Merchenthaler, 2006; Deltondo et al., 2008). Briefly, GHRH-IR perikarya are confined to the basal hypothalamus (Figs. 1 and 3). At the basal part of the infundibular region perikarya form four well-defined subdivisions (Fig. 3): (1) The majority of the GHRH-IR cell bodies can be observed in the infundibulum/median eminence and (2) in the basal part of the periventricular zone. (3) The dorsomedial subdivision of the ventromedial nucleus as well as (4) the basal perifornical area of the tuberal region also contains a group of neurons. GHRH neurons are generally fusiform in shape with processes emanating from the opposite ends of the cells. Only few
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Fig. 1. Stereoscopic images of the human diencephalon illustrating somatostatinergic perikarya (blue dots) and GHRH-IR perikarya (red dots). The overlapping sites between these systems are primarily located in the basal part of the infundibulum. Stereoscopic images can be seen using U or parallel vision. The eyes are relaxed to look into the distance until the pair of the images fuse, and then refocused by the brain. With this technique a 3D hypothalamus can be seen on the figure, appearing to float in front of the paper, with the immunolabeled perikarya (marked by black and red dots) discernible at different depth. If the separation is too great for the eyes (double vision), move the picture away and the required viewing angle will decrease. Stereoscopic magnifier is suggested to readers unfamiliar with U or parallel vision. Abbreviations: INF, infundibulum; MB, mammillary body; OCh, optic chiasm; PVN, paraventricular nucleus.
Fig. 2. Somatostatinergic neural elements in the human hypothalamus. (A) Infundibulum contains large numbers of somatostatin-IR perikarya. The pial surface is marked by asterisk. (B) Somatostatinergic perikarya are common in the paraventricular nucleus where their processes are often in close proximity of vessels. Asterisk denotes the ependyma lining the 3rd ventricle. (C) Fusiform somatostatin-IR neurons surround the fornix (asterisk). Somatostatin-IR cells are also located around the mammillary body (D). Occasionally, well-defined terminal fields can be observed in the periventricular region around neurons that are not immunoreactive for somatostatin (E). Magnification: (A–B): 200; (C–D): 400.
GHRH-IR perikarya can be detected in the medial preoptic area and in the posterior hypothalamus and none in the lateral hypothalamus. In the basal part of the infundibulum, GHRH-IR axonal varicosities form a dense network. GHRH-IR fibers can also be detected in the basal periventricular area and in the basal part of the medial hypothalamus, oriented parallel with the pial surface. Fibers can also be found in the perifornical area at the preoptic and tuberal regions. Few labeled axon varicosities can be detected in the lamina terminalis, in medial and lateral zones of the paraventricular nucleus, in the basal zone of the lateral hypothalamus and around the medial part of the mammillary body while no GHRH-IR fibers can be observed in the supraoptic nucleus. Juxtapositions between the somatostatinergic and GHRH systems By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya
received contacting somatostatinergic axonal varicosities in the arcuate nucleus (Fig. 4A–I). Here, the somatostatinergic axonal varicosities abutted GHRH perikarya in en passant fashion, often forming multiple contacts while passing by following the contours of the GHRH cell bodies and often the emanating axons as well. In contrast, GHRH fibers commonly avoided the close vicinity of the somatostatinergic perikarya (Fig. 4J) and abutted somatostatinergic neurons only rarely, forming only few contacts (Fig. 4K). These uncommon GHRH-somatostatinergic juxtapositions were restricted primarily to the basal part of the periventricular area.
DISCUSSION The release of GHRH from the hypothalamic infundibular nucleus (arcuate nucleus in rodents) regulates the secretion of GH from the anterior pituitary. The release of GHRH is antagonized by somatostatin synthesized in the anterior periventricular
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Fig. 3. Frontal section of the infundibular region of the human hypothalamus with Nissl staining, illustrating the location of the groups of GHRH neurons. The vast majority of the GHRH-IR perikarya are located in the (1) infundibulum/median eminence and in the (2) basal part of the periventricular zone. GHRH-IR cell bodies can also be found in the (3) ventromedial nucleus and in the (4) the basal perifornical area of the tuberal region. Abbreviations: Am, accessory magnocellular neurons; AN, arcuate nucleus; DM, dorsomedial nucleus; Fx, fornix; LHA, lateral hypothalamic area; OT, optic tract; PVNd, paraventricular nucleus, dorsal part; PVNm, paraventricular nucleus, magnocellular part; SON, supraoptic nucleus; VMd, ventromedial nucleus, dorsomedial subdivision; VMv, ventromedial nucleus, ventrolateral subdivision.
nucleus which project to the hypophyseal portal capillaries in the median eminence. While GHRH stimulates membrane depolarization and opens sodium channels leading to GH secretion, somatostatin activates and opens potassium channels leading to hyperpolarization and inhibition of GH secretion (Tsaneva-Atanasova et al., 2007). The released GH has direct effect on target cells or stimulates the production of insulin-like growth factor (IGF)-I. In addition to the antagonistic actions of GHRH and somatostatinergic systems on GH secretion, somatostatin regulates GH release by inhibiting GHRH secretion (Plotsky and Vale, 1985; Katakami et al., 1988). Indeed, in the present study, we have observed juxtapositions between the somatostinergic and GHRH-IR neuronal elements. The light microscopic analysis of these associations is not able to determine whether these contacts are functional synapses or not; however, the relatively long post mortem time of the cadavers does not make ultrastructural studies possible. Due to the rarity of the suitable samples, we were able to utilize a limited number of hypothalami. However, the abundance of the
juxtapositions we have observed between the GHRH and somatostatinergic elements suggests that these neurotransmitter systems communicate at the hypothalamic level directly by synaptic contacts. The secretion of GH from the pituitary is tightly regulated not only by GHRH and somatostatin in the hypothalamus but via negative feedback by GH and IGF-I at the level of the pituitary and hypothalamus. The negative feedback of GH and IGF-I at the level of the hypothalamus is predominantly acting to increase somatostatin secretion [see (Murray and Clayton, 2013) for a recent review] which is in turn decreases GHRH release. Indeed, the current morphological observations in the human hypothalamus support this view by describing primarily somatostatin to GHRH neuronal connections in the infundibulum. In contrast, the present study revealed that the GHRH to somatostatin connections are rare, indicating that GHRH does not influence the somatostinergic system significantly, and therefore, the negative feedback effect of GH and IGF-I targets the somatostatinergic system directly at the hypothalamic level and not via the GHRH system. These data are in consensus with previous studies describing more abundant somatostatin to GHRH juxtapositions than GHRH to somatostatin associations in rats (Willoughby et al., 1989). However, it is possible that GHRH may regulate somatostatin release indirectly by either hormonal routes or via other neurotransmitter systems and not via direct synaptic contacts. This negative feedback action of GH centered on increasing the somatostatin release may occur indirectly, via other neurotransmitter systems. The activity of GHRH and somatostatin is regulated by neurotransmitters, neuropeptides, and hormones. NPY, a potent orexigenic peptide, has been also recognized as an inhibitor of GH secretion. NPY stimulates hypophysiotropic somatostatin release and thereby inhibits GH secretion (Rettori et al., 1990). The indirect inhibitory role of NPY is also supported by the demonstration of synaptic connections between NPY-positive axon terminals and somatostatin-IR neurons in the periventricular nucleus (Hisano et al., 1990). GH is thought to exert a short-loop feedback action on the hypothalamic somatostatin- and GHRH-containing neurons. Indeed, GH receptors are found on somatostatin neurons in the periventricular nucleus, on NPY neurons in the arcuate nucleus, but not on GHRH neurons (Burton et al., 1992; Chan et al., 1996) which contradicts the claim that almost all NPY neurons also contain GHRH (Ciofi et al., 1987). We have shown that in the human hypothalamus GHRH-IR neurons have close contacts with NPY (Dudas and Merchenthaler, 2006), and therefore, NPY may play similar roles on GH secretion in humans and rodents. Another type of regulation of neuronal activity is paracrine or autocrine regulation is based on colocalization of multiple chemical messengers. GHRH and somatostatin neurons colocalize a variety of neuropeptides and neurotransmitters (Meister and Hokfelt, 1988; Sawchenko and Swanson, 1990; Fodor et al., 2006) but not each other. Colocalization of different
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Fig. 4. Somatostatinergic axonal varicosities (black) form intimate juxtapositions with the GHRH-IR perikarya (brown) in the human hypothalamus (A–I). The inserts J–K depict somatostatinergic perikarya in brown color and GHRH fibers in black. The majority of the somatostatinergic–GHRH associations are located in the infundibulum/median eminence where GHRH neurons receive contacting somatostatinergic axons in en passant fashion. Only few somatostatinergic neurons receive juxtaposing GHRH axons, mainly in the periventricular area (K); in the most frequent cases GHRH fibers do not contact somatostatinergic perikarya in the median eminence (J). The positions of the illustrated GHRH and somatostatinergic neurons are denoted by asterisks on the hypothalamic frontal sections at the corner of the inserts. Abbreviations: Fx, fornix; Ot, optic tract. Scale bar = 10 lm.
neurotransmitters and neuropeptides in neurohormonecontaining cells is functionally relevant, because it facilitates differential regulation of distinct subsets of cell populations and contributes to the fined-tuned regulation of their activities.
CONCLUSION The morphology and the abundance of the somatostatinGHRH juxtapositions indicate that these contacts between the somatostatinergic fibers and GHRH
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perikarya may be functional synapses, and may represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion in humans. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary in humans by regulating the hypothalamic GHRH secretion via direct synaptic contacts. In contrast, the relatively small numbers of GHRH-somatostatinergic juxtapositions we have detected indicate that GHRH does not have significant direct influence on the hypothalamic somatostatin secretion and may influence the somatostatinergic system mainly indirectly, via other neurotransmitter systems. The widespread distribution of the somatostatinergic elements in the human hypothalamus as well as the well-defined somatostatinergic terminal fields suggest that somatostatin, apart from its endocrine function to regulate GH secretion, may play a pivotal role in the regulation of other hypothalamic functions acting as a neurotransmitter/neuromodulator.
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(Accepted 24 March 2015) (Available online 1 April 2015)
