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Distribution and morphology of the juxtapositions between growth hormone-releasing hormone-(ghrh)-immunoreactive neuronal elements
Growth Hormone & IGF Research 20 (2010) 356–359
Contents lists available at ScienceDirect
Growth Hormone & IGF Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g h i r
Distribution and morphology of the juxtapositions between growth hormone-releasing hormone-(ghrh)-immunoreactive neuronal elements Daniel Anderson a, Matthew Baker a, George Grignol a, Walter Hu a, Istvan Merchenthaler b, Bertalan Dudas a,⁎ a b
Neuroendocrine Organization Laboratory (NEO), Lake Erie College of Osteopathic Medicine (LECOM), 1858 West Grandview Blvd, Erie, PA 16509, United States Department of Epidemiology & Preventive Medicine and Anatomy & Neurobiology, University of Maryland, Baltimore, MD 21201, United States
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 21 June 2010 Accepted 23 June 2010 Available online 31 July 2010 Keywords: Hypothalamus Human Growth Immunohistochemistry
a b s t r a c t Previous studies revealed that growth hormone-releasing hormone (GHRH)-immunoreactive (IR) neurons form a circumscribed cell group in the basal infundibulum/median eminence of the human hypothalamus. GHRH from these neurons is released into the hypothalamo–hypophyseal portal circulatory system in a pulsatile manner. It is a common consensus that the pulsatile release of GHRH is the main driving force behind the pulsatile release of growth hormone (GH) and may contribute to the regulation of other hypothalamic functions. The pulsatile release of GHRH requires synchronized activity of GHRH-IR neurons. However, the morphological basis of this synchronization between the GHRH-IR neural elements has not been elucidated yet. Since the utilization of electron microscopy combined with immunohistochemistry is virtually impossible in the human brain due to the long post mortem period, immunohistochemistry, evaluated with oil immersion light microscopy, was used in order to reveal the associations between the GHRH elements. Numerous GHRH–GHRH juxtapositions have been detected in the infundibular area/median eminence, where GHRH-IR axonal varicosities often formed multiple contacts with GHRH-IR perikarya. Examination of these associations with high magnification oil immersion light microscopy revealed (1) axonal swellings at the site of the contacts and (2) no gaps between the contacting elements suggesting that these juxtapositions may be functional synapses. The large number of GHRH–GHRH juxtapositions in the infundibular area/ median eminence suggests that these synapse-like structures may represent the morphological substrate of the synchronized activity of GHRH neurons that in turn may result in the pulsatile release of GHRH in human. © 2010 Growth Hormone Research Society Published by Elsevier Ltd. All rights reserved.
1. Introduction Growth hormone-releasing hormone (GHRH) is a 44 amino acid peptide that is the primary regulator of growth hormone (GH) secretion of the pituitary gland. Previous studies described GHRHimmunoreactive (IR) perikarya located primarily in the basal infundibulum/median eminence of the rat [1] and the human hypothalamus [2]. GHRH from these neurons is released into the hypothalamo–hypophyseal portal circulatory system, and influences the release of growth hormone (GH) from the pituitary gland. The pulsatile release of GHRH is a well known phenomenon [3–7], and has been previously believed to be the single major factor driving the pulsatile secretion of GH, that is released in 10–20 pulses in each 24h cycle. However, the pulsatile pattern seems to be species-dependent. In cattle, the majority of the GH pulses are correlated to a preceding GHRH pulse indicating the crucial role of GHRH in the frequency control of the GH release [3]. Passive immunization with somatostatin ⁎ Corresponding author. Tel.: + 1 814 866 8142; fax: +1 814 866 8411. E-mail address: [email protected] (B. Dudas).
antiserum does not alter pulsatile GH release, but immunization with antibodies raised against both somatostatin and GHRH abolishes GH bursts [8]. In contrast to these findings, patients with an inactivating defect of the GHRH receptor gene maintain the regular frequency of the GH release with a diminished total 24-h GH production rate, suggesting that GH pulses are under the control of intermittent somatostatin withdrawal while the amplitude of GH pulses is driven by GHRH [9]. The fundamental role of somatostatin in the GH pulse generation has been supported by previous data describing that most of the descending phases of somatostatin pulses are associated with the initiation of GH pulses in goats [5]. Although the exact function of the pulsatile release of GHRH remains to be elusive, there is a general consensus that this phenomenon is based on the synchronized activity of the GHRH-IR neurons, suggesting communication between the GHRH-IR neural elements, either directly, via synaptic connections, or indirectly, via other neurotransmitter systems. In the present study, we examined the possibility of direct synapse-like contacts between the GHRH-IR neurons that may represent one of the key elements responsible for the pulsatile release of GHRH in human. Since the utilization of electron microscopy
1096-6374/$ – see front matter © 2010 Growth Hormone Research Society Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ghir.2010.06.002
D. Anderson et al. / Growth Hormone & IGF Research 20 (2010) 356–359
357
Fig. 1. Distribution of the GHRH-IR neural elements in the human hypothalamus. GHRH-IR neurons are most abundant in the infundibulum/median eminence (A) and in the basal part of the periventricular area (B), where GHRH-IR perikarya are surrounded with dense GHRH-IR fiber network. The dorsomedial subdivision of the ventromedial nucleus (C) and the basal perifornical area (D) contain a smaller number of GHRH-IR perikarya and axonal varicosities. Cross section of a portal vessel is denoted by a five-pointed star while a sixpointed star marks the 3rd ventricle. Magnification: 100x.
combined with immunohistochemistry is virtually impossible in the human brain due to the long post mortem period, light microscopic immunohistochemistry was used in order to reveal the associations between the GHRH elements. Contacts between GHRH-IR structures were evaluated with high magnification (100×) and oil immersion.
1% silicotungstic acid and 0.2% formaldehyde for 3–4 min [12,13]. Olympus BX45 microscope with a 100× oil immersion objective was used for the evaluation of the immunohistochemical staining.
2. Methods
The terminology of the diencephalic structures was adapted from Braak and Braak [14] and Saper [15].
2.3. Terminology
2.1. Brain samples 3. Results Hypothalami (2 adult men and 1 adult woman, 67–81 years of age) were harvested from autopsies at 24–48 h post mortem period in accordance with the regulation and permission of the Ethics Board of Lake Erie College of Osteopathic Medicine and the University of Szeged, Hungary. The clinical records of the individuals did not indicate any neurological and neuroendocrinological disorders. The brain samples were fixed by immersion in buffered 4% paraformaldehyde at 4 °C for 2–8 weeks. The samples were cryoprotected with 30% sucrose in phosphate buffered saline (PBS) and then sectioned on a freezing microtome at 30 μm intervals in coronal planes. 2.2. Immunohistochemistry Immunohistochemistry was performed using streptavidin–biotin (ABC) methods combined with silver intensification introduced by Gallyas and coworkers [10,11]. The samples were pretreated with 10% thioglycolic acid for 30 min to suppress the endogenous tissue argentophilia, then with 0.2% Triton X-100 for 20 min to increase permeability of immunoglobulins, and finally with 10% normal goat serum (NGS) in PBS for 1.0 h at room temperature to block nonspecific staining. Thereafter, the sections were incubated in a rabbit anti-GHRH serum (Chemicon, Temecula, CA) at a dilution of 1:8000 containing 10% NHS, PBS and 0.1% sodium azide for 24 h. The sections were then incubated for 1.0 h with biotinylated goat anti-rabbit immunoglobulins (IgG) (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA) diluted 1:1000 in PBS. Following two washes in PBS for a total of 15 min, the tissue was incubated for 1.0 h in a solution of streptavidin–horseradish peroxidase (Vectastain ABC Elite kit) diluted 1:1000 in PBS. The cross-reactions of the secondary antibodies with the endogenous human IgGs were eliminated by the addition of 2% normal human serum to the secondary antiserum. The sections were washed 2 × 10 min in PBS and 10 min in Tris-buffered saline (TBS; pH 7.6) and then reacted with 3′–3′ diaminobenzidine tetrahydrochloride (DAB), using nickel ammonium sulfate intensification (20 mg DAB, 100 mg nickel ammonium sulfate and 7.0 μl 30% H202 in 40 ml TBS). The resulting DAB polymer was silver-intensified in a solution containing 0.1% silver nitrate, 0.1% ammonium–nitrate,
3.1. Human GHRH system The distribution and morphology of GHRH-IR neural elements have been described in our previous study [2]. Briefly, GHRH-IR neurons formed a dense, well-circumscribed cell group with the perikarya located almost exclusively in the basal part of the infundibular region (Fig. 1). Here, cell bodies formed four welldefined subdivisions: (i) the majority of the GHRH-IR perikarya were located in the infundibulum/median eminence (Fig. 1A) and (ii) in the basal part of the periventricular zone (Fig. 1B). (iii) A group of perikarya were observed in the dorsomedial subdivision of the ventromedial nucleus (Fig. 1C) and (iv) in the basal perifornical area of the tuberal region (Fig. 1D). The majority of GHRH neurons possessed fusiform cell bodies with processes emanating from the opposite ends of the cells. GHRH-IR fibers formed a dense network in the basal part of the infundibulum (Fig. 1A). GHRH-IR axon varicosities were also detected in the periventricular area (Fig. 1B) and in the basal part of the medial hypothalamus. 3.2. GHRH–GHRH juxtapositions Juxtapositions between the GHRH-IR neural elements were detected mainly in the infundibular area/median eminence (Fig. 2A and B) and in the basal part of the periventricular zone (Fig. 2C and D), where 32% of the GHRH-IR perikarya appeared to form multiple (2 or more) contacts with GHRH-IR axonal varicosities. The rest of the examined perikarya were lightly innervated (1 contact/cell; 21%) or did not form apparent juxtapositions with abutting GHRH-IR fibers. Few juxtapositions were detected in the dorsomedial subdivision of the ventromedial nucleus (Fig. 2E) and in the basal perifornical area of the tuberal region (Fig. 2F), where only 11% of the detected GHRH-IR perikarya received abutting GHRH fibers. These juxtapositions were morphologically similar to the ones found in the infundibular area/ median eminence and in the basal part of the periventricular zone. The GHRH–GHRH associations found in the human hypothalamus were typically en passant juxtapositions where GHRH-IR fibers
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D. Anderson et al. / Growth Hormone & IGF Research 20 (2010) 356–359
Fig. 2. Juxtapositions between the GHRH-IR elements in the human hypothalamus. These GHRH–GHRH associations are usually en passant juxtapositions where GHRH-IR fibers abut GHRH-IR perikarya forming multiple contacts and typically exhibiting axonal swellings at the site of the contacts. The GHRH–GHRH juxtapositions are most dense in the infundibulum/median eminence (A and B) and in the basal part of the periventricular area (C and D), while juxtapositions are less numerous in the dorsomedial subdivision of the ventromedial nucleus (E) and the basal perifornical area (F). Some of the juxtapositions are denoted by arrowheads. Magnification: 400×.
abutted GHRH-IR perikarya usually forming multiple contacts (Fig. 2). At the site of the contacts, the GHRH-IR axons typically exhibited axonal swellings (Fig. 2). Examination of these associations with high magnification oil immersion light microscopy revealed no gaps between the contacting elements. 4. Discussion The present paper is the first describing juxtapositions between GHRH-IR axonal varicosities and cell bodies in the human hypothalamus. Although the verification of these putative synapses with electron microscopy is virtually impossible in these human samples due to the long post mortem time, previous studies have shown that similar juxtapositions represent synapses at the ultrastructural level in rat [16–19]. In the present study we report that (1) close examination of these juxtapositions with high magnification failed to reveal any gap between the contacting elements and (2) at the site of the contact, GHRH-IR axons abutting on GHRH-IR perikarya exhibited a swelling, suggesting that these associations may indeed be functional synapses.
The pulsatile activity of luteinizing hormone-releasing hormone (LHRH)-IR neurons is a well-established phenomenon [20]. Since previous studies revealed numerous juxtapositions between LHRH neurons [21], it is a common consensus that these close associations may represent synaptic connections and they may be responsible for the pulsatile release of LHRH in numerous species. Similarly to the LHRH system, the juxtapositions described in the present paper may represent the morphological basis of the synchronized activity of the GHRH neurons. The abundance of the GHRH–GHRH juxtapositions in the human hypothalamus indicates the central role of the communication between GHRH-IR neurons in the physiology of growth. Indeed, previous studies suggested that the pulsatile release GHRH is crucial for maintaining the pulsatile GH release [3,8]. Preceding studies revealed that patients with inactivating defect of the GHRH receptor gene still maintain the regular frequency of the GH release with a diminished total 24-h GH production rate [9]. Although these data seem to indicate that somatostatin, instead of GHRH, is responsible for the pulsatile release of GH, more studies are required due to the heterogenity and limited number of the patient pool used. These former studies, however, raise the possibility that the GHRH
D. Anderson et al. / Growth Hormone & IGF Research 20 (2010) 356–359
juxtapositions described in the present study may also be responsible for coordinating various physiologic actions of GHRH that are not directly associated with growth regulation, including the modulation of sleep [22–24], mood, and behavior [25]. The present study revealed that the juxtapositions described between the GHRH neural elements were most abundant in the infundibulum/median eminence (Fig. 2A and B) and in the basal part of the periventricular zone (Fig. 2C and D) of the human hypothalamus. The abundance and morphology of these juxtapositions in these areas indicate that these regions may play a pivotal role in the pulsatile release of GHRH. Although stimulation of the periventricular nucleus attenuates GHRH-induced GH response [26], this phenomenon can also be explained by the release of somatostatin into the portal vessels as somatostatin-producing neurons are located in this hypothalamic region [27,28]. Consequently, the relatively small number of GHRH–GHRH associations found in the dorsomedial subdivision of the ventromedial nucleus (Fig. 2E) and in the basal perifornical area of the tuberal region (Fig. 2F) suggests that these subpopulations of GHRH neurons either (1) do not exhibit synchronized release or (2) may be synchronized via other neurotransmitter systems. In summary, the juxtapositions between GHRH axonal varicosities may be functional synapses and may represent the morphological substrate of the synchronized release of GHRH in the human hypothalamus. The pulsatile release of GHRH, in turn, may be a pivotal factor in modulating crucial physiological functions in human. Acknowledgement The authors are indebted to Prof. Andras Mihaly, Chair of Anatomy Department, University of Szeged, for providing the brain samples. References [1] I. Merchenthaler, S. Vigh, A.V. Schally, P. Petrusz, Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus, Endocrinology 114 (1984) 1082–1085. [2] J. Deltondo, I. Por, W. Hu, I. Merchenthaler, K. Semeniken, J. Jojart, B. Dudas, Associations between the human growth hormone-releasing hormone- and neuropeptide-Y-immunoreactive systems in the human diencephalon: a possible morphological substrate of the impact of stress on growth, Neuroscience 153 (2008) 1146–1152. [3] M.G. Thomas, M. Amstalden, D.M. Hallford, G.A. Silver, M.D. Garcia, D.H. Keisler, G. L. Williams, Dynamics of GHRH in third-ventricle cerebrospinal fluid of cattle: relationship with serum concentrations of GH and responses to appetiteregulating peptides, Domest. Anim. Endocrinol. 37 (2009) 196–205. [4] S. Nakamura, M. Mizuno, H. Katakami, A.C. Gore, E. Terasawa, Aging-related changes in in vivo release of growth hormone-releasing hormone and somatostatin from the stalk-median eminence in female rhesus monkeys (Macaca mulatta), J. Clin. Endocrinol. Metab. 88 (2003) 827–833. [5] K. Mogi, T. Yonezawa, D.S. Chen, J.Y. Li, M. Suzuki, K. Yamanouchi, T. Sawasaki, M. Nishihara, Relationship between growth hormone (GH) pulses in the peripheral circulation and GH-releasing hormone and somatostatin profiles in the cerebrospinal fluid of goats, J. Vet. Med. Sci. 66 (2004) 1071–1078. [6] L.A. Frohman, T.R. Downs, I.J. Clarke, G.B. Thomas, Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia, J. Clin. Invest. 86 (1990) 17–24.
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[7] P.M. Plotsky, W. Vale, Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial–portal circulation of the rat, Science 230 (1985) 461–463. [8] W.B. Wehrenberg, P. Brazeau, R. Luben, N. Ling, R. Guillemin, A noninvasive functional lesion of the hypothalamo-pituitary axis for the study of growth hormone-releasing factor, Neuroendocrinology 36 (1983) 489–491. [9] F. Roelfsema, N.R. Biermasz, R.G. Veldman, J.D. Veldhuis, M. Frolich, W.H. StokvisBrantsma, J.M. Wit, Growth hormone (GH) secretion in patients with an inactivating defect of the GH-releasing hormone (GHRH) receptor is pulsatile: evidence for a role for non-GHRH inputs into the generation of GH pulses, J. Clin. Endocrinol. Metab. 86 (2001) 2459–2464. [10] F. Gallyas, T. Gorcs, I. Merchenthaler, High-grade intensification of the endproduct of the diaminobenzidine reaction for peroxidase histochemistry, J. Histochem. Cytochem. 30 (1982) 183–184. [11] F. Gallyas, I. Merchenthaler, Copper–H2O2 oxidation strikingly improves silver intensification of the nickel–diaminobenzidine (Ni–DAB) end-product of the peroxidase reaction, J. Histochem. Cytochem. 36 (1988) 807–810. [12] F. Gallyas, T. Gorcs, I. Merchenthaler, High-grade intensification of the endproduct of the diaminobenzidine reaction for peroxidase histochemistry, J. Histochem. Cytochem. 30 (1982) 183–184. [13] F. Gallyas, I. Merchenthaler, Copper–H2O2 oxidation strikingly improves silver intensification of the nickel–diaminobenzidine (Ni–DAB) end-product of the peroxidase reaction, J. Histochem. Cytochem. 36 (1988) 807–810. [14] H. Braak, E. Braak, The hypothalamus of the human adult: chiasmatic region, Anat. Embryol. (Berl) 175 (1987) 315–330. [15] C.B. Saper, Hypothalamus, in: G. Paxinos (Ed.), The Human Nervous System, Academic Press, San Diego, 1990, pp. 389–413. [16] E. Hrabovszky, Z. Liposits, Adrenergic innervation of dopamine neurons in the hypothalamic arcuate nucleus of the rat, Neurosci. Lett. 182 (1994) 143–146. [17] Z. Liposits, W.K. Paull, G. Setalo, S. Vigh, Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis, Histochemistry 83 (1985) 5–16. [18] Z. Liposits, D. Sherman, C. Phelix, W.K. Paull, A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat, Histochemistry 85 (1986) 95–106. [19] Z. Liposits, I. Merchenthaler, W.K. Paull, B. Flerko, Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat, Histochemistry 89 (1988) 247–252. [20] W.C. Wetsel, M.M. Valenca, I. Merchenthaler, Z. Liposits, F.J. Lopez, R.I. Weiner, P.L. Mellon, A. Negro-Vilar, Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons, Proc. Natl Acad. Sci. USA 89 (1992) 4149–4153. [21] B. Dudas, A. Mihaly, I. Merchenthaler, Topography and associations of luteinizing hormone-releasing hormone and neuropeptide Y-immunoreactive neuronal systems in the human diencephalon, J. Comp. Neurol. 427 (2000) 593–603. [22] J.M. Krueger, F. Obal Jr., J. Fang, Humoral regulation of physiological sleep: cytokines and GHRH, J. Sleep Res. 8 (1) (1999) 53–59. [23] S. Mathias, K. Held, M. Ising, J.C. Weikel, A. Yassouridis, A. Steiger, Systemic growth hormone-releasing hormone (GHRH) impairs sleep in healthy young women, Psychoneuroendocrinology 32 (2007) 1021–1027. [24] F. Obal Jr., R. Floyd, L. Kapas, B. Bodosi, J.M. Krueger, Effects of systemic GHRH on sleep in intact and hypophysectomized rats, Am. J. Physiol. 270 (1996) E230–E237. [25] D.R. Rubinow, R.M. Post, C.L. Davis, A.R. Doran, Somatostatin and GHRH: mood and behavioral regulation, Adv. Biochem. Psychopharmacol. 43 (1987) 137–152. [26] M. Kato, M. Suzuki, T. Kakegawa, Inhibitory effect of hypothalamic stimulation on growth hormone (GH) release induced by GH-releasing factor in the rat, Endocrinology 116 (1985) 382–388. [27] L. Desy, G. Pelletier, Immunohistochemical localization of somatostatin in the human hypothalamus, Cell Tissue Res. 184 (1977) 491–497. [28] H. Langevin, P.C. Emson, Distribution of substance P, somatostatin and neurotensin in the human hypothalamus, Brain Res. 246 (1982) 65–69.
Contents lists available at ScienceDirect
Growth Hormone & IGF Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g h i r
Distribution and morphology of the juxtapositions between growth hormone-releasing hormone-(ghrh)-immunoreactive neuronal elements Daniel Anderson a, Matthew Baker a, George Grignol a, Walter Hu a, Istvan Merchenthaler b, Bertalan Dudas a,⁎ a b
Neuroendocrine Organization Laboratory (NEO), Lake Erie College of Osteopathic Medicine (LECOM), 1858 West Grandview Blvd, Erie, PA 16509, United States Department of Epidemiology & Preventive Medicine and Anatomy & Neurobiology, University of Maryland, Baltimore, MD 21201, United States
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 21 June 2010 Accepted 23 June 2010 Available online 31 July 2010 Keywords: Hypothalamus Human Growth Immunohistochemistry
a b s t r a c t Previous studies revealed that growth hormone-releasing hormone (GHRH)-immunoreactive (IR) neurons form a circumscribed cell group in the basal infundibulum/median eminence of the human hypothalamus. GHRH from these neurons is released into the hypothalamo–hypophyseal portal circulatory system in a pulsatile manner. It is a common consensus that the pulsatile release of GHRH is the main driving force behind the pulsatile release of growth hormone (GH) and may contribute to the regulation of other hypothalamic functions. The pulsatile release of GHRH requires synchronized activity of GHRH-IR neurons. However, the morphological basis of this synchronization between the GHRH-IR neural elements has not been elucidated yet. Since the utilization of electron microscopy combined with immunohistochemistry is virtually impossible in the human brain due to the long post mortem period, immunohistochemistry, evaluated with oil immersion light microscopy, was used in order to reveal the associations between the GHRH elements. Numerous GHRH–GHRH juxtapositions have been detected in the infundibular area/median eminence, where GHRH-IR axonal varicosities often formed multiple contacts with GHRH-IR perikarya. Examination of these associations with high magnification oil immersion light microscopy revealed (1) axonal swellings at the site of the contacts and (2) no gaps between the contacting elements suggesting that these juxtapositions may be functional synapses. The large number of GHRH–GHRH juxtapositions in the infundibular area/ median eminence suggests that these synapse-like structures may represent the morphological substrate of the synchronized activity of GHRH neurons that in turn may result in the pulsatile release of GHRH in human. © 2010 Growth Hormone Research Society Published by Elsevier Ltd. All rights reserved.
1. Introduction Growth hormone-releasing hormone (GHRH) is a 44 amino acid peptide that is the primary regulator of growth hormone (GH) secretion of the pituitary gland. Previous studies described GHRHimmunoreactive (IR) perikarya located primarily in the basal infundibulum/median eminence of the rat [1] and the human hypothalamus [2]. GHRH from these neurons is released into the hypothalamo–hypophyseal portal circulatory system, and influences the release of growth hormone (GH) from the pituitary gland. The pulsatile release of GHRH is a well known phenomenon [3–7], and has been previously believed to be the single major factor driving the pulsatile secretion of GH, that is released in 10–20 pulses in each 24h cycle. However, the pulsatile pattern seems to be species-dependent. In cattle, the majority of the GH pulses are correlated to a preceding GHRH pulse indicating the crucial role of GHRH in the frequency control of the GH release [3]. Passive immunization with somatostatin ⁎ Corresponding author. Tel.: + 1 814 866 8142; fax: +1 814 866 8411. E-mail address: [email protected] (B. Dudas).
antiserum does not alter pulsatile GH release, but immunization with antibodies raised against both somatostatin and GHRH abolishes GH bursts [8]. In contrast to these findings, patients with an inactivating defect of the GHRH receptor gene maintain the regular frequency of the GH release with a diminished total 24-h GH production rate, suggesting that GH pulses are under the control of intermittent somatostatin withdrawal while the amplitude of GH pulses is driven by GHRH [9]. The fundamental role of somatostatin in the GH pulse generation has been supported by previous data describing that most of the descending phases of somatostatin pulses are associated with the initiation of GH pulses in goats [5]. Although the exact function of the pulsatile release of GHRH remains to be elusive, there is a general consensus that this phenomenon is based on the synchronized activity of the GHRH-IR neurons, suggesting communication between the GHRH-IR neural elements, either directly, via synaptic connections, or indirectly, via other neurotransmitter systems. In the present study, we examined the possibility of direct synapse-like contacts between the GHRH-IR neurons that may represent one of the key elements responsible for the pulsatile release of GHRH in human. Since the utilization of electron microscopy
1096-6374/$ – see front matter © 2010 Growth Hormone Research Society Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ghir.2010.06.002
D. Anderson et al. / Growth Hormone & IGF Research 20 (2010) 356–359
357
Fig. 1. Distribution of the GHRH-IR neural elements in the human hypothalamus. GHRH-IR neurons are most abundant in the infundibulum/median eminence (A) and in the basal part of the periventricular area (B), where GHRH-IR perikarya are surrounded with dense GHRH-IR fiber network. The dorsomedial subdivision of the ventromedial nucleus (C) and the basal perifornical area (D) contain a smaller number of GHRH-IR perikarya and axonal varicosities. Cross section of a portal vessel is denoted by a five-pointed star while a sixpointed star marks the 3rd ventricle. Magnification: 100x.
combined with immunohistochemistry is virtually impossible in the human brain due to the long post mortem period, light microscopic immunohistochemistry was used in order to reveal the associations between the GHRH elements. Contacts between GHRH-IR structures were evaluated with high magnification (100×) and oil immersion.
1% silicotungstic acid and 0.2% formaldehyde for 3–4 min [12,13]. Olympus BX45 microscope with a 100× oil immersion objective was used for the evaluation of the immunohistochemical staining.
2. Methods
The terminology of the diencephalic structures was adapted from Braak and Braak [14] and Saper [15].
2.3. Terminology
2.1. Brain samples 3. Results Hypothalami (2 adult men and 1 adult woman, 67–81 years of age) were harvested from autopsies at 24–48 h post mortem period in accordance with the regulation and permission of the Ethics Board of Lake Erie College of Osteopathic Medicine and the University of Szeged, Hungary. The clinical records of the individuals did not indicate any neurological and neuroendocrinological disorders. The brain samples were fixed by immersion in buffered 4% paraformaldehyde at 4 °C for 2–8 weeks. The samples were cryoprotected with 30% sucrose in phosphate buffered saline (PBS) and then sectioned on a freezing microtome at 30 μm intervals in coronal planes. 2.2. Immunohistochemistry Immunohistochemistry was performed using streptavidin–biotin (ABC) methods combined with silver intensification introduced by Gallyas and coworkers [10,11]. The samples were pretreated with 10% thioglycolic acid for 30 min to suppress the endogenous tissue argentophilia, then with 0.2% Triton X-100 for 20 min to increase permeability of immunoglobulins, and finally with 10% normal goat serum (NGS) in PBS for 1.0 h at room temperature to block nonspecific staining. Thereafter, the sections were incubated in a rabbit anti-GHRH serum (Chemicon, Temecula, CA) at a dilution of 1:8000 containing 10% NHS, PBS and 0.1% sodium azide for 24 h. The sections were then incubated for 1.0 h with biotinylated goat anti-rabbit immunoglobulins (IgG) (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA) diluted 1:1000 in PBS. Following two washes in PBS for a total of 15 min, the tissue was incubated for 1.0 h in a solution of streptavidin–horseradish peroxidase (Vectastain ABC Elite kit) diluted 1:1000 in PBS. The cross-reactions of the secondary antibodies with the endogenous human IgGs were eliminated by the addition of 2% normal human serum to the secondary antiserum. The sections were washed 2 × 10 min in PBS and 10 min in Tris-buffered saline (TBS; pH 7.6) and then reacted with 3′–3′ diaminobenzidine tetrahydrochloride (DAB), using nickel ammonium sulfate intensification (20 mg DAB, 100 mg nickel ammonium sulfate and 7.0 μl 30% H202 in 40 ml TBS). The resulting DAB polymer was silver-intensified in a solution containing 0.1% silver nitrate, 0.1% ammonium–nitrate,
3.1. Human GHRH system The distribution and morphology of GHRH-IR neural elements have been described in our previous study [2]. Briefly, GHRH-IR neurons formed a dense, well-circumscribed cell group with the perikarya located almost exclusively in the basal part of the infundibular region (Fig. 1). Here, cell bodies formed four welldefined subdivisions: (i) the majority of the GHRH-IR perikarya were located in the infundibulum/median eminence (Fig. 1A) and (ii) in the basal part of the periventricular zone (Fig. 1B). (iii) A group of perikarya were observed in the dorsomedial subdivision of the ventromedial nucleus (Fig. 1C) and (iv) in the basal perifornical area of the tuberal region (Fig. 1D). The majority of GHRH neurons possessed fusiform cell bodies with processes emanating from the opposite ends of the cells. GHRH-IR fibers formed a dense network in the basal part of the infundibulum (Fig. 1A). GHRH-IR axon varicosities were also detected in the periventricular area (Fig. 1B) and in the basal part of the medial hypothalamus. 3.2. GHRH–GHRH juxtapositions Juxtapositions between the GHRH-IR neural elements were detected mainly in the infundibular area/median eminence (Fig. 2A and B) and in the basal part of the periventricular zone (Fig. 2C and D), where 32% of the GHRH-IR perikarya appeared to form multiple (2 or more) contacts with GHRH-IR axonal varicosities. The rest of the examined perikarya were lightly innervated (1 contact/cell; 21%) or did not form apparent juxtapositions with abutting GHRH-IR fibers. Few juxtapositions were detected in the dorsomedial subdivision of the ventromedial nucleus (Fig. 2E) and in the basal perifornical area of the tuberal region (Fig. 2F), where only 11% of the detected GHRH-IR perikarya received abutting GHRH fibers. These juxtapositions were morphologically similar to the ones found in the infundibular area/ median eminence and in the basal part of the periventricular zone. The GHRH–GHRH associations found in the human hypothalamus were typically en passant juxtapositions where GHRH-IR fibers
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Fig. 2. Juxtapositions between the GHRH-IR elements in the human hypothalamus. These GHRH–GHRH associations are usually en passant juxtapositions where GHRH-IR fibers abut GHRH-IR perikarya forming multiple contacts and typically exhibiting axonal swellings at the site of the contacts. The GHRH–GHRH juxtapositions are most dense in the infundibulum/median eminence (A and B) and in the basal part of the periventricular area (C and D), while juxtapositions are less numerous in the dorsomedial subdivision of the ventromedial nucleus (E) and the basal perifornical area (F). Some of the juxtapositions are denoted by arrowheads. Magnification: 400×.
abutted GHRH-IR perikarya usually forming multiple contacts (Fig. 2). At the site of the contacts, the GHRH-IR axons typically exhibited axonal swellings (Fig. 2). Examination of these associations with high magnification oil immersion light microscopy revealed no gaps between the contacting elements. 4. Discussion The present paper is the first describing juxtapositions between GHRH-IR axonal varicosities and cell bodies in the human hypothalamus. Although the verification of these putative synapses with electron microscopy is virtually impossible in these human samples due to the long post mortem time, previous studies have shown that similar juxtapositions represent synapses at the ultrastructural level in rat [16–19]. In the present study we report that (1) close examination of these juxtapositions with high magnification failed to reveal any gap between the contacting elements and (2) at the site of the contact, GHRH-IR axons abutting on GHRH-IR perikarya exhibited a swelling, suggesting that these associations may indeed be functional synapses.
The pulsatile activity of luteinizing hormone-releasing hormone (LHRH)-IR neurons is a well-established phenomenon [20]. Since previous studies revealed numerous juxtapositions between LHRH neurons [21], it is a common consensus that these close associations may represent synaptic connections and they may be responsible for the pulsatile release of LHRH in numerous species. Similarly to the LHRH system, the juxtapositions described in the present paper may represent the morphological basis of the synchronized activity of the GHRH neurons. The abundance of the GHRH–GHRH juxtapositions in the human hypothalamus indicates the central role of the communication between GHRH-IR neurons in the physiology of growth. Indeed, previous studies suggested that the pulsatile release GHRH is crucial for maintaining the pulsatile GH release [3,8]. Preceding studies revealed that patients with inactivating defect of the GHRH receptor gene still maintain the regular frequency of the GH release with a diminished total 24-h GH production rate [9]. Although these data seem to indicate that somatostatin, instead of GHRH, is responsible for the pulsatile release of GH, more studies are required due to the heterogenity and limited number of the patient pool used. These former studies, however, raise the possibility that the GHRH
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juxtapositions described in the present study may also be responsible for coordinating various physiologic actions of GHRH that are not directly associated with growth regulation, including the modulation of sleep [22–24], mood, and behavior [25]. The present study revealed that the juxtapositions described between the GHRH neural elements were most abundant in the infundibulum/median eminence (Fig. 2A and B) and in the basal part of the periventricular zone (Fig. 2C and D) of the human hypothalamus. The abundance and morphology of these juxtapositions in these areas indicate that these regions may play a pivotal role in the pulsatile release of GHRH. Although stimulation of the periventricular nucleus attenuates GHRH-induced GH response [26], this phenomenon can also be explained by the release of somatostatin into the portal vessels as somatostatin-producing neurons are located in this hypothalamic region [27,28]. Consequently, the relatively small number of GHRH–GHRH associations found in the dorsomedial subdivision of the ventromedial nucleus (Fig. 2E) and in the basal perifornical area of the tuberal region (Fig. 2F) suggests that these subpopulations of GHRH neurons either (1) do not exhibit synchronized release or (2) may be synchronized via other neurotransmitter systems. In summary, the juxtapositions between GHRH axonal varicosities may be functional synapses and may represent the morphological substrate of the synchronized release of GHRH in the human hypothalamus. The pulsatile release of GHRH, in turn, may be a pivotal factor in modulating crucial physiological functions in human. Acknowledgement The authors are indebted to Prof. Andras Mihaly, Chair of Anatomy Department, University of Szeged, for providing the brain samples. References [1] I. Merchenthaler, S. Vigh, A.V. Schally, P. Petrusz, Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus, Endocrinology 114 (1984) 1082–1085. [2] J. Deltondo, I. Por, W. Hu, I. Merchenthaler, K. Semeniken, J. Jojart, B. Dudas, Associations between the human growth hormone-releasing hormone- and neuropeptide-Y-immunoreactive systems in the human diencephalon: a possible morphological substrate of the impact of stress on growth, Neuroscience 153 (2008) 1146–1152. [3] M.G. Thomas, M. Amstalden, D.M. Hallford, G.A. Silver, M.D. Garcia, D.H. Keisler, G. L. Williams, Dynamics of GHRH in third-ventricle cerebrospinal fluid of cattle: relationship with serum concentrations of GH and responses to appetiteregulating peptides, Domest. Anim. Endocrinol. 37 (2009) 196–205. [4] S. Nakamura, M. Mizuno, H. Katakami, A.C. Gore, E. Terasawa, Aging-related changes in in vivo release of growth hormone-releasing hormone and somatostatin from the stalk-median eminence in female rhesus monkeys (Macaca mulatta), J. Clin. Endocrinol. Metab. 88 (2003) 827–833. [5] K. Mogi, T. Yonezawa, D.S. Chen, J.Y. Li, M. Suzuki, K. Yamanouchi, T. Sawasaki, M. Nishihara, Relationship between growth hormone (GH) pulses in the peripheral circulation and GH-releasing hormone and somatostatin profiles in the cerebrospinal fluid of goats, J. Vet. Med. Sci. 66 (2004) 1071–1078. [6] L.A. Frohman, T.R. Downs, I.J. Clarke, G.B. Thomas, Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia, J. Clin. Invest. 86 (1990) 17–24.
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