Brain Structure and Function (2020) 225:241–248 https://doi.org/10.1007/s00429-019-01990-x ORIGINAL ARTICLE Presence of substance P positive terminals on hypothalamic somatostatinergic neurons in humans: the possible morphological substrate of the substance P‑modulated growth hormone secretion Andrew Luu1 · Zachary Oberdoerster1 · George Grignol1 · Istvan Merchenthaler2 · Bertalan Dudas1 Received: 10 June 2019 / Accepted: 19 November 2019 / Published online: 5 December 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Substance P is an undecapeptide afecting the gastrointestinal, cardiovascular, and urinary systems. In the central nervous system, substance P participates in the regulation of pain, learning, memory, and sexual homeostasis. In addition to these efects, previous papers provided solid evidence that substance P exhibits regulatory efects on growth. Indeed, our previous study revealed that growth hormone-releasing hormone (GHRH) neurons appear to be densely innervated by substance P fbers in humans. Since growth hormone secretion is regulated by the antagonistic actions of both GHRH and somatostatin, in the present paper we have examined the possibility that SP may also afect growth via the somatostatinergic system. Therefore, we have studied the putative presence of juxtapositions between the substance P-immunoreactive (IR) and soma- tostatinergic systems utilizing double label immunohistochemistry combined with high magnifcation light microscopy with oil immersion objective. In the present study, we have revealed a dense network of substance P-IR axonal varicosities contacting the majority of somatostatin-IR neurons in the human hypothalamus. Somatostatinergic perikarya are often covered by these fber varicosities that frequently form basket-like encasements with multiple en passant type contacts, particularly in the infundibular nucleus/median eminence and in the basal periventricular area of the tuberal region. In addi- tion, numerous substance-P-somatostatinergic juxtapositions can be found in the basal perifornical zone of the tuberal area. If these contacts are indeed functional synapses, they may represent the morphological substrate of the control of substance P on growth. Indeed, the frequency and density of these juxtapositions indicate that in addition to the regulatory action of substance P on GHRH secretion, substance P also infuences growth by regulating hypothalamic somatostatinergic system via direct synaptic contacts. Keywords Substance P · Somatostatin · Juxtaposition · Immunohistochemistry · Human hypothalamus Introduction B (NKB), is a member tachykinin neuropeptide family and functions as a neurotransmitter and/or neuromodulator. Sub- Substance P is an eleven amino acid neuropeptide discov- stance P activates neurokinin 1 (NK1) receptors with high ered almost a century ago due to its ability to cause prompt afnity (Mantyh 2002), although it also exhibits low afnity intestinal contraction (von Euler and Gaddum 1931). Sub- to neurokinin 2 (NK2) and neurokinin 3 (NK3) receptors, stance P, along with neurokinin A (NKA) and neurokinin which are the targets of NKA and NKB, respectively. The role of substance P in nociception is well established (Zubrzycka and Janecka 2000), however, substance P is also * Istvan Merchenthaler [email protected] involved in the physiology and pathophysiology of mood disorders, anxiety, stress (Ebner and Singewald 2006), rein- 1 Neuroendocrine Organization Laboratory (NEO), Lake Erie forcement (Huston et al. 1993), neurogenesis (Park et al. College of Osteopathic Medicine (LECOM), 1858 West 2007), respiratory rhythm (Bonham 1995), neurotoxicity, Grandview Blvd, Erie, PA 16509, USA nausea/emesis (Hesketh 2001), vasodilation (Meeking et al. 2 Department of Epidemiology and Public Health 2000; Schrauwen and Houvenaghel 1980; Bossaller et al. and Anatomy and Neurobiology, University of Maryland Baltimore, 10 South Pine Street Mstf 977, Baltimore, 1992). Substance P also participates ininfammatory pro- MD 21201, USA cesses [for review see (O’Connor et al. 2004)] that appear Vol.:(0123456789)1 3 242 Brain Structure and Function (2020) 225:241–248 to be involved in the pathogenesis of Parkinson’s disease in female rhesus monkeys (Eckstein et al. 1980). In contrast, (Thornton and Vink 2015). Due to the presence of substance some studies report unafected somatostatin levels in the P in the hypothalamo-hypophyseal axis (Chawla et al. 1997; portal blood after intracerebroventricular administration of Dudas and Merchenthaler 2002, 2006), it is conceivable that substance P in rats under anesthesia (Abe et al. 1981). These substance P may also modulate neuroendocrine functions. results, coupled with fnding that administration of substance Indeed, our previously reported morphological fndings sug- P into the 3rd ventricle of conscious rats increases serum GH gest that substance P may be involved in the regulation of concentrations (Vijayan and McCann 1980) indicates that gonadal functions by direct synaptic contacts formed with the mechanism via somatostatin is involved in the substance- gonadotropin-releasing hormone (GnRH) neurons (Dudas P-regulated GH secretion is complicated and may depend on and Merchenthaler 2002). Similarly, substance P may also multiple factors [for review see (Aronin et al. 1986)]. The regulate growth, possibly at the hypothalamic levels, by fnding that neurokinin-1 (NK1) knockout mice reproduce afecting growth hormone (GH) secretion (Arisawa et al. and develop normally (Santarelli et al. 2001) raises the pos- 1989; Aronin et al. 1986; Chihara et al. 1978; Eckstein sibility of species diferences in this regulatory function. et al. 1980; Sheppard et al. 1979). Since substance P does To further investigate these apparently contradictory data, not afect basal or stimulated GH release from rat primary and explore the role of substance P on somatostatin release pituitary cells in vitro (Cheng et al. 1997), this regulatory in humans, in the present study we have examined the pos- action appears to involve the hypothalamus. Indeed, non- sibility that substance P fber varicosities directly innervate peptide substance P antagonist, CP-96,345, fails to infuence somatostatinergic neurons in the human hypothalamus. basal GH release at low concentrations in vitro (Houben These hypothetical juxtapositions, in accord with the previ- and Denef 1993). Moreover, incubation of synthetic sub- ously reported substance P-GHRH associations, may rep- stance P with dispersed anterior pituitary cells does not resent the morphological substrate of the involvement of afect GH release (Arisawa et al. 1989), further supporting substance P in the regulation of growth. the concept that substance P modulates GH secretion at the hypothalamic level, via growth hormone-releasing hormone (GHRH) and/or somatostatin secretion. Methods In our recent studies, we provided evidence that sub- stance P communicates with GHRH neurons in the human Hypothalamic samples hypothalamus and therefore, may regulate GH secretion via modulating GHRH neuronal activity (Uhlman et al. 2019). Human hypothalami of 1 adult man and 3 women, This conclusion was also supported by physiological fnd- 75–87 years of age, were harvested from cadavers within ings indicating that substance P modulates GHRH secre- less than 12 h post mortem period, in accordance with the tion in numerous species (Bitar et al. 1991; Lemamy et al. regulations of the Institutional Review Board of Lake Erie 2012; Coiro et al. 1992). However, it is conceivable that College of Osteopathic Medicine (LECOM). None of the substance P may also exert regulatory function on growth individuals had neurological or neuroendocrinological dis- via the somatostatinergic system. Indeed, substance P dose- orders in their clinical records. dependently increases somatostatin release from hypotha- lamic blocks in vitro (Sheppard et al. 1979). Decreased Tissue preparation plasma GH levels following intracerebroventricular admin- istration of substance P (Arisawa et al. 1989; Aronin et al. Hypothalami were fxed by immersion in 0.1 M phosphate- 1986; Chihara et al. 1978; Eckstein et al. 1980; Sheppard bufered 4% formaldehyde at 4 °C for 2–8 weeks. Then, the et al. 1979) can also be explained with the efect of sub- tissue blocks were trimmed, and the diencephalon was cut stance P on the periventricularly arranged somatostatiner- in half along the midsagittal line. The blocks were cryo- gic neurons via volume transmission. Administration of a protected with 30% sucrose in phosphate bufer containing substance P antiserum or a specifc substance P antagonist, 0.9% sodium chloride (PBS) and 0.15% sodium-azide. After [D-Pro2,D-Trp7,9]-substance P into the 3rd ventricle consid- the blocks were sunk in the sucrose solution, they were sec- erably elevates GH secretion in rats, while intracerebroven- tioned on a freezing microtome (American Optical Corp. tricular or intravenous injection of synthetic substance P Model 880) and 35 μm thick sections were prepared. The signifcantly decreases plasma GH levels (Arisawa et al. sections were collected in sequential order into 24-well plas- 1989). Moreover, the suppressive efect of substance P on tic tissue culture plates containing PBS with 0.2% sodium- serum GH levels, when administered to the lateral ventricle azide then stored at 4 °C until processing. Adjacent sections of anesthetized rats, is blocked by somatostatin antiserum were processed as follows: (1) single label immunohisto- (Chihara et