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Investigación Clínica ISSN: 0535-5133 Universidad del Zulia

Duque-Díaz, Ewing; Martínez-Rangel, Diana; Ruiz-Roa, Silvia in the human Investigación Clínica, vol. 59, no. 2, 2018, April-June, pp. 161-178 Universidad del Zulia

DOI: https://doi.org/10.22209/IC.v59n2a06

Available in: https://www.redalyc.org/articulo.oa?id=372960175007

How to cite Complete issue Scientific Information System Redalyc More information about this article Network of Scientific Journals from Latin America and the Caribbean, Spain and Journal's webpage in redalyc.org Portugal Project academic non-profit, developed under the open access initiative Invest Clin 59(2): 161 - 178, 2018 https//doi.org/10.22209/IC.v59n2a06

Neuropeptides in the human brainstem.

Ewing Duque-Díaz1, Diana Martínez-Rangel1 and Silvia Ruiz-Roa1,2. 1Universidad de Santander UDES. Laboratory of Neuroscience, School of Medicine, Bucaramanga, Colombia. 2Universidad de Santander UDES. Laboratory of Neuroscience, Nursing Program, Bucaramanga, Colombia. Key words: neuropeptides; mesencephalon; ; ; human.

Abstract. The physiological importance of the brainstem has made it one of the most studied structures of the central nervous system of mammals (in- cluding human). This structure receives somatic and visceral inputs and its neurons send motor efferences by means of the cranial , which innervate the head, and sensory organs, and it mediates in several actions such as movement, pain, cardiovascular, respiratory, salivary, sleep, vigil and sexual mechanisms. Most of these actions are mediated by neuroactive substances denominated neuropeptides, which are short chains widespread dis- tributed in the nervous system, that play a role in neurotransmission, neuro- modulation (paracrine and autocrine actions), and act as neurohormones. In- creased study of these substances has taken place since the 1980s to shed light on both their potential role and the way that they mediate in the organism’s different activities. Thus, our aim is a detailed review of available morphologic and physiologic data regarding some neuropeptides in the human brainstem. To such end, we will discuss aspects like: 1) the distribution of neuropeptides in the human brainstem; 2) their possible physiological actions in the human brainstem; 3) coexistences in the human brainstem; and 4) fu- ture research in neuropeptides in the human brainstem.

Corresponding author: Ewing Duque Díaz. Universidad de Santander UDES. Escuela de Medicina. Labotario de Neurociencias. Campus Lagos del Cacique CP 680006. Bucaramanga, Colombia. E-mail: [email protected] 162 Duque-Díaz et al.

Neuropéptidos en el tronco del encéfalo humano. Invest Clin 2018; 59 (2): 161 - 178

Palabras clave: neuropéptidos; mesencéfalo; puente; medulla oblongata; humano.

Resumen. La importancia fisiológica del tronco del encéfalo la ha conver- tido en una de las estructuras más estudiadas del sistema nervioso central de mamíferos (incluido el humano). Esta estructura recibe impulsos somáticos y viscerales, además de enviar eferencias motoras mediante los nervios craneales que inervan la cabeza, el cuello y órganos sensoriales, mediando en diferentes acciones tales como movimiento, dolor, mecanismos cardiovasculares, respira- torios, salivación, sueño y vigilia, como también mecanismos sexuales. Muchas de esas acciones son mediadas por sustancia neuroactivas denominadas neuro- péptidos, los cuales son cadenas cortas de aminoácidos ampliamente distribui- dos en el sistema nervioso que juegan un papel en la neurotransmisión, neuro- modulación (acciones paracrina y autocrina)y actúan como neurohormonas. A partir de los años ochenta se incrementó el estudio de estas sustancias que han permitido ampliar el conocimiento sobre papel y la forma en que estas median en las diferentes actividades del organismo. Por lo tanto, nuestro objetivo es realizar una revisión detallada de los datos morfológicos y fisiológicos disponi- bles respecto a algunos neuropéptidos en el tronco del encéfalo humano. Para tal fin discutiremos aspectos como: 1) la distribución de neuropéptidos en el tronco del encéfalo humano; 2) las posibles acciones fisiológicas en el tronco del encéfalo humano; 3) la coexistencia de neuropeptidos en el tronco del en- céfalo humano; y 4) investigaciones futuras de neuropeptidos en el tronco del encéfalo humano.

Recibido: 25-05-2017 Aceptado: 02-03-2018

INTRODUCTION methionin-, -enkephalin, beta-endorphin and adrenocorticotropic A wide variety of techniques have been hormone in non-human primates (Macaca used to study the distribution of substances fascicularis and Saimirís ciureus) brainstem as neuropeptides, vitamines and D- amino- (1, 8).These studies have reported pioneer- acids in the nervous system of vertebrates, ing data about the presence of fiber and/or including humans. These methods include cell bodies containing these substances. immunocytochemistry, radioimmunoassay, The term neuropeptides refers to short chromatography, in situ hybridization and amino acid chains synthesized in the nervous autoradiography, among others (1-23). Our system, classified as non-classical transmit- research group has been developing work in ters and working generally in synergy with neuropeptide mapping in the brainstem of other , a fact that makes it mammals since 2007. Thus, the distribution difficult to determine their post-synaptic ef- of substances such as alpha-neo-endorphin fects. However, their importance in the mod- and in the human brainstem ification of the effect of other neurotrans- has been studied, besides the presence of im- mitters on post-synaptic cells has been munolabeled structures containing neuroki- established (24). Biochemically, neuropep- nin B, , -28 [1-12], tides synthesize from the mRNA of the pre-

Investigación Clínica 59(2): 161 - 178, 2018 Neuropeptides in the brainstem 163 pro-, originating in turn from DNA. neuropeptide binds to receptors placed in This mRNA is translated by the ribosomes the pre-synaptic component, controlling its and the action of endopeptidases transforms release; 3) a action, where it into a pro-peptide, which, due to post- the neuropeptide binds to receptors in the translational factors mediated by peptidases postsynaptic component, favoring chemical and other chemical modifications (glycosyl- synapsis; and 4) an action distant from the ation, phosphorylation, and/or sulfonation), place where it was released (blood or cere- finally forms neuropeptides, which are re- brospinal fluid) acting as a neurohormone. leased into the extracellular space by means Moreover, it is know that neuropeptides ap- of exocytosis (Fig. 1) (25). Once released, pear to be the major signal for volume trans- neuropeptides can perform different actions: mission, a concept introduced twenty years 1) a paracrine action (neuromodulator), ago concerning cellular communication in where the neuropeptide binds its receptors the central nervous system that operates located in the post-synaptic component near via the diffusion of chemical signals in the to the place where it was released; 2) an au- extracellular fluid pathways of the brain to tocrine action (neuromodulator), where the reach affinity membrane receptors (24, 25).

Fig. 1. Pathways for biosynthesis of opioid . These arise from three genes. Gene 1 (above) give rise the precursor molecule termed pro-opiomelanocortin (POMC). Posttranslational processing produces the peptides shown: α-, β- and γ- melanophore stimulating hormone (MSH); β-endorphin, adrenocorti- cotropic hormone (ACTH) and corticotropin-like intermediate lobe peptide (CLIP). The (below), arise from two other genes that code for A and B: M, met-enkephalins(Tyr- Gly-Gly-Phe-Met); L: leu-enkephalin (Tyr-Gly-Gly-Phe-Leu); MAGL (M-Arg-Gly-Leu); MAP: (M-Arg-Phe); α-neo-endorphin (α-NE); dinorphin-A (DN-A); dinorphin-B (DN-B); dinorphin 32 (D-32). I.L., Interme- diate lobe of pituitary; A.L., anterior lobe of pituitary (Based on Shepherd, 1989).

Investigación Clínica 59(2): 161 - 178, 2018 164 Duque-Díaz et al.

Different techniques have been devel- NEUROPEPTIDES DISTRIBUTION oped to demonstrate the presence of neu- IN THE HUMAN BRAINSTEM ropeptides, one of which is the immuno- cytochemical method, used to detect the The distribution of some neuropeptides presence and distribution of these or other in the human brainstem is shown in Table I neuroactive substances in fibers and/or cell and Figs. 2 and 3). Nomenclature of the dif- bodies, an advantage over other methods ferent nuclei and/or tracts was carried out through which tissue preservation is not pos- according to Haines’ atlas of the human sible (1). Additionally, immunocytochemis- brain (28), and the same atlas was used for try can be complemented with other tech- the terminology of the brainstem regions. niques, like in situ hybridization, allowing comparison of the location where a substance Opiate Peptides is synthesized and its site of expression, en- There are three families of opioid pep- abling different immunocytochemical mark- tides, classified according to their precur- ings in the same region, facilitating estab- sors: pro-opiomelanocortin, proenkephalin lishment of the possible coexistence and/or and pro- (7, 29, 30). Alpha- and colocalization of several substances, which gamma-endorphin, beta-endorphin and me- could, in turn, yield an explanation of the thionine-enkephalin (metenk) are produced relation between them. Consequently, the from pro-opiomelanocortin; met-enk, methi- immunocytochemical method allows detec- onine-enkephalin-Arg6-Phe7, - tion of several neuroactive substances under enkephalin-Arg6-Gly7-Leu8 and leucine- conditions of experimentation. enkephalin (leu-enk) are produced from In humans, the study of the presence of pro-enkephalin; leu-enk, , dynor- neuropeptides has been developed in differ- phin B (rimorphin) and alpha- and beta-neo- ent regions of the brainstem (2-6, 26, 27), endorphinare produced from pro-dynorphin one of the most widely studied structures. (8, 31-34). The non-opiate peptide adreno- This structure is anatomically divided in corticotropin hormone (ACTH) is exclusive- three portions: medulla oblongata, pons and ly derived from pro-opiomelanocortin, and mesencephalon (from caudal to rostral re- hence this hormone is a good marker for the gion). It has been implicated in sleep, vocal- pro-opiomelanocortin system (8). ization, , analgesia and heart In general, opiate receptors have been rate, as well as in sexual, visual, attentive, found in the central nervous system of mam- auditive and motor mechanisms (6). It also mals, of which three types have been identi- receives somatic and visceral inputs, and its fied: mu, delta, and kappa. While enkepha- neurons send motor efferences by means of lins are also known to be very active in mu the , which innervate the head, and delta receptors, dynorphin has been de- neck and sensory organs (1). termined to be a relatively selective agonist According to the previous, the aim of to receptor kappa (31), reinforcing the the- this work is a detailed description of the ory about the importance of opiate peptides distribution of some neuropeptides in the and their receptors in the phenomenon of human brainstem, taking into account the analgesia, given that the presence of these different techniques previously described substances in regions mediating pain trans- based on a bibliographic search. Also, previ- mission has been demonstrated (35). ous results published by our research group Use of radioimmunoassay and immuno- regarding the experience in the distribution cytochemistry techniques has led to detec- of these neuroactive substances in the brain- tion of two POMC-derived peptides in the stem of mammals, including that of humans, human brainstem: β-endorphin and α-MSH will be considered. 36-39).These studies have not only evidenced

Investigación Clínica 59(2): 161 - 178, 2018 Neuropeptides in the brainstem 165 ------F + + (R) (R) (R) (R) OXY ------+ CB (R) ------F (R) (R) FF2 ------+ CB ------CF ORX ------CB ------CF CRH ------+ CB ------+ + + + + + CF (R) (R) (R) (R) (R) +(R) SOM ------+ + + CB (R) +++ +++ ------F + + + + + + +++ +++ +++ NPY ------+ + + + + + CB ------F + + + + ++

NKB ------+ NP CB ------F TABLE I TABLE NK ------+ + + CB ------F + + + + SP ------+ + + + + CB ------F + + + + + ++ +++ +++ +++ +++ +++ -neo-E ------α + + CB DISTRIBUTION OF NEUROPEPTIDES IN THE BRAINSTEM ------F + DYN ------CB ------F + + + + + + + + + + ------Met-E CB ------F + + + + + + + + + + ------Leu.E CB ------F + -End ------β CB SO PO ML LVN SCP DLF RetF MVN MAO SolTr CeGy RaNu HyNu NuGr SCPL LRNu NuCu LCNu DCNu SolNu FacNu NuAm DMNu Nuclei

Investigación Clínica 59(2): 161 - 178, 2018 166 Duque-Díaz et al. ------F (R) (R) OXY ------CB ------F (R) FF2 ------CB -neo-E), α ------+ + CF ORX ------CB ------CF CRH ------+ CB ------+ + + + + CF (R) SOM ------+ + + + + CB +++ ------F + + + + + + ++ +++ +++ +++ NPY ------+ + + + + CB ++ ) Presence (+ Low density; ++ moderate density; +++ High den Presence ------F (+) + + + + + ++ NKB ------+ NP CB ------Continuation F ( ++ ++ ++ Cell Bodies, NK ------CB + + CB ------F + + + TABLE I. TABLE SP ------+ + + + + CB ------F + + + + + ++ +++ +++ -neo-E ------α + + + + CB ------F DYN ------CB ------F + + + + + + + (R) ------Met-E -End), leucine-enkephalin (Leu-E), metionine-enkephalin (Met-E), dinorphin (DYN), alfa-neo-endorphin ( (Leu-E), (Met-E), dinorphin (DYN), -End), leucine-enkephalin metionine-enkephalin CB β ------F + + + + + + + (R) Receptor. ------Leu.E CB (R) -endorphin ( ------F β -End ------β CB Absence; (-) LL SN SC SVN ISNu IPNu NuPp SpVN OcNu SCNu TrapB VesNu TroNu IC, LZ VTegA Nuclei SpTNu LocCer IC, CNu TriMoNu Distribution of Hormone (CRH), (ORX), Neuropeptide Release Y (NPY), Somatostatin (SOM), Corticotropin B (NKB), Neuropeptide (NK), Neurokinin (SP), Neurokinin FF2 (FF2) y (OXY). The first column contains the abbreviations of nuclei in the brainstem. sity);

Investigación Clínica 59(2): 161 - 178, 2018 Neuropeptides in the brainstem 167

Fig. 2. (A-H). Schematic drawings of coronal section (rostral to caudal region) depicting the distribution of alpha-neo-endorphin (A-D) and neurokinin B (E-H) immunoreactivity in the human brainstem. The low, moderate and high distribution of fibers and cell bodies containing these neuropeptides is repre- sented by symbols ( ) and ( ) respectlly.Density of immunoreactive perikarya was conside- red to be higher when more than 10 cell bodies/section were found; density was moderate when 5-10 cell bodies/section were observed, and low when fewer than 5 cell bodies/section were visualized. Aditionally, immunoreactive fibers were considered short (< 90 mm), medium (90-120 mm) or long in length (> 120 mm) and cell bodies were considered small (diameter below 15 µm), medium-sized (15-25 µm), and large (above 25 µm). This figure is redrawn from the maps of alha-neo-endorphin and neurokinin B published in reference (6), with permission of Springer.

Investigación Clínica 59(2): 161 - 178, 2018 168 Duque-Díaz et al.

Fig. 3. (A-F). Neuropeptides immunoreactivity in the human brainstem. (A-C) Alpha-neo-endorphin fibers in the spinal trigeminal , reticular formnation and matter. These nuclei contain low (B), moderate (C) and high (A) density of fibers (arrows). (D-F) shown cell bodies and fibers (arrows) containing neurokinin B in the periaqueductal gray matter, and . Scale bar= 100 µm for (A, C, D, E, F); 50 µm for (B). Panels A, C and F are provided from Ewing Duque-Díaz. Panels B, D and E are from Duque E, Mangas A, Salinas P, Díaz-Cabiale Z, Narváez JA, Coveñas R. Mapping of alpha-neo-endorphin-and neurokinin B-immunoreactivity in the human brainstem. Brain StructFunct. 2013; 218(1): 131-149 with permission of Springer.

Investigación Clínica 59(2): 161 - 178, 2018 Neuropeptides in the brainstem 169 the presence of high levels of β-endorphin in tary tract, cuneate nucleus, superior olivary the middle regions of the , and of nucleus, , among others moderate levels of same in the regions of (Table I). pons and medulla oblongata, but also iden- On the other hand, Fordor et al. (38), tified β-endorphin-labeled cell bodies in the evidenced the presence of B-dynorphin-la- lateral and middle cuneate nucleus, as well beled fibers in the rostral region of the lateral as fibers in the lateral parabrachial nucleus, parabrachial nucleus. Additionally, a recent jointly labeled with β-endorphin and α-MSH. immunocytochemical study demonstrated Additionally, the presence in the human the presence of immunoreactive structures mesencephalon of fibers labeled with deri- by another Pro-dynorphin derivate, alfa-neo- vates of Pro-enkephalins such as met-enk, endorphin, in several nuclei and regions of met-8 andleu-enk has been demonstrated the human brainstem (6) with a low density (5, 9, 11,12), located in the substantia nig- of cell bodies in the medullar periaqueduc- ra, , between the bra- tal gray matter and the spinal trigeminal chium conjuntivum and the lemniscus me- nucleus (gelatinous pars), as well as high, dialis, lateral parabrachial nucleus adjacent moderate and low densities in zones like the to the trochlear root, periaqueductal gelatinous and magnocellular portion of the gray matter pontine region, , spinal trigeminal nucleus, principal senso- reticular formation, and inferior olivary nu- rial nucleus, dorsal motor nucleus of vagus, cleus, suggesting modulatory mediation in , , me- different actions like respiratory and motor dial vestibular nucleus, , mechanisms. In addition, methionine-en- cuneate nucleus and reticular formation kephalin and leucine-enkephalin receptors (Table I). have been reported in the of the human brainstem and diencephalon a) Tachykinins during aging (12); however, the results did Substance P (SP) is one of the best not show significant changes in the levels known members of this family, and one of of these neuropeptides in this region with the first to be isolated. This peptide can be respect to zones like the pallidum and the obtained from a pro-hormone or in associa- caudate. These findings suggest that during tion with substance K (also denominated the phenomenon of aging, enkephalins in ) of a precursor derived from these regions can modulate the action of do- the gen of per-pro-tachykinin A (30, 31, 40, paminergic neurons, given the close relation 41); among its best studied actions is its between enkephalins and these pathways, es- mediation in nociceptive events, including pecially the pathways affecting receptors D1 pain. On the other hand, neurokinin B de- and D2, resulting in motor effects. rives from the gen of pro-tachykinin B (30). Coveñas et al. (5) demonstrated the Other members of the tachykinin family ex- presence of Arg6-Phe7-Leu8 (Met-enk-8)-me- ist, like: , and fisalaemin. thionine-enkephalin-labeled cell bodies and/ was isolated in amphibians, and or fibers in the whole human brainstem. This its homologue in mammals is -releas- study evidenced different densities of immu- ing peptide (GRP), in which both molecules nolabeled structures (high, moderate and have the same carboxi-terminal heptapep- low density) in regions such as the reticu- tide (30). lar formation, (medulla oblongata, pons and Studies have detected immunoreactive mesencephalon), medial vestibular nucleus, structures for various tachykinin-derived solitary nucleus, dorsal motor nucleus of peptides in the human brainstem by means vagus, periaqueductal gray matter, spinal of different immunocytochemical methods, trigeminal nucleus, ambiguus nucleus, soli- demonstrating the presence of SP in regions

Investigación Clínica 59(2): 161 - 178, 2018 170 Duque-Díaz et al. of the brainstem (13, 14, 27, 39). Cell bod- sponse to and food intake (44), the ies immunostained with SP were detected in reason why its study focuses on detection of superior colliculus, reticular formation (me- this substance – receptors and cellular struc- dulla oblongata, pons and mesencephalon) tures– in regions of the cortex and the di- and nucleus, as well as immu- encephalon (15-17, 45-48). The presence of noreactive fibers containing SP in the infe- cell bodies and/or fibers containing NPY has rior colliculus, braquium conjuntivum, para- been described in the human brainstem (49, brachial nucleus, locus coeruleus, spinal 50) from the utmost rostral to the utmost trigeminal nucleus, dorsal motor nucleus of caudal region, in nuclei like: periaqueductal vagus, medial vestibular nucleus and cune- gray matter, reticular formation, substantia ate nucleus. Also, and by means of radioim- nigra (pars lateralis), inferior colliculus, lo- munoassay and autoradiography, high levels cus coeruleus, tegmental dorsal tegmental of SP were observed in the mesencephalon, nucleus of Gudden, trigeminal spinal nucle- pons and medulla oblongata (42, 43). us, lateral reticular nucleus, medial and infe- On the other hand, the presence of neu- rior vestibular nucleus, dorsal motor nucleus rokinin and neurokinin B-labeled cell bodies of vagus, and , inter- and/or fibers in the human brainstem has peduncular nucleus and nucleus laterodor- been demonstrated (4, 6). Although these salistegmenti. studies evidenced ample distribution of these tachykinins, the density of immunoreactive c) Somatostatin structures shows some differences. Thus, Somatostatin is a cyclic tetradecapep- Coveñas et al. (4) described a high density of tide that originally isolated of the ovine cell bodies labeled for neurokinin in regions on the basis to inhibit the such as periaqueductal gray matter, reticu- release of from anterior pi- lar formation, dorsal motor nucleus of vagus, tuitary cells (25). Several molecular forms dorsal longitudinal fascicle, and inferior col- of this peptide exist, such as somatosta- liculus, and Duque et al. (6) observed a low tin-14, somatostatin-28 and a fragment of density of neurokinin B cell bodies labeled this latter, somatostatin-28 [1-12] (51). in the periaqueductal gray matter, superi- Studies have been conducted in the human or colliculus and pons reticular formation. brainstem of the distribution of receptors Both studies determined a similar pattern in for somatostatin, allowing observation of a the density of fibers containing neurokinin- relative density (low, moderate, high) in the and-neurokinin B, where immunoreactive fi- different regions (medulla oblongata, pons, bers were described in similar nuclei: in the mesencephalon), and locating high levels of spinal trigeminal nucleus, substantia nigra, somatostatin receptors in the gracilis nucle- reticular formation, and solitary tract nucle- us, cuneate nucleus, inferior olivary nucleus, us. Such differences might arise from the an- hypoglossal nucleus, dorsal motor nucleus tibody specificity, considering the similarity of vagus, and solitary tract nucleus. In con- of methodology applied. trast, lower levels of somatostatin recep- tors were observed in the lateral vestibular b) nucleus and the medial vestibular nucleus Neuropeptide Y (NPY) is a molecule of (11). By using the immunocytochemistry 36 amino acids derived from a pre-pro-NPY technique, Chigr et al. (27) observed differ- and belonging into a family that includes ent densities of immunoreactive structures polypancreatic peptide (PP) and peptide YY containing somatostatinin the - (PYY). This peptide is highly conserved in stem. In this study, cell bodies labeled with mammals and it is catalogued as a neuro- this neuropeptide were observed in the su- protector, additionally to its role in the re- perior colliculus, supratrochlear nucleus,

Investigación Clínica 59(2): 161 - 178, 2018 Neuropeptides in the brainstem 171 , , in- nucleus neurons, with fibers projecting to- terpeduncular nucleus, periaqueductal gray wards the medulla, where they develop no- matter (pons), medial vestibular nucleus, ciceptive functions (24). A study conducted and reticular nucleus. Additionally, fibers by Goncharuk and Jhamandas (55) detected containing somatostatin were found in in- the presence of structures (cell bodies and/ terpeduncular nucleus, periaqueductal gray or fibers) immunolabeled for receptor of FF2 matter, cuneate nucleus, substantia nigra, in different structures of the encephalon, superior central nucleus, locus coeruleus, among them regions of the brainstem such parabrachial nucleus, dorsal motor nucleus as the rostral part of the dorsal motor nucle- of vagus, trigeminal spinal nucleus, ambigu- us of vagus, solitary tract nucleus and ventral us nucleus, reticular formation (medulla ob- tegmental area. It must be highlighted that longata), inferior olivary nucleus and acces- along this work, the highest receptor den- sory olivary nucleus. Also, Bouras et al. (52) sity was observed in the hypothalamus, sug- identified immunoreactive structures (cell gesting a mediating role of this substance in bodies and fibers) containing somatosta- neuroendocrine regulation. tin-14 and somatostatin-28 (1-12) in the hu- The use of in vitro light microscopic au- man brainstem. This work allowed observa- toradiography allowed to demonstrate the tion of not only a high density of cell bodies presence of oxytocin receptors in the pars in the periaqueductal gray matter, oculomo- compact of the substantia nigra, the gelati- tor nucleus, hypoglossal nucleus, trochlear nous layer of the spinal trigeminal nucleus, nucleus, but also moderate and low density medio-dorsal region of the solitary tract of cell bodies in nuclei like the ventral teg- nucleus and hypoglossal nucleus (56). Ad- mental area, locus coeruleus, superior and ditionally, Boccia et al. (57) demonstrated lateral vestibular nucleus, through immunohistochemistry the pres- and facial nucleus. ence of oxytocin receptors in solitary tract nucleus, ambiguus nucleus and hypoglossal d) Other neuropeptides and nucleus cell bodies and fibers, but not in the neuroactive substances inferior olivary. By using the radioimmunoassay tech- Besides neuropeptides, the presence of nique, increased level of corticotrophin-re- neuroactive substances (classic neurotrans- leasing hormone (CRH) in different pontine mitters) has been evidenced by means of nuclei of the brainstem of depression-diag- diverse immunolabeling techniques in dif- nosed suicide men was observed (53). This ferent regions of the human brainstem. work evidenced 30 and 45% CRH increase Presence was detected of cell bodies and with respect to raphe nucleus controls in its fibers labeled for receptors in the caudal and dorsal nuclei portions, as well as dorsal motor nucleus of vagus, hypoglossal locus coeruleus. Hasegawa et al. (54) dem- nucleus, solitary tract nucleus, geniculated onstrated the presence of fibers containing nucleus, trigeminal spinal nucleus, cuneate oxerin B associated with dopaminergic neu- nucleus, locus coeruleus, raphe dorsal nu- rons in the ventral tegmental area and the cleus, olivary complex, inferior olivary and substantia nigra of the post-mortem human lateral reticular nucleus (58, 59). brainstem. These findings suggest that the Finally, Fig. 4 shows the percentage of orexinergic mechanism may play an impor- nuclei with immunoreactive structures (cell tant modulating role in the reward process. bodies and fibers) for the different neuro- Although neuropeptide FF2 is an 8-ami- peptides. It evidences that the highest per- no-acid peptide regularly located in the ven- centage of nuclei with labeled cell bodies tromedial and dorsomedial hypothalamus, corresponds to NPY and somatostatin, with it has also been observed in solitary tract 29%, while the highest number of nuclei

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Fig. 4. Percentage of nuclei with immunolabeled structures (cell bodies and / or fibers) with the different neuropeptides studied in the human brainstem. with immunolabeled fibers corresponds to neuropeptides neuromodulate nociceptive NPY, with 45%. Nuclei with immunolabeled mechanisms (pain and temperature), as well cell bodies suggest these regions may have as visceral sensitivity (5, 6). On the other a role as regulating centers in the release of hand, the presence of immunoreactive struc- these substances and are capable of project- tures containing alfa-neo-endorphin in the ing axons towards other areas of the brain- superior colliculus suggests this substance stem, while those nuclei containing fibers can have a modulating action in visual mo- labeled for the different neuropeptides are tor coordination (6). part of the projections of diverse origins. Evidence has been established of the important role of some peptides derived PHYSIOLOGICAL ACTIONS from pro-tachykinins, like NKA, NKB and OF NEUROPEPTIDES IN THE HUMAN substance P, whose biological interaction is BRAINSTEM mediated by three receptors: NK-1, NK-2, and NK-3. Besides, tachykinins are known In general, the neuroactive substances to be involved in different actions as saliva- specified in this review show an ample distri- tion, smooth muscle contraction regulation, bution; they are located all along the whole depolarization of potential action in central structure of the human brainstem, suggest- neurons, hyperactivity, activation of behav- ing that diverse neuropeptides may be in- ioral mechanisms, , etc. (4, 41). volved in a wide range of physiological ac- Such data are in accordance with previous tions. One example of such is the presence results where cell bodies and fibers con- of fibers immunolabeled with Pro-enkepha- taining neurokinin-and-neurokinin B were lin-derived opiates, like methionine-enkeph- observed in regions like trigeminal spinal alin-8 and pro-dynorphin, like alfa-neo-en- nucleus, periaqueductal gray matter and dorphin in regions like the trigeminal spinal , regions involved nucleus, periaqueductal gray matter, and in nociceptive and antinociceptive mecha- solitary tract nucleus, suggesting that these nisms, and in the solitary tract nucleus and

Investigación Clínica 59(2): 161 - 178, 2018 Neuropeptides in the brainstem 173 parabrachial nucleus, involved in respiratory (hypocretins) are widely stud- and cardiovascular mechanisms. Their pres- ied neuropeptides in the central nervous sys- ence in the dorsal motor nucleus of vagus tem of mammals, including that of humans. has been related to smooth muscle contrac- These substances have been reported to be tion mechanisms, substantia nigra, associ- involved in mechanisms like sleep and alert- ated with motor mechanisms, and finally ness , alimentary disorders the inferior colliculus, involved in auditory and energetic balance (54, 62, 63). mechanisms (4, 6, 60). Finally, it is important to highlight that NPY, in principle synthesized in the ar- the ample distribution of neuropeptides in cuate nucleus, projects itself to many other the brainstem suggests their participation nuclei where it shows an effect on multiple in various physiological actions controlled systems, thus indicating a possible neuro- by this structure. modulating action on the central nervous system (48). As previously mentioned, most COEXISTENCE OF NEUROPEPTIDES studies on the distribution of NPY in humans IN THE HUMAN BRAINSTEM have involved mostly hypothalamic regions; however, two studies of the human brainstem Presence of different neuropeptides in (49, 50) evidenced the presence of this neu- nuclei and/or tracts of the human brain- ropeptide in caudal regions of the brainstem stem (Table I) suggests possible interaction (medulla oblongata), suggesting that this among these substances, and an elaborate region receives projections with NPY from modulating action in physiological func- the hypothalamus, which allows it posterior tions. The location of neuropeptides in vari- and diverse autonomic and neuroendocrine ous similar nuclei hints of their coexistence control functions. in the same neuron, which is why some stud- Presence of NPY has been associated ies evidence a possible coexistence of methi- with an inhibitory role in the hypothalamic onine-enkephaline-8 and substance P in the hormonal synthesis of somatostatin (27). solitary tract nucleus and in the locus coeru- However, other studies of the human brain- leus (9, 11, 12). stem suggest other possible actions. Pres- Taking into account the distribution ence of somatostatin-immunolabeled struc- and peptidergic characteristics of immuno- tures in the periaqueductal gray matter and labeledcell bodies, a possible coexistence trigeminal spinal nucleus suggests a modu- can be described between different neuroac- lating effect on nociceptive activities. On tive substances summarized below: the other hand, their presence in the locus • Neurokinin B and somatostatinin the coeruleus can influence the noradrenergic reticular formation of the pons region system, given that this nucleus is involved (2, 3, 6). in stress, major depression, and paradoxical • Somatostatin and substance P in the sleep response mechanisms (52, 61). The spinal trigeminal nucleus (2, 3, 13, 14, presence of structures immunolabeled for 27, 52). somatostatin in the dorsal motor nucleus • Neurokinin B and alfa-neo-endorphin in of vagus, as well as solitary tract nucleus the periaqueductal gray matter (6). may suggest that this neuropeptide par- • β-endorphin, dynorphin A, , ticipates in regulation of some autonomous enkephalins, SP, NPY, CCK in the para- functions, based on results from previous brachial nucleus and the locus coeru- studies in rats, where the intraventricular leus (39). administering of somatostatin stimulated Given that, the coexistences herein pro- intestinal motility and gastric acid secre- posed are described taking into account cell tion (2, 3). bodies labeling, double-labeling immunocy-

Investigación Clínica 59(2): 161 - 178, 2018 174 Duque-Díaz et al. tochemical studies should be conducted to ent projections of peptidergic systems in a demonstrate them. structure like the brainstem. This would pro- vide wider, more detailed knowledge of the FUTURE RESEARCH connections that can be established in the IN NEUROPEPTIDES IN THE HUMAN different areas of the human brainstem, as BRAINSTEM well as the level of influence that a region can have on another. The previous suggests involvement of Thus, the results deriving from combin- neuropeptides in many nervous central sys- ing the different techniques mentioned can tem actions. Given that data available in the contribute to in-depth comprehension of the field of the human brainstem are occasion- functions of the different neuroactive sub- ally incomplete, the need arises for in-depth stances –among them neuropeptides– in the studies of aspects that will shed light on the human brainstem. detailed distribution of these substances and their physiological implication. ACKNOWLEDGEMENTS Use of diverse methodologies can pro- vide more detailed information on the syn- This work has been supported by the Re- thesis of these neuroactive substances. For search Commission and the School of Medi- example, hybridization in situ would allow cine of the Universidad de Santander UDES. comparison between mRNA distribution and the final molecule of a neuropeptide, ABBREVIATION evidencing if a given substance is produced in the same region where it acts, or, on the CeGy: Periaqueductal contrary, it is produced in another region CSNu: Central Superior Nucleus and transported to its site of action. Such DCNu: Dorsal studies can be supplemented by conducting DLF: Dorsal Longitudinal Fasciculus tracking of not only the receptor of a neuro- DMNu: Dorsal Motor Nucleus of Vagus peptide, but also its activity. FacNu: Facial Nucleus On the other hand, the distribution of HyNu: Hypoglossal Nucleus peptidases would also allow supplementary IC, CNu: Inferior Colliculus, Central Nucleus study of the action pathways of a neuropep- IC, LZ: Inferior Colliculus, Lateral Zone tide. In studying the cellular communication IPNu: Interpeduncular Nucles pathway where neuropeptides mediate, it is ISNu: Inferior Salivatory Nucleus important to develop electronic microscopy LCNu: Lateral Cuneate Nucleus and immunocytochemistry techniques (sim- LL: Lateral Lemniscus ple and/or double labeling) to demonstrate LocCer: Locus Coeruleus whether a neuropeptide is released alone or LRNu: Lateral reticular Nucleus together with other neuroactive substances. LVN: Lateral Vestibular Nucleus Given the knowledge of the possible co- MAO: Medial AccsesoryOlivary Nucleus existence of neuropeptides, double-or-triple- ML: Medial Lemniscus labeling immunofluorescence studies must MVN: Medial Vstibular Nucleus be implemented, aided by studies by means NuAm: Nucleus Ambiguu of confocal microscopy in order to analyze NuCu: Cuneate Nucleus if co-localization exists between neuropep- NuGr: Gracile Nucleus tides. NuPp: The combination of methods such as OcNu: immunocytochemistry and trac-tracing PO: Inferior Olivary Nucleus would allow knowledge of afferent and effer- RaNu: Raphe Nucleus

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