Morphological Relationships between the Cholinergic and Somatostatin-28(1-12) Systems in the Alpaca ( Lama pacos ) Brainstem

: In the alpaca brainstem, the distribution of the cholinergic system by the immunohistochemical detection of the enzyme choline acetyltransferase (ChAT) has been described, and its relationship with the distribution of somatostatin-28(1-12) is analyzed by double-immunostaining techniques. Overlapping distribution patterns for both substances were observed in many brainstem regions, suggesting that interactions between them may occur in the reticular formation, nucleus ambiguus or laterodorsal tegmental nucleus. Colocalization of the two substances in the same cell bodies was only observed in restricted areas, such as the nucleus of the solitary tract, reticular formation and nucleus ambiguus. In addition, in several regions, an apparent high innervation of the peptidergic ﬁbers on cholinergic neurons has been observed. The results suggest that chemospeciﬁc interactions could be crucial for the control of speciﬁc cardiorespiratory and/or digestive functions in alpacas. These interactions may represent brain-adaptive mechanisms to particular environments and have a potential therapeutic use in respiratory disorders.


Introduction
Cetaceans (dolphins, whales) and artiodactyls (even-toed ungulates, e.g., sheep, giraffe) belong to the order Cetartiodactyla. The family Camelidae is part of artiodactyls, and the alpaca (Lama pacos) is included in this family [1][2][3][4][5]. Alpacas are important animals for the economy of numerous South American countries due to the excellent quality of their wool, and in this sense, numerous studies focused on their maintenance and reproductive cycles have been performed [6,7]. The members of the Camelidae family have specific morphological characteristics, such as long necks and seven cervical vertebrae, and moreover alpacas can live at sea level and at 5000 m above sea level [6][7][8][9][10][11][12]. These characteristics suggest the existence of important and unique adaptation mechanisms, mainly related to cardiovascular and respiratory mechanisms, which are controlled by the central nervous system, specifically brainstem centers. Other members of the Certiodactyla order also exhibit brain specializations that help them to survive within their respective environments [1][2][3][4][5].
Since all these physiological functions are regulated by neuroactive substances, several studies have reported the distributions of different classical neurotransmitters and neuropeptides by means of immunohistochemical methods in the alpaca brain [6][7][8][9][10][11][12]. These studies complement previous works regarding the mapping of neuro-modulatory systems in the brain of Artiodactyla and confirm that the nuclear complement of neurotransmitters, such as acetylcholine or catecholamines, detected in the alpaca brainstem and diencephalon Anatomia 2022, 1 55 was similar to that found in other members of the same order, and this finding supports Manger's hypothesis [13]. In addition, the distribution of some neuropeptides has been studied in the alpaca brainstem [6][7][8][9]12] and diencephalon [10,11]. Moreover, the morphological relationship between neuropeptides and neurotransmitters has been reported, and double-immunolabeling for tyrosine hydroxylase, which is the rate-limiting enzyme of the catecholaminergic synthesis, and somatostatin-28(1-12) (Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)) has been carried out in the diencephalon of the alpaca [11]. In the brainstem of this species, a double-labeling for choline acetyl transferase (ChAT, a marker for the cholinergic system) and calcitonin gene-related peptide (CGRP), as well as for CGRP and tyrosine hydroxylase [9,12], has been described. According to the distribution of double-labeled perikarya, the results observed in the brainstem in these studies suggest that CGRP might interact more with the catecholaminergic system than with the cholinergic system. However, although the physiological interactions between somatostatin and catecholamines have been reported in the literature [13], no double-labeled neurons for these two substances were detected in the alpaca diencephalon [11]. A possible explanation may be that the interaction between the two substances may occur with another somatostatin fragment different from the one studied in that work.
Somatostatin, in addition to inhibiting the growth hormone, acts as a neuromodulator in numerous physiological functions, blocking the release of noradrenaline and stimulating the release of serotonin and acetylcholine [14]. The precursor named pro-somatostatin is cleaved into somatostatin-28, somatostatin-12 (corresponding to the first 112 amino acids of somatostatin-28) and somatostatin-14 [15]. These fragments can elicit different responses in relation to the same mechanism, such as the cardiovascular regulation [15], probably due to the activation of different somatostatin receptor subtypes [16]. In addition, the interaction of somatostatin with other neurotransmitters such as acetylcholine is well-known [16][17][18][19][20][21], but the studies that described such interactions were more focused on memory processing.
Studies previously performed in the alpaca brainstem suggest a similar distribution pattern for CGRP and Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) [7,8]. The presence of CGRP and ChAT in the same perikarya was reported in some of the alpaca brainstem nuclei; thus, the goal of the present work was to know whether there is a neuroanatomical basis for possible interactions between Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and ChAT in the alpaca brainstem. Moreover, due to the participation of these two substances in cardiovascular and respiratory functions, another aim of this study is to know whether these interactions can constitute a morphological basis for the control of cardiovascular and/or respiratory mechanisms in this species. The results obtained will help to understand the distinctive control mechanisms that exist in the alpaca as physiological adaptations to living in such different habitats in terms of altitude, which leads to changes in air quality and composition. Knowledge of these adaptive mechanisms and their morphological basis will contribute to a better understanding of the neuroanatomy and physiology of the alpaca. Comparison with the results obtained in other mammalian species will allow to assess whether these mechanisms may constitute therapeutic targets for the possible treatment of cardiovascular, respiratory and/or digestive disorders.

Animals
As reported previously in similar studies [8,12], six adult male alpacas (Lama pacos) (70-80 kg; 5-8 years) were used here. From birth to the perfusion day, animals were maintained at 0 m on the sea level and kept under standard conditions of temperature and light and with free access to water and food. The study was performed following the guidelines of the ethical and legal recommendations of the Spanish legislation [8,12].

Single Immunolabeling for Som-28(1-12) and ChAT
The distribution of Som-28(1-12)-and ChAT-immunoreactive structures was studied according to the anatomical description performed by de Souza et al. [8] and Marcos and Coveñas [12]. Since the detailed mapping of the distribution of the immunoreactive structures containing Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and ChAT in the alpaca brainstem has been performed previously in these two studies, only a brief description will be reported in the present work, more focused on the coexistence of ChAT and Som-28(1-12) in nerve cells. As in previous studies performed using a similar methodology, the brown precipitate (for Som-28(1-12)) and the blue staining (for ChAT) are easily distinguishable. Som-28(1-12)-immunoreactive profiles showed a typically peptidergic morphology, with Som-28(1-12)-positive cell bodies containing visible secretion granules and immunoreactive fibers of varicose appearance. In contrast, profiles containing ChAT showed no varicose labeling and a more homogeneous precipitate.

Discussion
The distribution of immunoreactive profiles for ChAT and Som-28(1-12) is widespread in the alpaca brainstem. According to their respective distributions, these neuroactive substances could participate in the regulation of nociceptive, motor, autonomic, visual, auditory, cardiovascular or respiratory mechanisms [8,12]. The distribution of cholinergic cell bodies suggests that acetylcholine might be implicated, among others, in the regulation of rapid eye movement (REM) sleep and wakefulness as well as in motor functions, since ChAT was observed in the nuclei of several motor cranial nerves and in the pedunculopontine and laterodorsal tegmental nuclei [12]. Moreover, neurons containing Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) detected in the brainstem could be the main source for the somatostatinergic innervation of the alpaca diencephalic and other brainstem nuclei [8,10,11].
Although immunoreactive cell bodies for ChAT or Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) were observed in several regions of the alpaca brainstem, the presence of the two substances in the same cell bodies was only detected in the nucleus ambiguus, reticular formation and nucleus of the solitary tract, suggesting that the interaction between the cholinergic and the somatostatinergic systems in the same neurons is restricted in the alpaca brainstem. The relation between the two systems in the mammalian brain is well-known, mainly in memory processes due to the important role played by acetylcholine in this function: somatostatin stimulates the release of acetylcholine from cholinergic terminals [18,19], and somatostatin administered intracerebrally improved memory failures in rodents showing brain cholinergic deficits [21]. In addition, a monosynaptic relationship between cholinergic forebrain neurons and somatostatin-containing axons has been described [41]. The results reported in the present study suggest that the intracellular interaction between acetylcholine and somatostatin could be mainly related with the regulation of cardiovascular, respiratory, gastrointestinal and gustatory systems [20,42]. This has been reported in other mammals [43] such as cats [20,42] and rodents [18,19,44,45]. A study performed in rats described the relationship between acetylcholine and several somatostatin molecules in the solitary tract complex (including the nucleus of the solitary tract and dorsal motor nucleus of the vagus) and reported that Som-28 and Som-14 could be considered as inhibitory neurotransmitters in the solitary tract complex, but neither Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) nor Som-28(1-10) hyperpolarized the same cells that showed Som-28-or Som-14-evoked hyperpolarization in this region [45]. This suggests that the different somatostatin fragments might exert different actions and that several somatostatin receptors could participate in these responses. It has been reported that cholinergic parasympathetic neurons in the dorsal motor nucleus of the vagus are immunoreactive for somatostatin receptor subtypes 2A, 2B, 4 and 5 [16]. The results obtained in the alpaca brainstem displayed neurons containing Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and ChAT in the nucleus of the solitary tract but not in the dorsal motor nucleus of the vagus, although the two substances were detected in this later region in separated cellular populations. In cats, it has been suggested that the source of cholinergic elements for the nucleus of the solitary tract could arise from the dorsal motor nucleus of the vagus [42]. The methodology used in the present work does not allow to know whether the immunoreactive neurons detected in the alpaca brainstem are projection neurons or not. However, the results presented here suggest that the interactions between somatostatin and acetylcholine could be different in both regions, and that acetylcholine might be related to Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in the nucleus of the solitary tract and/or to other somatostatin fragment(s) in the dorsal motor nucleus of the vagus. In other brain regions, different distribution patterns have been reported for distinct somatostatin fragments, at least in humans [43], and this could also be the case in alpaca.
The coexistence of somatostatin and acetylcholine in centers involved in autonomic regulation suggests that they could also be implicated in the regulation of the gastrointestinal tract. Esophageal afferents terminate in the nucleus of the solitary tract, which participates in deglution, eliciting the entire sequence of muscle activity. This nucleus projects to esophageal motoneurons located in the rostral portion of the nucleus ambiguus. It has been reported that this projection contains somatostatin, which can inhibit the neuronal firing in this pathway [19,46]. On the other hand, acetylcholine is present in motoneurons of the nucleus ambiguus that project to the esophagus and stimulates the contraction of the striated regions of the esophagus [46]. Somatostatin also participates in the generation of ambigual excitatory postsynaptic potential [19]. According to the results obtained in the brainstem of the alpaca, it can be suggested that acetylcholine and somatostatin might interact on neurons of the nucleus of the solitary tract and nucleus ambiguus, since both regions displayed double-labeled cells, and then could play a role in the central control of deglution and esophageal motility. In this sense, it has been reported that the somatostatinergic neurons of this circuit modulate viscerosensory signaling and provide a strong postsynaptic inhibition of this signal [44]. However, this regulatory function has been assigned to the somatostatinergic neurons containing GABA located in the nucleus of the solitary tract connected to neurons of the dorsal motor nucleus of the vagus that project to the antrum [44]. The direct stimulation of the latter nucleus increases phasic contractions and gastric tone, effects that are independent from changes in heart rate and blood pressure, which are also regulated by these regions of the brainstem [47,48]. The results obtained in the alpaca agree with these findings, since Som-28(1-12) has been detected in very few cholinergic neurons of the nucleus of the solitary tract and no colocalization has been observed in the dorsal motor nucleus of the vagus. Thus, although the interaction between both substances cannot be discarded, it seems more likely that the regulation of the gastrointestinal tract was mainly related to the somatostatin-GABAergic neurons and/or other somatostatin fragments different from Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). According to the literature, the results presented here emphasize the complex regulatory mechanisms carried out in the alpaca by brainstem structures, since the same neuroactive substances detected in the same regions might exert different regulatory roles on distinct physiological functions. Further studies are needed to confirm whether these complex regulatory mechanisms are common to several species or whether they are specific adaptations of the alpacas to their habitat, since these animals can live at sea level and at altitudes of more than 5000 m. Life in these environments, together with the special morphological characteristics of the alpaca, make it a good candidate to study the brain morphological characteristics underlying these adaptations.
The results obtained in the present paper describe an abundant somatostatinergic innervation in the alpaca brainstem. In two regions, the nucleus ambiguus and the medial division of the facial nucleus, these peptidergic fibers are especially numerous surrounding the perikarya of the cholinergic neurons, suggesting that these cell bodies may be strongly innervated by somatostatinergic terminals. Supporting this interaction, the colocalization of somatostatin receptors 2A in cholinergic neurons in the nucleus ambiguus of rats has been reported [16], as well as the innervation of the vagal motoneurons of the nucleus ambiguus by somatostatin in cats [46]. Below the facial nucleus, a region displaying numerous peptidergic and cholinergic fibers in the alpaca brainstem, a small group of neurons called the retrotrapezoid nucleus, are activated by increases in CO 2 levels and regulate the breathing cycle [49]. These neurons are mainly glutamatergic and receive cholinergic inputs as well as somatostatinergic innervation from the pre-Bötzinger complex, and it has been reported that this could be a neuroanatomical substrate to interact with the chemosensory control of breathing [50,51]. This region is unknown in the alpaca brainstem, but the abundance of fibers containing Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and ChAT as well as the possible somatostatinergic innervation on cholinergic cells detected in this area support the suggestion of a neuroanatomical basis for the chemosensory control of breathing, where somatostatin and acetylcholine might interact. Somatostatin has been involved in the chemosensory drive to breathe [49], and it should be clarified whether these results are again something common in several mammalian species or whether they are adaptive mechanisms of alpacas to their particular living conditions.
The opposite pattern (peptidergic neurons innervated by ChAT-immunoreactive fibers) was more difficult to observe in the alpaca brainstem. This was probably due to the appearance of the fibers containing the enzyme, that are usually harder to visualize near the Som-28(1-12)-immunoreactive cell bodies. The distinct appearance of cholinergic and peptidergic terminals has been reported in optical and electron microscopy [42], but the possibility of methodological aspects to this difficult visualization cannot be ruled out. It may be possible that the DAB precipitate prevents the correct visualization of cholinergic terminals when they are very thin, since the fibers containing ChAT of a larger diameter are easily visible.
The cholinergic projection from the laterodorsal tegmental nucleus links the forebrain limbic circuit with the limbic midbrain [17], and the presence of ChAT in the laterodorsal tegmental and pedunculopontine nuclei has been involved in the control of sleep, especially important in Artiodactyls to modulate the initiation of REM sleep, but not REM sleep maintenance [1]. It has been reported that putative sleep factors have hypnotic properties that could be related to changes in blood pressure, such as urotensin II [52], but this does not seem to be the case for Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in the alpaca brainstem considering the results reported in the present study, since apparently the peptide was detected in a different population than acetylcholine. However, in the alpaca brainstem, the regulation of the initiation of REM sleep carried out by the cholinergic neurons of the laterodorsal tegmental nucleus seemed to be more related to CGRP, since double-labeled cholinergic neurons containing this peptide have been detected in this region [12]. Other regions such as the reticular formation and the nucleus ambiguus also displayed neurons double-labeled with CGRP and ChAT. It may be possible that some cholinergic neurons of the reticular formation and/or the nucleus ambiguus containing Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) are also immunoreactive for CGRP, and thus a possible influence of the two peptidergic systems (somatostatin and CGRP) on cholinergic cell bodies can be suggested associated with the regulation of cardiovascular, digestive and respiratory functions, and this could be related to other regions of the brainstem. In this regard, the pedunculopontine tegmental nucleus modulates breathing by releasing acetylcholine into the retrotrapezoid nucleus in rats [49]. As discussed previously, in the alpaca, these latter regions showed immunoreactive profiles for ChAT, CGRP and Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), suggesting the involvement of these neuroactive substances in the regulation of the breathing homeostasis. The deep knowledge of the morphological basis underlying these mechanisms may potentially have a therapeutic use for the treatment of respiratory control problems, especially those associated with disorders of breathing during sleep [49]. In this regard, the morphological study of the alpaca brainstem can be very useful for the comprehension of cardiorespiratory control mechanisms, given the anatomophysiological peculiarities of these animals.

Conclusions
Although the distributions of Som-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and ChAT in the alpaca brainstem are similar, the colocalization of both substances in the same cell bodies is very scarce, suggesting a very limited interaction at the intracellular level. However, the abundant somatostatinergic innervation detected in some regions containing cholinergic cells points to a possible regulation of these neurons by the peptide, which may be related to respiratory control. Nevertheless, results obtained in previous studies suggest that the interaction between ChAT and CGRP would be more important than the interaction between ChAT and somatostatin in the alpaca brainstem, although ChAT, CGRP and somatostatin could be involved in the regulation of the sleep cycle. The detailed knowledge of these mechanisms may contribute to the development of therapies related to respiratory problems, especially those related to breathing disorders affecting some patients during sleep.
Funding: This research received no external funding.
Institutional Review Board Statement: Experimental design, procedures and protocols were carried out under the guidelines of the legal and ethics recommendations of the Spanish legislation. Ethical review and approval were waived for this study due to the use of histological sections from a previous study.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.