Morphology and immunohistochemical characteristics of the otic ganglion in the chinchilla (Chinchilla laniger Molina)

The available literature provides relatively little information on the morphology of the autonomic head ganglia in rodents including their neurochemical codding. The present study was thus designed to investigate the morphology and neurochemical properties of the otic ganglion in the chinchilla. The results will contribute to our knowledge of the organization of the autonomic nerve system in mammals. Morphological investigations of the otic ganglion were performed using the modified acetylcholinesterase method. The cellular structure was investigated with histological techniques and neurochemical properties were studied with double-labelling immunofluorescence method Macromorphological investigations allowed the otic ganglion to be identified as a compact, oval agglomeration of neurons and nerve fibers located inside the skull on the medial surface of the mandibular nerve, just above the oval foramen. Multidimensional cross-sections revealed densely arranged neuronal perikarya and two populations of nerve cells differing in size were distinguished. The large cells (40–50 μm) accounted for about 80% of the neurons in the otic ganglion cross-sections. Moreover, a small number of intraganglionic nerve fibers was observed. Immunohistochemical staining revealed that over 85% of the neuronal cell bodies in the otic ganglion contained immunoreactivity to VAChT or ChAT. VIP-immunoreactive perikarya comprised approximately 10% of the ganglionic cells. Double staining revealed the presence of VAChT and NOS-positive neurons which amounted to about 45% of the nerve cells in the otic ganglion. NOS-positive only perikarya comprised approx. 15% of all the neurons. Immunoreactivity to enkephalin, substance P, somatostatin and galanin was expressed in single nerve cell bodies and nerve fibres except numerous SP-positive intraganglionic nerve fibres. Some of them stained also for CGRP. Single neurons stained for TH. The present results, compared with previous findings, suggest the existence interspecies differences in the morphology, cellular structure and immunohistochemical properties of the head autonomic ganglia in mammals.


Introduction
The use of a modified classical histochemical method to localize cholinesterase activity has allowed autonomic cranial ganglia, including the otic ganglion, to be identified in many species of mammals [1,2]. Acetylcholinesterase (AChE) positive neurons were found in the otic ganglion in a number of animal species, such as dog [3], cat [4], spotted suslik [5], Egyptian spiny mouse [6], and rat [7]. Comparative anatomical studies performed in mammals indicate the presence of a significant relationship between the morphology, topography, and cellular structure of the parasympathetic head ganglia and the systematic position of the species investigated. Moreover, these ganglia display pronounced immunohistochemical differentiation between species. The specific immunohistochemical markers of parasympathetic structures are the enzyme that synthesizes acetylcholine, choline acetyltransferase (ChAT), and the specific neurotransmitter transporter protein, vesicular acetylcholine transporter (VAChT), responsible for loading acetylcholine into secretory vesicles in neurons, making it available for secretion. The presence of these two substances has been demonstrated in the otic ganglion of the rat [8][9][10][11] and pig [12]. Moreover, the expression of other biologically active substances, such as neuropeptide Y, vasoactive intestinal polypeptide, substance P, and calcitonin gene-related peptide, has been found in the otic ganglion of humans, pigs, and rats [13,8,12,9,10].
The available literature contains no information on the morphology and neurochemical properties of the otic ganglion in the chinchilla. The present study was thus designed to investigate this. The results will contribute to our knowledge of the organization of the autonomic nerve system in mammals.

Material and methods
The investigations were carried out on twelve chinchilla of both sexes (Chinchilla laniger Molina). These studies were approved in accordance with the appropriate Polish statute law on protection of research animals (Ustawa z dnia 15 stycznia 2015 r. o ochronie zwierząt …); studies on tissues obtained post-mortem do not require an approval of the Ethics Committee. The material was collected immediately after industrial slaughter at a chinchilla fur farm. The mandibular nerves were exposed under a stereomicroscope, rinsed in physiological solution, and fixed for 30 min. in 10% formalin. Four individuals were used for morphological investigations using the modified acetylcholinesterase method [1,2]. Another four animals were used for the histological study. Once fixed in formalin, the mandibular nerves were embedded in paraffin (Paraplast plus) and in Historesine® (70-2218-501 Mounting Medicum), cut with a microtome (Mikrotom 335) for 3-5 µm slides, stained according to methylene blue and the H&E methods, and silvered method. Morphometry and photographic documentation were performed using a Nikon Digital Sight SD-L1 System and Nis-Elements 3.22 software with Nikon Eclipse 90i microscope.
The final four animals used for the immunohistochemical investigations were transcardially perfused with 0.4 l of 4% ice-cold buffered paraformaldehyde, and the mandibular nerves were collected as described above. The tissues were postfixed by immersion in the same fixative for 2 hours, rinsed with phosphate buffer and transferred to 30% buffered sucrose solution for storage until further processing. The ganglia were cut into 12 μm -thick cryostat sections, which were processed for double-labelling immunofluorescence method on slide-mounted sections. The sections were washed thrice for  (Table 1) diluted in PB containing 0.25% Triton X-100. After incubation with primary antiserum, the sections were washed three times for 10 min. in PB and further incubated with secondary antisera for 1 h in RT. After incubation, the sections were washed three times for 10 min. in PB, coverslipped with buffered glycerol, and examined under a confocal microscope (Zeiss, LSM 700). Every seventh section was used to avoid double counting of the same neurons and sixty histological sections were used for the measurement. Staining specificity was controlled by preabsorbtion of a diluted antiserum with 20 µg/ml of an appropriate antigen, which abolished the specific immunoreaction completely. In addition, experiments were carried out in which the primary antiserum was replaced by nonimmune serum or by PBS, in order to verify the specificity of particular immunoreactions.

Results
The histochemical investigations revealed extensive acetylcholinesterase (AChE) staining in the otic ganglion of the chinchilla. This allowed the ganglion to be identified as a compact, oval agglomeration of neurons and nerve fibers located inside the skull on the medial surface of the mandibular nerve, just above the oval foramen ( Fig 1A). The ganglion was 3-5 mm long, 2-3 mm wide and about 0.6 mm thick. The small petrosal nerve, as the parasympathetic radix, reaches the caudal part of the ganglion. In its course, a small aggregation (1 mm long and 0.4 mm wide) of neurocytes was observed. The maxillary artery ran below the ganglion crossing the mandibular nerve more often on the medial side (70% cases), and rarely on the lateral side ( Fig 1A).
Postganglionic fibers leaving the otic ganglion intensely stained for AChE. A delicate bundle of fibers left the superior surface of the structure and entered the trigeminal ganglion ( Fig 1A). A small aggregation of neurons in the course of this nerve was frequently observed.
The histological investigations confirmed the close contact between the otic ganglion and the mandibular nerve (Fig 1A-D). Multidimensional cross-sections revealed densely arranged neuronal perikarya. Two populations of nerve cells differing in size could be distinguished: the large neurons with a diameter of 40-50 µm, and the small cells with a diameter of 28-25 µm (Fig 1B, C). The large cells accounted for about 80% of the neurons in the otic ganglion cross-sections. The ganglionic neurons were surrounded by satellite cells with intensely stained nuclei ( Fig 1C). Moreover, a small number of intraganglionic nerve fibers was observed (Fig 1D). The entire chinchilla otic ganglion was surrounded by thin connective tissue capsule.
Immunohistochemical staining revealed that over 85% of the neuronal cell bodies in the otic ganglion had immunoreactivity to VAChT (Fig 2A), and a comparable number of the perikarya stained for ChAT ( Fig 2C). These cells were mainly of the large type. VIPimmunoreactive (IR) perikarya made up approximately 10% of the ganglionic cells; these were small or medium-sized neurons (Fig 2A). Double staining revealed that approximately 20% of the VIP-IR neurons were VAChT-negative (Fig 2A). Intraganglionic VAChT-IR nerve fibers were numerous and often formed basket-like structures surrounding all the neuronal perikarya (Fig 2A). Immunoreactivity to enkephalin was expressed in the nerve cell bodies and nerve fibers. Staining against Met-ENK revealed only 3-4 nerve cell bodies per section that were immunoreactive to or this peptide ( Fig 2C); approximately half of these were simultaneously ChAT-positive (Fig 2C). Intraganglionic Met-ENK-IR nerve fibers were moderate in number ( Fig 2C). Leu-ENK-immunoreactivity was found in the cell bodies of one or two neurons per section. These perikarya were simultaneously VAChT-positive. The nerve fibers were very scarce.

GAL-IR neurons also occurred only individually (3-4 per section) and stained for
ChAT. GAL-positive nerves were very scarce and delicate. Few SOM-positive only nerve cell bodies were encountered in the ganglion (Fig 2D). Intraganglionic SOM-IR nerves were solitary and delicate. SP-positive intraganglionic nerve fibers were numerous and some of them also stained for CGRP (Fig 2E). SP-IR nerves formed basket-like structures surrounding nerve cell bodies. CGRP-immunoreactivity was found in nerve fibers which were less numerous than those which stained for SP. The vast majority of these were also SP-positive; individual nerve fibers contained only CGRP-immunoreactivity. Immunoreactivity to SP was also found in 1-2 neurons per section ( Fig 2E). Also, 3-4 neurons per section stained for TH; these were small, intensely stained and scattered throughout the ganglion (Fig 2F).
Our results did not reveal any sex differences.

Discussion
Our analysis of the topography of the otic ganglion in mammals revealed a close relationship between two anatomical structures: the mandibular nerve and the maxillary artery [14,3]. In most small mammalian species that have been investigated, the maxillary artery runs along the medial size of the mandibular nerve. In this case, the otic ganglion was located on the medial surface of the mandibular nerve or had direct contact with the maxillary artery [15,3,[16][17][18]. The otic ganglion in the chinchilla is an intracranial structure found on the medial surface of the mandibular nerve, and only postganglionic fibers run parallel to the maxillary artery.
Sometimes the otic ganglion forms a ring-shaped structure encircling the maxillary artery [18]. In some species, the otic ganglion consists of several interconnected nerve cell clusters. This morphological form has been described in the wild pig [22], dog [3], goat, and sheep [23], whereas in cats the ganglion is composed of one main cluster of neurons and about ten small additional ganglia [4]. The otic ganglion in the chinchilla represents a single agglomeration of neurons and only one small additional ganglion was found, associated with the small petrosal nerve.
This relatively small ganglion is an important source of nerve fibers contributing to the innervation of many organs, including the parotid gland, buccal glands, and the blood vessels supplying the brain, lower lip, and gingiva [24,25]. Previous studies have shown that the vast majority of neurons in the otic ganglion are cholinergic in nature [12]. Correspondingly in the chinchilla, 85% of these neurons are immunoreactive to ChAT or VAChT. The presence of catecholaminergic (TH-positive) neurons within this parasympathetic ganglion may be considered somewhat surprising. This small neuronal population probably represents a kind of the "developmental relic", consisting of not fully functional adrenergic nerve cells. This has been suggested in relation to catecholaminergic neurons in the porcine ciliary and pterygopalatine ganglia [26,27], and in the pterygopalatine ganglion of the chinchilla [28].
Our study showed that VIP-positive neurons constituted 10% of all ganglionic neurons. Few VIP-immunoreactive neurons have been found in the porcine and rat otic ganglion [12,8], while in the human otic ganglion over 90% of neurons are VIP-positive [32].
In the chinchilla pterygopalatine ganglion, a large population (40%) of VIP-immunoreactive neurons was found [28], whereas in the pig, all neurons in this ganglion were VIP-positive [27]. Our study found that many (60%) ganglionic neurons stained for NOS. A large (80%) population of NOS-positive neurons have been found in the human otic ganglion [32]. The pterygopalatine ganglion in the chinchilla also makes up a large population (42%) of NOS-IR neurons [28], whereas all the neurons are NOS-positive in the porcine pterygopalatine ganglion [27].
This study has revealed small populations of neurons immunoreactive to Metenkephalin and Leu-enkephalin. The expression of opioid peptides in otic ganglion neurons has been studied in the guinea pig [33,34] and, unlike our results on the chinchilla, a large (35%) subpopulation of the opioidergic neurons was found [34]. Individual GAL-and SOM-IR neurons were found in the porcine otic ganglion [ 12]. These findings correspond with our results.
In our study, we observed a dense network of VAChT-immunoreactive nerve terminals, frequently forming basket-like structures surrounding the nerve cell bodies. The nerve fibers that were immunoreactive to SP and CGRP were also relatively numerous. It can