Morphological Distribution Patterns and Neuroimmune Communication of Ganglia in Molly Fish ( Poecilia sphenops, Valenciennes 1846)

: Twenty-four adult molly ﬁsh ( Poecilia sphenops , Valenciennes 1846) were collected to study the morphology and distribution of ganglia using histological, immunohistochemical, and electron microscopy and focusing on their relation to the immune cells. The ganglia were classiﬁed spatially into cranial and spinal, and functionally into sensory and autonomic. Spinal ganglia (dorsal root ganglia, DRG) contained large close ganglionic cells, enclosed by satellite cells, as well as bundles of both myelinated and non-myelinated nerve ﬁbers. There are glial cells, immune cells and telocytes close to the ganglion. In addition, oligodendrocytes were closely related to myelinated axons. Glial ﬁbrillary acidic protein (GFAP) expression was conﬁned to the glia cells and the nerve ﬁbers in the cervical ganglia next to the gills, and surprisingly, in the large ganglionic cells of the DRG. The vestibular ganglia were large, connected to the hind brain, and contained numerous neurons packed in columns. The cervical ganglia were large and observed around the pseudobranch, head kidney, and thymus. Their neurons are randomly distributed, and nerve ﬁbers are peripherally situated. CD3-positive T-lymphocytes, dendritic cells, and CD68-positive macrophages were in close contact with the ganglia. Furthermore, the ganglia around the head kidney showed positive Iba1-expressing cells. Most ganglion cells and nerve ﬁbers in the DRG, autonomic, and vestibular ganglia showed moderate to strong S-100 immunoreactivity. The enteric glia, CD68-expressing macrophages, and acetylcholine (Ach)-expressing neurons were observed along the muscular layer of the intestinal wall. In conclusion, different ganglia of molly ﬁsh displayed direct


Introduction
A ganglion is a cluster or group of nerve cells found in the peripheral nervous system (PNS) [1]. One characteristic that distinguishes vertebrates is the presence of neural crest cells. These multipotent neural crest cells can differentiate into a wide range of tissues and

Sample Collection
The materials employed in this study consisted of randomly obtained 24 adult molly fish (Poecilia sphenops, Valenciennes 1846). The fish were purchased from an ornamental fish shop. The specimens were 4.40 ± 4.0 cm in standard length and 10.20 ± 1.50 gm in body weight.

Histological and Histochemical Analysis
Tissues were dissected from fish immediately after death at 1 × 1 × 0.5 cm and fixed in Bouin's fluid for 22 h. The fixed tissues were dehydrated with ethanol and cleared by methyl benzoate, then embedded in paraffin wax. Serial sagittal and transverse (5 µm thick) paraffin sections were taken and stained with Harris hematoxylin and Eosin, Grimilus silver stain, and Cresyl violet [11].

Semithin Sections and TEM
Small specimens of the ganglia were fixed in a solution of 2.5% paraformaldehydeglutaraldehyde and left overnight for fixation. Then, they were washed in 0.1 Mol/L phosphate buffer and osmicated with 1% osmium tetroxide in 0.1 Mol/L sodium-cacodylate buffer at pH 7.3. After that, the specimens were dehydrated by ethanol followed by propylene oxide and embedded in Araldite. One micrometer-thick semithin sections were stained with toluidine blue and examined under a light microscope. Ultrathin sections (70 nm) were obtained using Ultrotom-VRV (LKB Bromma, Stegen, Germany) and were stained with lead citrate and uranyl acetate. TEM images were captured with JEOL-100CX II electron microscope.

Immunohistochemical Analysis
Sections of fish were prepared for immunohistochemical analysis using an UltraTek HRP Anti-Polyvalent (DAB) Staining System (ScyTek Laboratories, West Logan, UT, USA, AMF080). The sections were deparaffinized with xylene, rehydrated in graded ethanol, and washed with distilled water. The sections were heated for 10 min in a sodium citrate buffer (0.01 M, pH 6.0) to increase epitope exposure. The sections were cooled at room temperature for 30 min and washed with PBS. The endogenous peroxidase activity was quenched with 3% H 2 O 2 in distilled water for 15 min at RT followed by washing with PBS (2 × 5 min). The sections were blocked with the blocking solution of the kit for 5 min at RT. The sections were incubated overnight at 4 • C with the diluted (1:100) primary antibodies against S100 protein (Z0311, Dako, Glostrup, Denmark), rabbit polyclonal anti-CD3 (1:200; Abcam, Cambridge, UK, ab828), mouse monoclonal anti-CD68 (1:100; Santa Cruz, sc-17832), mouse monoclonal anti-Olig2 (1:100; Santa Cruz, Dallas, TX, USA, sc-515947(, rabbit polyclonal Nicotinic Acetylcholine R alpha 7 NACHRA7 (1:100; ABclonal, Wuhan, China, A7844), and the polyclonal glial fibrillary acidic protein (GFAP) (PA5-16291, Thermo Fisher Scientific, Waltham, MA, USA). In parallel, tissue specimens, in which S100 protein primary antibody was omitted and replaced with buffer, served as negative controls (Figures S1 and S2). Sections were rinsed (three times and 5 min each) with PBS and were incubated for 15 min with the secondary Ultra Tek HRP Anti-polyvalent antibody (goat anti-mouse, rat, rabbit and guinea pig IgG) purchased from Scy Tek, (TX, USA). Following that, the slides were washed three times for 3 min each with a wash buffer, and the tissues were incubated with the HRP for 15 min and then washed three times for 3 min each with a wash buffer. The visualization of the reaction was carried out with Diaminobenzodiazibin DAB chromogen diluted with DAB substrate (provided within the same Scy Tek Detection kit) according to the manufacturer protocol for 10-15 min until the desired staining was achieved and counterstained with Harris hematoxylin and mounted with mounting media, DPX.

Results
The results are summarized in Table 1. The ganglia were distributed all over the body of molly fish that could be classified according to location into spinal ganglia, cranial ganglia, cervical, and enteric ganglia. Spinal ganglia appear as swellings in the dorsal roots of the spinal nerves, close to the spinal cord ( Figure 1A,B). Microscopically, the cell bodies of spinal ganglia neurons are characterized by large sizes, close together and enclosed by small nuclei of the flattened supporting satellite cells ( Figure 1C). The neurons showed a positive reaction for cresyl violet ( Figure 1D). The semi-thin section stained with toluidine blue showed that the ganglion is ensheathed by a capsule of dense connective tissue that branches internally into trabeculae to give a framework for the neurons. It was possible to see immune cells and telocytes close to the ganglion ( Figure 1E). The cell bodies of this neuron were round to oval in shape with round vesicular nuclei. The cytoplasm of these neurons contained large amounts of Nissl's granules. The cell body of neurons was enveloped with satellite glial cells (SGC) ( Figure 1E). showed a positive reaction for cresyl violet ( Figure 1D). The semi-thin section stained with toluidine blue showed that the ganglion is ensheathed by a capsule of dense connective tissue that branches internally into trabeculae to give a framework for the neurons. It was possible to see immune cells and telocytes close to the ganglion ( Figure 1E). The cell bodies of this neuron were round to oval in shape with round vesicular nuclei. The cytoplasm of these neurons contained large amounts of Nissl's granules. The cell body of neurons was enveloped with satellite glial cells (SGC) ( Figure 1E).  Bundles of both myelinated and non-myelinated nerve fibers could be demonstrated predominantly in the center of the ganglion (Figure 2A,B). Moreover, large ganglionic cells were demonstrated in the DRG which were arranged in rows (Figure 2A,B). Schwann cells ensheathed myelinated nerve fibers were distinct ( Figure 2B). SGCs could be also observed around the neurons ( Figure 2C). By electron microscopy, the ganglion cells showed large round cell bodies with large nuclei and distinct nucleolus. The cytoplasm of ganglion cells contained large amounts of rER, ribosomes, and mitochondria ( Figure 2C). We observed that the satellite glia cells were completely enveloping the ganglion cell body. It has a heterochromatic nucleus and it sends cell processes that contained mitochondria ( Figure 2C). the largest ganglion close to the hind brain QUENNIEneurons arranged in many compact columns S-100 ovoid or round vary in size QUENNIEglial cells a few glia cells could be identified distributed between neurons QUENNIEnerve fibers scanty within the vestibular ganglion arranged in between the neurons Myelinated axons in both longitudinal and cross sections can be demonstrated within the ganglion ( Figure 2C,D). It was possible to see that oligodendrocytes were closely related to myelinated axons. They were characterized by their large vesicular nucleus and sparse cytoplasm, which included ribosomes and mitochondria ( Figure 2C). Myelinated axons in both longitudinal and cross sections can be demonstrated within the ganglion ( Figure 2C,D). It was possible to see that oligodendrocytes were closely related to myelinated axons. They were characterized by their large vesicular nucleus and sparse cytoplasm, which included ribosomes and mitochondria ( Figure 2C). GFAP (glial fibrillary acidic protein) expression could be observed in both satellite glia cells and nerve fibers ( Figure 3A,B). Surprisingly, the expression of GFAP could be detected in the large ganglion cells of the DRG ( Figure 3C,D).
However, the expression of GFAP was confined to the glia cells ( Figure 4A-C) and the longitudinally oriented nerve fibers in the cervical ganglia that were observed near the gills ( Figure 4D,E). The enteric glia could be observed along the muscular layer of the intestinal wall. The nerve fibers and these glia cells showed strong immunoreactivity for GFAP ( Figure 5A-D).
Macrophages expressed CD68 around the glia cells in the tunica muscularis of the intestine ( Figure 6A,B). In addition, Ach was expressed in the neurons in the intestinal muscularis layer ( Figure 6C,D). GFAP (glial fibrillary acidic protein) expression could be observed in both satellite glia cells and nerve fibers ( Figure 3A,B). Surprisingly, the expression of GFAP could be detected in the large ganglion cells of the DRG ( Figure 3C,D).
However, the expression of GFAP was confined to the glia cells ( Figure 4A-C) and the longitudinally oriented nerve fibers in the cervical ganglia that were observed near the gills ( Figure 4D,E). The enteric glia could be observed along the muscular layer of the intestinal wall. The nerve fibers and these glia cells showed strong immunoreactivity for GFAP ( Figure 5A-D).      Macrophages expressed CD68 around the glia cells in the tunica muscularis of the intestine ( Figure 6A,B). In addition, Ach was expressed in the neurons in the intestinal muscularis layer (Figure 6C,D).   Macrophages expressed CD68 around the glia cells in the tunica muscularis of the intestine ( Figure 6A,B). In addition, Ach was expressed in the neurons in the intestinal muscularis layer (Figure 6C,D).   The vestibular ganglia were observed close to the hind brain ( Figure 7A). They are the largest ganglia with the vestibular ganglion cells arranged in numerous compact columns ( Figure 7B). The vestibular ganglion cells are either ovoid or round and have central or eccentric nuclei. These cells vary considerably in size and a few glial cells were distributed between them. The nerve fibers are scanty within the vestibular ganglion ( Figure 7C,D). The vestibular ganglia were observed close to the hind brain ( Figure 7A). They are the largest ganglia with the vestibular ganglion cells arranged in numerous compact columns ( Figure 7B). The vestibular ganglion cells are either ovoid or round and have central or eccentric nuclei. These cells vary considerably in size and a few glial cells were distributed between them. The nerve fibers are scanty within the vestibular ganglion ( Figure 7C,D).  The cervical ganglia were large ganglia observed around the pseudobranch (Figure 8), head kidney, and thymus ( Figure 9). These are characterized by randomly distributed neurons and peripherally situated nerve fibers ( Figure 8A-E). The ganglia located beside the immune organs (thymus and head kidney) show abundant neurons ( Figure 9A-C).
The ganglia along the trunk kidney exhibit cell bodies more widely spaced than in spinal and vestibular ganglia because of the presence of numerous neurites (axons and dendrites) in between ( Figure 10A-D) that most of them showed positive reaction for silver stain ( Figure 10E). The semi-thin sections to these ganglia revealed the presence of many types of immune cells around the ganglia. Numerous lymphocytes, dendritic cells, and macrophages could be identified in close contact with the ganglia (Figure 11A-C). Moreover, telocytes with distinct telopodes were oriented around the ganglia ( Figure 11D). These distributed T-lymphocytes around the ganglia expressed CD3 (Figure 12A,B).
Additionally, the macrophages expressed CD68 that were distributed around the ganglia of the kidney ( Figure 13A) and ovary ( Figure 13B). On the other hand, the neurons and nerve fibers in the ganglia around the trunk kidney expressed Ach ( Figure 13C,D). Furthermore, the ganglia around the head kidney showed positive expression of Iba1 in microglia and macrophages around the ganglia ( Figure 14D). Most ganglion cells and nerve fibers in the DRG, autonomic, and vestibular ganglia showed moderate to strong immunoreactivity for S-100 protein ( Figure 15).   The ganglia along the trunk kidney exhibit cell bodies more widely spaced than in spinal and vestibular ganglia because of the presence of numerous neurites (axons and dendrites) in between ( Figure 10A-D) that most of them showed positive reaction for silver stain ( Figure 10E). The semi-thin sections to these ganglia revealed the presence of many types of immune cells around the ganglia. Numerous lymphocytes, dendritic cells, and macrophages could be identified in close contact with the ganglia (Figure 11A-C).       Additionally, the macrophages expressed CD68 that were distributed around the ganglia of the kidney ( Figure 13A) and ovary ( Figure 13B). On the other hand, the neurons and nerve fibers in the ganglia around the trunk kidney expressed Ach ( Figure 13C,D). Furthermore, the ganglia around the head kidney showed positive expression of Iba1 in microglia and macrophages around the ganglia ( Figure 14D). Most ganglion cells and nerve fibers in the DRG, autonomic, and vestibular ganglia showed moderate to strong immunoreactivity for S-100 protein ( Figure 15).

Discussion
In the current work, we used histomorphological, immunohistochemical, and ultrastructural approaches to characterize the ganglion throughout the body of the molly fish. We were able to identify a variety of ganglion types observed in molly fish, such as the DRG, enteric ganglia, and autonomic ganglia. The precise coordination of the various cellular components that make up the gut wall requires the enteric nervous system (ENS),

Discussion
In the current work, we used histomorphological, immunohistochemical, and ultrastructural approaches to characterize the ganglion throughout the body of the molly fish. We were able to identify a variety of ganglion types observed in molly fish, such as the DRG, enteric ganglia, and autonomic ganglia. The precise coordination of the various cellular components that make up the gut wall requires the enteric nervous system (ENS), which provides local control of the gastrointestinal tract. In most fish species ENS is capable of carrying out a number of CNS-independent tasks, including gut peristalsis, homeostasis, hormone secretion from the stomach, and osmoregulation [12,13]. In mammals, the enteric ganglia are made up of two major layers: an outer myenteric plexus and an inner submucosal plexus [14]. However, Zebrafish and the currently investigated molly fish only have one myenteric plexus [15]. ENS ganglia exist as a collection of neurons and glia that are arranged in a series of plexuses throughout the gastrointestinal tract. The main enteric nerve cell types, sensory, inter, and motorneurons, are located in the myenteric plexus [16]. Enteric glia, astrocyte-like cells that surround enteric neurons, regulate enteric neurons' output. Indeed, there are molecular similarities between astrocytes and enteric glia as they both express similar proteins, such as the intermediate filament glial fibrillary acidic protein (GFAP), and have similar electrophysiological properties [17]. GFAP is a major constituent of glial intermediary filaments that are expressed in astrocytes of the central nervous system and in the peripheral nervous system, and enteric glial cells [18,19]. In mammals and birds, glial cells outnumber neurons by an average of up to four.
Glial cells have a variety of functions in the mammalian gut; including protecting and supporting the neurons and they can also participate in neurotransmission [20]. Recent investigations have shown putative glial cells which are immunoreactive to GFAP in the gut of various teleost species [21]. In fish, the ENS ganglia contain neurons that are sparsely distributed along the surface of the gut and these neurons do not cluster to form ganglia as mammals [16]. The same observation has been reported in zebrafish [22] and the currently investigated study. We investigated that these neurons express the neurotransmitter choline acetyltransferase (ChAT). Nonetheless, in zebrafish, the enteric neurons express several neurochemical and neurotransmitters markers; including serotonin (5HT), tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calbindin (CB), calretinin (CR), choline acetyltransferase (ChAT) and nitric oxide (NO) [23][24][25][26]. Both sympathetic and sensory ganglia contain satellite glia and Schwann cells, two prominent glial cell types that are closely related to their neuronal neighbors and significantly affect a wide range of neuronal processes [27]. Schwann cells are connected to peripheral axons and play well-known functions in myelination, axon regeneration, the trophic and metabolic support of neurons [28,29].
The sensory ganglia found in the dorsal roots of the spinal cord are made up of afferent neurons, and satellite glial cells (SGCs) that encase them [30]. SGCs and DRG neurons are intimately associated, forming a unique structural unit that supports strong bidirectional communication. SGCs control the ionic and neurotransmitter concentrations, stimulate neuronal morphogenesis, control synaptic transmission, and serve as a functional substitute for the deficient blood-brain barrier, just as astrocytes do in the CNS [30][31][32][33]. Additionally, they are capable of forming perikaryal myelin sheaths and even participating in phagocytic activity, which is characteristic of oligodendrocytes and microglia, respectively [34,35]. In the neurons of the DRG and sympathetic ganglia of molly fish, we demonstrated the expression of S-100 protein, which constitutes the largest subgroup of calcium-binding proteins. A similar observation has been shown in a subpopulation of sensory and sympathetic neurons, and it was not observed in the enteric nervous system of adult zebrafish [36]. Since S-100 protein has primarily been found in the satellite glial cells of both sympathetic and sensory ganglia in higher vertebrates, especially mammals, the expression of S-100 protein in molly fish and zebrafish clearly differs from that in these species [37].
It is suggested that the immune system of zebrafish is very close to mammals and has innate and adaptive immune cells such as B cells, T cells, macrophages, and neutrophils.
In addition to immune mediators like cytokines and complement proteins [38]. Moreover, the sensory ganglia, including the DRG, host several types of immune cells allowing for local neuroimmune communication which is important for neuropathic pain [39]. It is well-known that in most vertebrates, the immune system contributes to the normal nervous system development from cellular to behavioral levels. At the cellular level, neuronal apoptosis is a critical mechanism promoting normal neural connectivity in the development of the nervous system [40].
A study investigating human ganglia found a population of local macrophages around 5-20% of the total cells present. In addition to low numbers of CD3 and CD8 lymphocytes were detectable in sensory and autonomic ganglia [41]. It was observed that human trigeminal ganglia-resident SGC (TGSGC) equally expressed the common leukocyte marker CD45 and the macrophage markers CD14, CD68, and CD11b, as well as dendritic cell (DC) marker CD11c, the T-cell costimulatory molecules CD40, CD54, CD80, and CD86 and MHC class II [34].
A study using zebrafish embryos as a model host for Mycobacterium lepra (M. leprae) infection detected bacterial, glial, neural and immune cell interactions during M. leprae infection. M. leprae were able to alter the myelin structure of glial cells surrounding axons within the spinal cord by stimulating macrophages production of reactive nitrogen species causing mitochondrial and axonal damage in both myelinated and non-myelinated axons [42].
Dendritic cells are part of innate immune cells that play a key role in linking innate and adaptive immunity. They recognize and respond to pathogen-associated and dangerassociated signals, and shape the acute inflammatory response [43]. It was found that following peripheral nerve injury, there is an accumulation of dendritic cells (DCs) within the dorsal root leptomeninges (DRLs).
A study in mice infected with Herpes simplex virus type 1 (HSV-1), which is a type of infection, is controlled mainly by the immune response within the trigeminal ganglia (TG). This study results that dendritic cells (DCs) and macrophages were the key sources of Interleukin 1 beta (IL-1β) and Inducible nitric oxide synthase (iNOS), respectively which both are essential for the immune response against HSV-1 [44].
Several studies indicated several functions of T-cells in fish, such as activating macrophages to initiate their microbicidal activity and B-cells to produce antibodies, in addition to enhancing cell-mediated immunity [45]. A study characterized the immune response in ganglia after infection of primary simian varicella virus (SVV) in African green monkeys (AGMs) found that local reduction in viral load within ganglia was correlated with increased infiltrating of T cells, suggesting intragSupplanglionic immunity involves controlling SVV proliferation [46].
We noted that the vestibular ganglion in molly fish is large and the vestibular ganglion cells arranged in a very compact, linearly arranged mass of cells similar to that described in cats [47]. The large size of the ganglion and the high density of its cells is related to a greater number of hair cells in the inner ear, a larger nerve and a greater size of the primary central area [48]. It has been reported from a pioneer study on cats, guinea pigs and squirrel monkeys that each cell size receives fibers from specific parts of the vestibular sensory regions [49]. Upon injury, monocyte/macrophage /microglia are recruited to the ganglia. Both M1 and M2 macrophages, which secrete IL-1β and Arginase 1, respectively, were reported [50].

Conclusions
Different ganglia of molly fish displayed direct communication with immune cells which support and maintain healthy ganglionic cells. This study enhances our understanding of the possible cells underlying the normal support and the possible peripheral neuropathy events following injury or infection.