Serotonergic and FMRF-amidergic neurons in the nerve ganglia of Haliotis asinina Linnaeus.

ABSTRACT The neurons containing serotonin and FMRF-amide were immunohistochemically localized in the cerebral, pleuropedal and visceral ganglia of Haliotis asinina. The large (~10 x 20 [micro]m in diameter) and medium size (~7 x 10 [micro]m in diameter) neurons of these three ganglia contained immunoreactivities to both antibodies against serotonin and FMRF-amide, whereas the more numerous small size neurons (<6 x 8 [micro]m in diameter) did not show these immunoreactivities. The large neurons had oval-shaped nuclei containing mostly euchromatin and long cytoplasmic processes, whereas the medium size neurons, which could be neuroseeretory cells, contained round nuclei with patches of heterochromatin and lack processes. The 5-HT immunoreactive cells were concentrated in the upper half of the medial edge of the cerebral ganglion, the edge of ventral and dorsal horns of the pleuropedal ganglion, and the fight latero-ventral edge of the visceral ganglion; whereas the FMRF-amide immunoreactive neurons were concentrated in the dorsal and ventral edges of the cerebral ganglion, the edge of dorsal horn and the dorsal edge of the body of the pleuropedal ganglion, and the left latero-ventral and latero-dorsal edge of the visceral ganglion. The 5-HT cells were most numerous in the cerebral (~70/2,300 cells per section), and the pleuropedal ganglia (~120/2,700 cells per section), whereas they were least numerous in the visceral ganglion (~50/400 cells per section). The FMRF-amide cells were most numerous in the cerebral (~76/2,000 cells per section), and the pleuropedal ganglia (~130/2,500 cells per section), whereas they were least numerous in the visceral ganglion (~60/1,050 cells per section). The present study suggests that serotonin and FMRF-amide are major neurotransmitters in the neural ganglia, with only the large- and medium-sized neurons involved in the production and storage of these two neurotransmitters.

KEY WORDS: Haliotis asinina, serotonin, FMRF-amide, immunohistochemistry, nerve ganglia

INTRODUCTION

Serotonin (5-hydroxytryptamine; 5-HT) and tetrapeptide Phe-Met-Arg-Phe-amide (FMRF-amide) are neurotransmitters that play many important roles in controlling the physiologic and behavioral processes of invertebrates (Kuang et al. 2002, Mercier et al. 2003). In molluscs, 5-HT controls heartbeat (Skelton et al. 1992), locomotion (McClellan et al. 1994, Satterlie & Norekian 1995), feeding (Kupfermann & Weiss 1981), memory and learning (Nelson & Alkon 1997), and reproduction (Ram et al. 1996, Juneja & Koide 1996). FMRF-amide can evoke muscle tension (Schot & Boer 1982, Buckett et al. 1990), modulate cardiac output (Buckett et al. 1990), and reproduction (Lehman & Greenberg 1987). By immunolocalization methods, 5-HT and FMRF-amide have been observed in many ganglia of the central nervous system (CNS) of gastropod molluscs (Audesirk 1985, Elekes 1992, Croll et al. 2001, Fickbohm et al. 2001), especially in the pulmonate snails, Helix pomatia and Helix aspersa (Griffond et al. 1986, Lehman & Price 1987, Hernadi et al. 1989, Elekes & Nassel 1990, Hernadi 1992). Other gastropods that have also been investigated are a sea hare Aplysia kurodai, a land snail Achatina fulica (Fujii & Takeda 1988), an aquatic snail Lymnaea stagnalis (Audesirk 1985), and an opisthobranch Phestilla sibogae (Croll et al. 2001). The 5-HT and FMRF-amide immunoreactive neurons have been identified in the CNS of all these species with specific distribution patterns. For example, in Aplysia kurodai and Achatina fulica, the 5HT-immunoreactive cells and neuropils were observed in all ganglia except the left pleural ganglia. The sizes of these neurons are generally large, and some reach up to 50-70 [micro]m in diameter. FMRF-amide immunoreactive neurons have been located throughout the central ganglia (cerebro-pleural, parietal, pedal, and visceral ganglia) of Phestilla sibogae, and about 65-100 intensely labeled neurons were observed in these ganglia, whereas another 40-45 neurons were weakly or variably stained (Croll et al. 2001). A similar study has been done in an aquatic snail, Lymnaea stagnalis, in which FMRF-amide immunoreactive neurons and neuropils were consistently found in the cerebral, pedal, right parietal, and visceral ganglia (Audesirk 1985). Moreover, the colocalization of 5-HT and FMRF-amide has also been performed in a chiton, Lepidopleurus asellus, which is a primitive mollusc species (Moroz et al. 1994). Both types of neurons are distributed in characteristic patterns in the CNS (i.e., the 5-HT immunoreactive neurons are concentrated in the ventral cords while FMRF-amide immunoreactive neurons in the lateral cords). In addition, a tight network of 5-HT and FMRF-amide immunoreactive nerve fibers were found in the body wall and the foot muscle (Moroz et al. 1994). The prevalence of serotonergic and FMRF-amidergic neurons in the CNS in gastropods implies that they are important neurotransmitters in these animals.

In contrast to the higher gastropods, very few works along this line have been carried out in the primitive gastropods. Only two studies reported the presence of 5-HT in the cerebral ganglion of developing larvae of red abalone, Haliotis rufescens (Kataoka et al. 1987, Barlow & Truman 1992) and 5-HT-immunoreactive fibers in the outer zone of the retinal plexiform layer and the optic nerve trunk (Kataoka et al. 1987). In the present study, we have examined and compared the distribution of 5-HT and FMRF-amidergic neurons in the neural ganglia of the CNS of Haliotis asinina, a tropical abalone commonly found in Thai coastal waters.

MATERIALS AND METHODS

Tissue Collection

Adult male H. asinina (age >16 mo) were collected from a land-based aqua culture system at Coastal Aquaculture Development Center, Department of Fisheries, Prachaubkirikhun Province, Thailand. They were kept in concrete tanks housed in the shade and well flushed with mechanically circulated sand-filtered seawater and provided with an air delivery system to maintain the stable controlled environment. The optimum level of salinity is about 22.5-32.5 ppt and temperature ~22[degrees]C to 26[degrees]C (Singhagraiwan & Doi 1999). They were fed with macroalgae (usually Gracilaria spp. and Laminaria spp.), supplemented with artificial food.

Antibodies Against 5-HT and FMRF-Amide

The rabbit polyclonal antibody against 5-HT was purchased from Zymed Laboratories Inc. For antibody against FMRF-amide, the peptide (Sigma Company) was dissolved in deionized water at 0.5 mg/mL. This solution was mixed with keyhole lympet hemocyanin (KLH) (10 mg/mL) in 1:1 ratio. Then, glutaraldehyde was added up to 0.375% with 1 M glycine-HCl. After that, the solution was mixed with 0.1 M phosphate buffered saline (PBS) containing complete Freund's adjuvant in 1:1 ratio for subcutaneous injection into experimental mice (300 [micro]L/mouse). After 14 days, a similar dose of FMRF-KLH mixed with incomplete Freund's adjuvant was injected to boost the immune response of the animals. On day 49, the blood was collected from the heart bleeding and centrifuged at 5,000g, and the serum was collected, mixed with 20 folds in volume of KLH solution to preabsorb antiKLH in the antiserum. Then, the dot blot ELISA was used to detect the specificity of the mouse antiFMRF-amide polyclonal antibody, and make sure that antiKLH was completely absorbed. The preabsorbed antiserum was used to stain the sections of cerebral, pleuropedal and visceral ganglia.

Immunoperoxidase Method

Ten adult male H. asinina weighing more than 20 g were anesthesized with 5% Mg[Cl.sub.2], then the shells were removed. Cerebral, pleuropedal, and visceral ganglia were dissected out and fixed in Bouin's solution at 4[degrees]C for 12-16 h. Specimens were washed in 70% ethanol for removal of the fixative. Then, they were dehydrated through a graded series of ethanol (70% to 100%) for 30-45 min each depending on the size of the specimens, cleared with dioxane, infiltrated and embedded in paraffin wax, and sectioned at 5-[micro]m thick. For immunostaining, the paraffin sections of each ganglion were deparaffinized with xylene, rehydrated through a graded series of ethanol (100% to 80%), and finally in 70% ethanol containing 1% Lithium carbonate (LiC[O.sub.3]). The endogenous peroxidase was blocked by treating the sections with 3% [H.sub.2][O.sub.2] in methanol for 30 min. Then, the sections were covered with 4% bovine serum albumin (BSA) in 100 mM phosphate buffered saline, containing 0.25% triton-X 100, pH 7.4 (PBST), for 30 min. Following the blocking step, the consecutive sections were incubated in either the rabbit anti5-HT or mouse antiFMRF-amide serum as the primary antibodies, for 1 h, at 37[degrees]C. After incubation, the sections were washed three times with PBST, then incubated in the HRP-conjugated goat antirabbit or anti mouse IgG as the secondary antibodies, for 30 min, at 37[degrees]C. Finally, the sections were immersed in the substrate solution containing 0.03% w/v DAB, 0.3% Ni[Cl.sub.3] and 0.1% [H.sub.2][O.sub.2] in 50 mM Tris buffer pH 7.2, washed several times with distilled water, counter-stained with Hematoxylin, and mounted in the permount solution. The sections were observed and photographed by a Nikon microscope equipped with digital camera DXM 1200. In addition, the numbers of serotonergic and FMRF-amidergic neurons in each ganglion of each abalone were counted and estimated as number of positive cells per total number of cells per section, which were taken at the middle of each ganglion.

RESULTS

Immunohistochemical Localization 

By using immunoperoxidase technique enhanced with nickel chloride (Ni[Cl.sub.2]) we were able to localize two separate sets of neurons containing 5-HT and FMRF-amide in the cerebral, pleuropedal and visceral ganglia. The patterns of distribution of these two types of neurons in each ganglion are described below.

Cerebral Ganglion

The 5-HT cells are concentrated in the upper half of the cortex of medial edge of the ganglion (Figs. 1B, C, Fig. 7A), whereas there are few widely scattered 5-HT cells in the lateral edge of the ganglion. FMRF-amide cells are concentrated in the cortex of the dorsal and ventral edges of the ganglion whereas the lateral edge and the dorso-medial edge have only few widely scattered FMRF-amide cells (see Figs. 3A, B, Fig. 7A). Both 5-HT and FMRF-amide immunoreactive nerve fibers were also observed throughout the neuropils of the ganglia. The 5-HT immunoreactive nerves branching out from the ganglia were also observed around the head and buccal areas of the abalone, but fewer FMRF-amide immunoreactive fibers were found (Fig. 1B, see Fig. 4A later). Based on the histologic characteristics, the serotonergic neurons could be classified into 2 types (i.e., the large and medium size neurons). The large oval or pyrimidal shape neurons (about 10 x 20 [micro]m) contain the oval-shaped nuclei with complete euchromatin, and usually large nerve processes (Figs. 1E, F), whereas the medium size neurons (about 7 x 10 [micro]m), which could be neurosecretory cells, contain round nuclei with patches of heterochromatin (Upatham et al. 1998, Kruatrachue et al. 1999, Thongkukiatkul et al. 2000) and lacking processes (Fig. 1G). However, there was no immunoreactivity of 5-HT in the cell bodies of small neurons (<6 x 8 [micro]m) (Figs. 1F, G). Similarly, from the histologic characteristics, FMRF-amidergic neurons are also mostly the large size neurons and medium size neurons, with no staining in the small neurons (Figs. 4C, D). The number of 5-HT cells are about 70/2,300 cells per section in comparison to 76/2,000 cells per section for FMRF-amide cells.

[FIGURES 1,3-4,7 OMITTED]

Pleuropedal Ganglion

In pleuropedal ganglia, the 5-HT cells were found concentrated in the upper and lower one-third part of the dorsal and ventral horns of the ganglion (Fig. 2A to C, Fig. 7B) with widely scattered 5-HT cells on the lateral edge of the ventral horn (Fig. 2A, Fig. 7B). FMRF-amide cells were found concentrated in the cortex of the upper one-third of the dorsal horn and the dorsal edge cortex of the body, with few widely scattered positive cells in the ventral horn and the ventral edge of the body (Fig. 5A). The immunoreactivity of both types of nerve fibers were also found throughout the neuropils of the ganglia, especially in the dorsal and ventral horn regions, (Fig. 5A, C). Histologically, there are only two types of immunoreactive 5-HT and FMRF-amide neurons with similar characteristic as those found in the cerebral ganglia (Figs. 2D, E, Fig. 5D). The numbers of both types of these neurons are approximately equal and they are evenly distributed in specific areas of the ganglia as mentioned. There are about 120/2,700 cells per section for sertonergic neurons in the ganglion, and about 130/2,500 cells per section for FMRF-amide cells.

[FIGURES 2,5 OMITTED]

Visceral Ganglion

The 5-HT neurons are concentrated on the right lateral and latero-ventral edge of the ganglion (Figs. 3A to C, Figure 7C), while the FMRF-amidergic neurons are concentrated on the left latero-ventral and latero-dorsal edge (Fig. 6A, Fig. 7C). A few immunoreactive nerve fibers containing 5-HT and FMRF-amide were also observed in the neuropil of the ganglion (Figs. 3A to C). The two types of immunoreactive neurons for both 5-HT and FMRF-amide could also be identified, with similar characteristics as in the cerebral and pleuropedal ganglia (Figs. 3D, E, Fig. 6B to D). The number of serotonergic neurons is about 50/400 cells per section, whereas that of FMRF-amide cells is about 60/1,050 cells per section.

[FIGURES 6 OMITTED]

DISCUSSION

The distribution of 5-HT and FMRF-amide, the major neurotransmitters, have been widely studied in a number of invertebrates (Beltz & Kravitz 1983, Nassel et al. 1985, Fujii & Takeda 1988, Too & Croll 1995, Raikova et al. 2000) including the gastropod molluscs (Audesirk 1985, Elekes 1992, Croll et al. 2001, Fickbohm et al. 2001). However, most previous studies have been done in the opisthobranch and pulmonate species, the higher gastropods. Up to now, there are few works on the prosobranch including abalone, which belong to the most primitive subclass of gastropods (Barlow & Truman 1992).

In the present study, we have studied the distribution patterns of these two types of neurotransmitters in the central ganglia of H. asinina. The results showed the presence of immunoreactive 5-HT and FMRF-amide neurons in all three ganglia, with the distinctive distribution patterns in each ganglion as summarized in Figure 7. Both serotonergic and FMRF-amidergic neurons also showed a tendency to be grouped into clusters as in higher gastropods, whereas in species of the lower classes; such as, platyhelminthes, annelida, and arthropoda, serotonergic neurons tend to be more widely scattered (Fujii & Takeda 1988).

H. asinina possesses a fairly high number of both serotonergic and FMRF-amidergic neurons in all three ganglia, especially in the pleuropedal ganglia. Fujii and Takeda (1988) found that the number of serotonergic neurons in invertebrates tends to increase in the higher phyla. Furthermore, it was found that one of the main factors that controls the number of 5-HT cells is the physiologic condition. In a snail Helix pomatia, it was reported that the number of serotonergic cells in the central ganglia changed with the physiologic conditions of the animals (i.e., they increased during active phase and decreased during hibernation) (Hiripi & Salanki 1973).

While the immunoreactive 5-HT and FMRF-amide neurons were observed in all three ganglia, the immunoreactive nerve fibers containing 5-HT were found concentrated in the neuropils of the ganglia, as well as in musculature of the head, buccal, and foot regions, whereas FMRF-amide fibers were only found in the neuropils and around the hemolymph sacs. These characteristics were similar to the higher gastropod molluscs (Schot & Boer 1982, Fickbohm et al. 2001). Therefore, it is possible that serotonergic neurons may play a primary role in controlling the muscle contraction for head and foot movement and feeding, as in other gastropods, whereas FMRF-amidergic neurons may function mainly as a neuromodulator in the CNS.

The sizes of both serotonergic and FMRF-amidergic neurons are different from those in other gastropods, especially in a pulmonate and opisthobranch molluscs, also in which the positive neurons showed remarkable size range (Fickbohm et al. 2001). In H. asinina, both immunoreactive neurons are confined to the large nerve cells with processes and the medium size nerve cells with no processes. The large neurons could be equivalent to the large neurons as classified by Kruatrachue et al. (1999), which seem to be the motor neurons controlling the musculature of head, buccal, and foot region via their long axon. The medium size cells have all characteristics of neurosecretory cells (Kruatrachue et al. 1999). Interestingly all small size nerve cells, which are believed to be association or interneurons, are nonimmunoreactive. Thus, the strong presence of 5-HT and FMRF-amide in the CNS of H. asinina suggests that both neurotransmitters have important roles in controlling the physiologic and behavioral responses of this animal as in the other gastropods.

ACKNOWLEDGMENTS

This research was supported financially by the Thailand Research Fund (Senior Research Scholar Fellowship to Prasert Sobhon, and the Royal Golden Jubilee Ph.D. Scholarship to Sasiporn Panasophonkul).

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SASIPORN PANASOPHONKUL, (1) PRAPEE SRETARUGSA, (1) * NARUMOL ANUNRUANG, (1) SOMJAI APISAWETAKAN, (1) PORNCHARN SAITONGDEE, (1) SUCHART E. UPATHUM, (2) TANES POOMTONG, (3) PETER J. HANNA, (4) AND PRASERT SOBHON (1)

(1) Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; (2) Department of Medical Science, Faculty of Science, Burapha University, Chonburi 20130, Thailand; (3) The Coastal Aquaculture Development Center, Department of Fisheries, Klongwan, Prachaubkirikhun Province 77000, Thailand; (4) The School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria 3174, Australia

* Corresponding author: E-mail: [e-mail]