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Distribution of GABA in the nerve ganglia of Haliotis asinina linnaeus.

ABSTRACT Gamma-aminobutyric acid (GABA) is a major neurotransmitter and effective settlement inducer in abalone aquaculture. This study aimed to explore the distribution of GABA within neural tissues of Haliotis asinina. Gammaaminobutyric acid was found in neuronal cell type 1 of 3 major ganglia (i.e., cerebral, pleuropedal, and visceral ganglia) of both sexes. The distribution of GABA-immunoreactive (-ir) cells in the cerebral ganglion was concentrated mostly in the cortex region of the dorsal horn, whereas it was scattered throughout the pleuropedal ganglion, with more in the upper half. Gammaaminobutyric acid-ir nerve fibers were found throughout the neuropils of the ganglia. The visceral ganglion had the least numbers of GABA-ir neurons compared with the other 2 ganglia. The cells were distributed mainly in the dorsal horn. We also observed GABA to be colocalized with 2 other neurotransmitters: serotonin (5-HT) and dopamine (DA). In the cerebral ganglion, fluorescence double staining of GABA and 5-HT, and GABA and DA showed immunoreactivity in separate cells and was also colocalized in the same cells. In the pleuropedal ganglion, the staining pattern was similar to the cerebral ganglion, but positivestaining cells were less numerous. In the visceral ganglion, GABA and DA, and GABA and 5-HT were colocalized in the same cell types. Overall, we found that GABAergic cells were most numerous in the cerebral ganglion of H. asinina. Further studies are required to determine the functions of these neurotransmitters in relation to their distribution.

KEY WORDS: gamma-aminobutyric acid, Haliotis asinina, ganglia, immunohistochemistry, localization


Gamma-aminobutyric acid (GABA) is well known as an inhibitory neurotransmitter in the vertebrate central nervous system (CNS) (Roberts et al. 1976), whereas in the CNS of molluscs, GABA has been found to have both excitatory effects (Kovacs & Erdelyi 1995, Norekian 1999) and inhibitory effects (Alkon et al. 1992, Collin et al. 1992). Osborne (1971) was the first to report the presence of GABA (0.9 [micro]moles/g wet weight) in extracts of cerebral ganglia of the adult common garden snail Helix aspersa, and in later investigations, GABA was also shown to be present in cerebral ganglia extracts of the East African land snail Achatina fulica (Takeuchi et al. 1977, Ku et al. 1985).

Gamma-aminobutyric acid, as well as GABA homologs and analogs, have been shown to the induce settlement and metamorphosis in abalone (Morse et al. 1979, Morse & Morse 1984, Morse 1992, Roberts & Nicholson 1997, Bryan & Qian 1998, Gapasin & Polohan 2004). Gamma-aminobutyric acid and 5-AVA (a GABA analog), induced Haliotis asinina larvae to settle on plastic sheets, at 70% and 60%, respectively (Stewart et al. 2008). In abalone hatcheries, GABA treatment has been used to settle competent larvae onto tank surfaces (Searcy-Bernal et al. 1992, Roberts & Watts 2010). In addition, abalone mucus is known to contain GABA, which can induce settlement of abalone larvae (Laimek et al. 2008), and this is thought to be associated with mucus-containing pheromones (Kuanpradit et al. 2012). Gamma-aminobutyric acid is also known to mediate communications between plants and animals, fungi, bacteria, and other plants, and plays a functional role as a messenger in regulating sponge feeding behavior (Shelp et al. 2OO6).

There is a lack of information pertaining to the distribution of GABA in the nervous system of abalone. One group reported GABA to be present in the sensory papillae of adult Haliotis asinina (Wanichanon et al. 2004). The papillae have neuroepithelial cells that are widely distributed on the surface of cephalic and epipodial tentacles, and exhibit intense staining for GABA, along with their innervating nerve fibers. In other molluscs, including Helix pomatia (Hernadi 1994), Lymnaea stagnalis (Hatakeyama & Ito 2000), Haliotis aspersa (Ierusalimsky & Balaban 2001), and Aplysia californica (Jing et al. 2003), GABA-like immunoreactivity has been found in the CNS, and with specific distribution patterns. For example, in all species studied, with the exception of L. stagnalis, cells showing positive GABA immunoreactivity have been found in the buccal, cerebral, and pedal ganglia. In L. stagnalis, GABA immunoreactive (-ir) cells have been detected in all ganglia (Hatakeyama & Ito 2000). Gamma-aminobutyric acid-ir regions have been observed in the viscera-parietal-pleural ganglion complex of many molluscs (Richmond et al. 1991, Norekian 1999, Hernadi 1994, Dyakonova et al. 1995, Arshavsky et al. 1993, Cooke & Gelperin 1988). Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes extend into the connectives and commissures of the ganglia, implying an important central integrative role of GABA-ir neurons (Hernadi 1994, Diaz-Rios et al. 1999). In the current study, we examined the distribution of GABA in nerve ganglia of H. asinina, and then examined the colocalization of this neurotransmitter with that of serotonin (5-HT) and dopamine (DA), both of which play important roles in controlling reproduction of this animal.


Tissue Preparation

Mature Haliotis asinina were cultured in a land-based aquaculture system at the Coastal Aquaculture Development Center, Department of Fisheries, Prachaubkirikhun Province, Thailand. Ten male and 10 female H. asinina weighing more than 20 g each were used. Cerebral, pleuropedal, and visceral ganglia were dissected out, fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), held overnight at 4[degrees]C, and were processed routinely for paraffin embedding. Five-micrometerthick serial frontal sections were cut with a rotary microtome and mounted on glass slides precoated with 3-aminopropyl triethoxysilane solution (Sigma-Aldrich Co., Saint Louis, MO) in preparation for immunohistochemistry.

Polyclonal antibody production Against GABA

An immunogen was produced by conjugating GABA (Sigma) with bovine serum albumin (BSA fraction V, 67 kD; Sigma), which was used as high-molecular weight carrier protein. Briefly, 100 [micro]mol GABA was mixed with 12 mg BSA in 0.1 M PBS, pH 7.4, and 100 [micro]mol glutaraldehyde (Electron Microscopy Sciences) was added to the reaction mixture, which was then stirred for 30 min at room temperature. The immunogen was then mixed with Freund's complete adjuvant (Sigma) and was used to immunize 6-wk-old ICR male mice intraperitoneally with 100 [micro]L GABA-BSA conjugate (5 mg protein in 0.1 mL PBS). The mice were obtained from the Animal Care Unit, Mahidol University, and were used with the approval of the animal ethics committee. Three booster injections were performed at intervals of 2 wk, using the same immunogen, but mixed in incomplete Freund's adjuvant (Sigma). Retro-orbital plexus blood was then collected and centrifuged to collect antiserum, which was stored at -20[degrees]C. To determine antibody specificity to GABA, each antiserum was preabsorbed in 2.5 mg/mL BSA in PBS at a dilution of 1:50 v/v antiserum:BSA, overnight at 4[degrees]C, and its antibody titer was determined by ELISA.

Localization of GABA in the Nerve Ganglia Using an Immunoperoxidase Technique

Paraffin sections of ganglia were deparaffinized with xylene and then rehydrated through a graded series of ethanol up to 70% ethanol containing 1% lithium carbonate. The sections were immersed in 3% [H.sub.2][O.sub.2] in methanol, then 4% BSA in 0.05 M PBS, containing 0.4% Triton-X 100 (PBST), pH 7.4, for 30 min each to block endogenous peroxidase and nonspecific protein binding, respectively. The sections were incubated with anti-GABA, preabsorbed with BSA, at a dilution of 1:400 in PBS, for 1 h at room temperature. After washing 3 times with PBS, the antibody binding was then detected by the addition of HRP goat antimouse IgG (Zymed Laboratories), at a dilution of 1:500 in PBST, for 30 min at room temperature. The color was detected by incubation in aminoethyl carbazole (AEC-red substrate kit; Zymed Laboratories). The sections were then counterstained with Meyer's hematoxylin and observed under a Nikon E600 microscope equipped with a DXM 1200F digital camera. In controls, the sections were processed by the same protocol, but using preimmune mouse serum instead of the primary antibody. Using Image Pro software, the number of GABA-ir neurons was counted in 20 sections of each abalone ganglion, and recorded as the mean number of positive cells per total number of cells (neurons and neurosecretory cells).

Gomoris Aldehyde-Fuchsin (GAF) Staining for Neurosecretory Cells

The staining methods of Panasophonkul et al. (2009) were used to identify the neurosecretory cells within sections that were prestained with immunoperoxidase for GABA detection. After taking images of immunoperoxidase-stained sections, the sections were rehydrated through a graded series of ethyl alcohol, oxidized in potassium permanganate solution (0.3% potassium permanganate and 0.3% concentrated sulfuric acid) for 1 min, and rinsed in distilled water. They were then bleached in 2.5% sodium bisulfate solution until permanganate color was removed, then washed in running tap water for 5 min before transferring into 70% ethanol for 2 min. For detection of neurosecretory cells, the sections were stained in GAF solution for 10 min, rinsed in 95% ethyl alcohol, and further differentiated in fresh 95% ethanol until free stain was removed. The sections were then transferred to 70% ethyl alcohol for 2 min, rinsed in distilled water for 1 min, and stained in Halmi solution (0.2% light green SF, 1% orange G, and 0.5% Chromotrope 2R). Last, the sections were differentiated in 95% ethyl alcohol containing 0.2% acetic acid for 3 min, rinsed in fresh 95% ethanol, dehydrated in absolute alcohol, cleared in xylene, and mounted with Permount.

Immunofluorescence Localization of GABA, 5-HT, and DA in Nerve Ganglia

Slides containing tissue sections were deparaffinized in xylene and rehydrated in a descending ethanol series. The sections were then incubated in 0.1% glycine in PBS for 5 min, then washed with PBST (PBS containing 0.1% Tween 20). Nonspecific binding of proteins was blocked by incubating the sections in 2% normal goat serum with 4% BSA in PBST for 2 h, followed by incubation in mouse polyclonal anti-GABA, at a dilution of 1:400 in PBST, for 2 h at room temperature. Sections were rinsed 3 times in PBST and incubated for 2 h in second primary antibody--namely, rabbit polyclonal anti-5HT (Zymed Laboratories) or rabbit polyclonal anti-DA (Zymed Laboratories)--diluted 1:200 in PBST. The sections were washed 3 times with PBST and then incubated for 30 min in secondary antibodies--Alexa Fluor 594 conjugated goat anti-mouse IgG and Alexa Fluor 488 conjugated goat antirabbit IgG (Molecular Probes)--diluted 1:500 in PBST. Last, the sections were washed with PBST and subsequently mounted in VECTA shield fluorescent mounting medium before viewing under a confocal laser scanning microscope (Olympus FV1000). In controls, the sections were processed using the same protocol, but preimmune mouse and normal rabbit serum were used instead of the primary antibodies.


Distribution of GABA in the Cerebral Ganglia

Gamma-aminobutyric-immunoreactive neurons in the cerebral ganglion were detected in the medial edge of the cortex of the dorsal horn of the ganglion in both sexes (Fig. 1A-C), whereas there were only a few widely scattered GABA-ir neurons in the lateral edge of the lower half of the ganglion. These neurons were stained moderately with anti-GABA, and the numbers of these cells per all cells in the same section were approximately 22/1,535 in both sexes. The neuropils of the ganglion stained intensely with anti-GABA, especially at the medial edge of the dorsal horn and the lateral edge of the ventral horn of the ganglion in male abalone (Fig. 1A, B). There was no positive staining of GABA in control sections.


Distribution of GABA in the Pleuropedal Ganglia

The GABA-ir neurons, which were stained intensely, were numerous throughout the cortex region of the ganglion, especially in the upper and lower third of the dorsal and ventral horns of the ganglion (Fig. 1D), whereas they were widely scattered in the ventral edge of the body of the ganglion. The number of GABA-ir neurons was approximately 55/3,880 cells per section in both sexes. The ir nerve fibers were detected throughout the neuropils of the ganglion, especially in the dorsal and ventral horns (Fig. 1E, F).

Distribution of GABA in the Visceral Ganglia

The immunoreactivity patterns were similar for both male and female abalone. Gamma-aminobutyric acid-ir neurons were densely scattered in the cortex region of the dorsolateral and dorsomedial parts of the visceral ganglion of both females and males (Fig. 1G-I). The number of positive neurons per all cells was approximately 26/435 per section in both sexes. There was no positive staining of GABA in the control sections.

Immunoperoxidase and GA F Staining

Gamma-aminobutyric acid immunoreactivity was detected in the soma of neuronal cells located in the cortex of cerebral, pleuropedal, and visceral ganglia (Fig. 1). Based on histological characteristics described by Upatham et al. (1998), the GABA-ir neurons could be classified as type 1 neuronal cells (NR1), which are the largest neurons having an oval or pyramidal shape (diameter, about 10 x 20 [micro]m), and contain an oval-shaped nucleus with complete euchromatin, and large nerve process. Specific staining for neurosecretory cells was performed using GAF dye to produce a dark-violet color, and there was no positive GAF staining of the GABA-ir cells (Fig. 2). However, GAF stained neuronal types 2 and 3, which are neurosecretory cells concentrated in the cortex area of cerebral, pleuropedal, and visceral ganglia (Fig. 2B, D, F).


Colocalization of GABA-ir and 5-HT-ir or DA-ir in Neural Tissues by immunofluorescence

Immunofluorescence staining showed that GABA immunoreactivity and 5-HT immunoreactivity were present in different cells, but were also both present in some cells, of the cerebral, pleuropedal, and visceral ganglia. In addition well, GABA immunoreactivity and DA immunoreactivity were also found in similar distributions within these ganglia.

In the cerebral ganglion, all 3 neurotransmitters stained positively within cells of the cortex and fibers in the medulla (Fig. 3), but the 5-HT-ir fibers were particularly intense (Fig. 3C). The fluorescence staining of GABA and 5-HT in cerebral ganglia mainly occurred in separate cells, but were colocalized in some cells (Fig. 3E, G). The GABA-ir neurons were concentrated primarily in the dorsal horn of each ganglion, and the number of GABA-ir neurons was approximately 25 cells per section. There were about 29 cells per section that stained positively for 5-HT only, and these were located mainly in the ventral horn of the ganglion, whereas there were 40 cells per section with colocalized GABA and these were scattered throughout the middle part of the ganglion. There were a few positively stained GABA-ir fibers in the medulla region of ganglion (Fig. 3B), whereas many 5-HT-ir fibers were detected in the same region of the ganglion (Fig. 3C, E). No DA-positive fibers were observed (Fig. 3D, F), but 24 cells per section stained positively for DA only and these were scattered throughout the cortex in both sexes. Gamma-aminobutyric acid and DA are colocalized in the same cells (Fig. 3F, H).

In the pleuropedal ganglion, fluorescence staining showed cells with GABA immunoreactivity with DA immunoreactivity (Fig. 4E, G), and GABA immunoreactivity with 5-HT immunoreactivity (Fig. 4D, F). There were about 12 GABA-ir, 4 5-HT-ir, and 2 DA-ir cells per section, whereas the numbers of cells showing both GABA 5-HT and GABA--DA were 35 and 33 per section, respectively, in both sexes. All these double-positive staining cells were observed throughout the cortex of the pleuropedal ganglion. In addition, only a small number of DA-ir nerve fibers were detected throughout the neuropils in the ventral horn of the pleuropedal ganglion (Fig. 4B, E), but there was no positive staining of GABA and 5-HT in nerve fibers (Fig. 4A, B, D).

In the visceral ganglion, there were about 5 GABA-ir, 2 5-HT-I,r and only 1 DA-ir cells per section, respectively; however, there were only 2 cells with colocalization of GABA with the other 2 neurotransmitters (Fig. 5).


The Distribution of GABA in Neural Ganglia of Adult Haliotis asinina

Cells showing GABA immunoreactivity have been documented in the buccal, cerebral, and pedal ganglia in gastropods, including Helisoma trivolvis (Richmond et al. 1991), Helix pomatia (Hernadi 1994), Helix aspersa (Ierusalimsky & Balaban 2001), Cepaea nemoralis (Dyakonova et al. 1995, Clione limacina (Arshavsky et al. 1993, Norekian 1999), Limax maximus (Cooke & Gelperin, 1988), and Aplysia californica (Diaz-Rios et al. 1999). However, the majority of GABA-ir cell bodies were located predominantly in the pleuropedal ganglia, with few GABA-ir cells found in the buccal ganglia (Hernadi 1994). In the current study, we found GABA-ir cells not only in the pleuropedal ganglia, but in all 3 major ganglia, including the cerebral and visceral ganglia, about 25 cells and 5 cells per section, respectively. This distribution of GABA-ir cells is similar to that shown in Lymnaea stagnalis, where GABA-ir cells were detected in all ganglia (Hatakeyama & Ito 2000). It is also similar in H. aspersa, where GABA-ir neurons are found throughout the cellular layer in the pedal and buccal ganglia, and are mostly located on the dorsal surface in the cerebral ganglia (Ierusalimsky & Balaban 2001).



We have shown that GABA-ir cells are located in parts of the cerebral and throughout the pleuropedal ganglia. In the visceral ganglion, GABA immunoreactivity was found in the left lateral and ventromedial parts, with ir nerve fibers containing GABA concentrated in the neuropils of the ganglia. This observation supports the findings of varicose GABA-ir fibers in the neuropil areas and in the cell body layers of the ganglia in Helisoma trivolvis, Clione limacine, Helix pomatia and Limax maximus (Cooke & Gelperin 1988, Richmond et al. 1991, Arshavsky et al. 1993, Hernadi 1994, Norekian 1999), and the majority of GABA-ir axonal processes extend into the connectives and commissures of the ganglia, which indicates an important central integrative role of GABA-ir neurons. Interestingly, GABA was detected only in large neurons but not in neurosecretory neurons, the granules of which were stained with GAF. Because most large neurons are motor cells (Kruatrachue et al. 1999), it is likely that GABA in the CNS is more concerned with controlling muscle contraction rather than neurosecretion. In the nematode Caenorhabditis elegans, GABA is released from the ventral cord neurons and inhibits contraction of body wall muscles during locomotion (McIntire et al. 1993). In addition, exogenous GABA causes cessation of swimming and foot exploration behavior in abalone (Akashige et al. 1981, Barlow 1990). Together, these data imply that GABA inhibition of muscle and cilia movement is a main factor in larval settlement.


Colocalization of GABA with the Other Neurotransmitters

Close relationships have been observed between 5-HT and GABA in vertebrates, such as the rabbit (Osborne & Beaton 1986) and the cat (Wassle & Chun 1988), with double-labeling studies demonstrating the coexistence of these 2 neurotransmitters in amacrine cells of the retina. In amphibians, double-labeling experiments with antibodies against 5-HT and GABA have determined the coexistence of GABA and 5-HT in neurons of the caecilian retina (Watt & Florack 1992, Main et al. 1993, Zhu & Straznicky 1993). In Xenopus laevis, double labeling of amacrine cells for GABA and DA showed 52% of contained DA at stage 42 of embryonic development, and only 20% at stage 44, whereas double labeling for 5-HT and DA identified only 4% of the DA cell population (Zhu & Straznicky 1993). In a study of the retina of Bufo marinus, some 5-HT-ir cells had GABA immunoreactivity, whereas other cells remained unlabeled for GABA (Main et al. 1993). However, it was also shown in double-labeling experiments that neurons accumulating 5HT also synthesize DA and that in some neurons DA is colocalized with GABA, thus it is similar to the result in Xenopus. This suggests that there are 3 types of amacrine cells--namely, DA only, GABA and DA, and 5-HT and DA cells. Each type is found more in the anterior and dorsal regions rather than the posterior and ventral regions, and their overall distribution patterns are indistinguishable statistically (Huang & Moody 1998).

Neurons displaying coexistence of 5-HT and GABA in the midbrain raphe have also been reported (Harandi et al. 1987, Gao et al. 1993), and GABA-ir cell bodies were less numerous than those for 5-HT (Harandi et al. 1987, Panasophonkul et al. 2004). It was observed that in cell bodies of rat somatic nerves, 40% of cells containing GABA also contained 5-HT, whereas 30% of neurons containing 5-HT also showed GABA immunoreactivity (Harandi et al. 1987). Hence, it was proposed that some neurons could be both GABAergic and serotonergic. In a more recent study, it was found that DA mediates divergent and convergent rapid excitatory postsynaptic potentials from 2 influential central pattern generator interneurons in Aplysia californica, and l of these interneurons contained GABA-ir cells (Diaz-Rios & Miller 2005). Therefore, from these previous studies, there is a definite coexistence of 2 transmitters, one possibly in a higher concentration and having a functional role and the other in a lower concentration without a definite function (Leslie & Osborne 1984). We also observed colocalization of GABA with DA and with 5-HT within the same cells of all ganglia. These were all NR1. The coexistence between an amine and/or an amino acid and 1 or more neuropeptides is common because these substances are stored in separate vesicles and are released differentially, depending on the frequency of stimulation (Von Bohlen und Halbach & Dermietzel 2002). Therefore, under specific conditions, 1, 2, or more compounds may be released from a single neuron. However, it is still unclear why there is a coexistence of multiple messengers. It has been proposed that 2 or more of the neurotransmitters DA, 5-HT, and GABA are transported into hypophyseal vessels through a neurotransmitter neurotransmitter, peptide-peptide, or a neurotransmitter peptide interaction resulting from colocalization and corelease from a neuron (Hokfelt et al. 1994). It is also possible that colocalization is a consequence of evolutionary divergence whereby neurotransmitters evolved as a gradual evolutionary transition from one neurotransmitter to another. Therefore, one might expect to find certain neurons still harboring more than one transmitter. Neurons in Haliotis asinina, which is a lower form of invertebrate, may still have trace amounts of a second neurotransmitter coexisting with the true transmitter. It is most likely that the presence of 2 transmitter-type molecules in a neuron does not reflect any functional significance; probably, one of the transmitters has a vestigial presence simply because suppression of a certain gene did not proceed to its end point. Alternatively, it is possible that the proportions of each compound colocalizing in a neuron depend on the age or physiological requirements of the organism (Takayama & Inoue 2004, Gacia-Lavandeira et al. 2005). This is now part of our continuing studies.


This research was supported by a Distinguished Research Professor Grant (cofunded by the Thailand Research Fund, the Commission on Higher Education, and Mahidol University) to P. Sobhon, and a Commission on Higher Education PhD scholarship to P. Stewart.


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(1) Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani 12121, Thailand; (2) Genecology Research Group, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD 4558, Australia; (3) Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; (4) Pro Vice-Chancellor's Office, Faculty of Science and Technology, Deakin University, Locked Bag 20000, Geelong, VIC 3220, Australia

* Corresponding author. E-mail:

DOI: 10.2983/035.032.0111
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Author:Soonklang, Nantawan; Stewart, Michael J.; Wanichanon, Chaitip; Stewart, Praphaporn; Hanna, Peter J.;
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:8AUST
Date:Apr 1, 2013
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