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Characterization of Neurons in the Visceral Ganglia of the Green-lipped Mussel (perna Canaliculus) Using Antibodies Raised Against Neuropeptides and Neurotransmitters

S. Mahmud
Chittagong Veterinary and Animal SciencesUniversity, Chittagong-4202, Bangladesh

P. V. Mladenov
Department of Physiology, School of Medicine, University of Otago, Dunedin, New Zealand

P. Sheard
Department of Physiology, School of Medicine, University of Otago, Dunedin, New Zealand

S. C. Chakraborty
Department of Fisheries Technology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Central neurons in the visceral ganglia of both male and female Green lipped mussel, Perna canaliculus were characterized by immunohistochemical techniques. We used mollusc antibodies raised against neuropeptides and neurotansmitters known to control reproduction and spawning. Anti-ELH and anti-APGWamide showed very strong immunoreactivity in small type of neurons. Anti-5-HT and anti-DA immunoreactivity was mostly in large type of neurons. The labelled neurons are consistent with descriptions of neurosecretory cells implicated in the control of reproduction and spawning on the basis of earlier histological staining techniques used in this species. The use of selective immunological markers for peptides and amines appears to be a promising tool for further Characterization of neurosecretory cells. To isolate and characterise neuropeptides and other biologically active materials involved in the control of reproduction in Perna canaliculus.

Keywords: Characterization, Visceral ganglia, Green lipped mussel, Antibodies, Neuropeptides

Location: Department of Physiology at the University of Otago, Dunedin, New Zealand

Start Date: 00-00-2008

End Date: 00-00-2008

Program Area: Animal Health and Management

Commodity: Diseases

Objectives:

1. To locate and identify the neurons containing neurotransmitters or egg-laying hormones in the green-lipped mussel using immunohistochemistry.

Methodology:

Collection of mussels, fixation and dissection of ganglia - The green-lipped mussels, Perna canaliculus, were collected from an exposed rocky shore at Purihurihu Point, near Blueskin Bay, in the South Island of New Zealand. Collection of ganglia of both sexes for immunohistochemistry was done shortly after transporting the mussels to the laboratory of the Department of Physiology at the University of Otago, Dunedin, New Zealand. The visceral ganglia were collected from both sexes. Individual tissues were placed gently in the bottom of an aluminium foil boat containing pre-cooled Tissue-Tek™ O.C.T. compound and then the foil boat was filled with O.C.T. compound. The tissue was snap frozen by partial immersion of the foil boat into isopentane cooled in liquid nitrogen. Individual tissues were preserved at –70°C for sectioning. Antibodies used for immunohistochemistry - Four antisera were used in this study, all produced in rabbits: (i) Anti-ELH was raised against a synthetic peptide representing the N-terminal fragment (ISINQDLKAITDML) from the egg laying hormone of Aplysia. This antibody was produced by G. T. Nagle and J. E. Blankenship (University of Texas Medical Branch), and its Characterization and specificity were described by Ram et al., (1998), (ii) Anti-APGWamide (CHEMICON International, Inc. 28835 Single oak Drive, Temecula, CA 92590), (iii) Anti-Dopamine (CHEMICON International, Inc. 28835 Single Oak Drive, Temecula, CA 92590), and (iv) Anti-Serotonin was obtained from Dept. of Zoology, University of Otago, Dunedin, New Zealand. The unlabelled goat anti-rabbit secondary antibody was obtained from Cappel Research Products (Durham, North Carolina) and the peroxidase-antiperoxidase complex employing rabbit antibodies was obtained from Sigma Chemical Co. (Mississauga, Ontario). Immunocytochemistry protocol - Serial sections (two sets - one for experimental and another for control) were cut at 10 μm in a cryostat at -18°C and approximately 8-10 sections were mounted on each slide for immunohistochemistry. The dried sections were fixed for 10 minutes in freshly prepared 4% paraformaldehyde and were washed in PBS. Primary antiserum were then applied and left overnight at 4°C. Antiserum dilutions of between 1:400 and 1:100 were used in an immunodiluent (ID) solution of 2% normal goat serum (Sigma Chemical Co.) and 0.2% Triton X-100 (Sigma Chemical Co.) in PBS. Next day, secondary antibody was added to all slides after washing in PBS and was left for an hour at room temperature. The secondary antiserum was diluted 1:200 in ID. After another several washes in PBS the slides were kept for another one-hour incubation in peroxidase-antiperoxidase diluted 1:400 in ID. After incubation, slides were washed off again in PBS and were developed for 2-3 minutes using diaminobenzidine (DAB)- hydrogen peroxide. Slides were dehydrated in graded ethanols washed in xylene, and mounted in DPX. One set of serial sections from each ganglion was processed as described above, with the elimination of the incubation in primary antibody as a negative control. Slides were viewed through an Olympus BX50 Microscope and photographed digitally.

Source of Information: Bang. J. Anim. Sci. 2008, 37 (1) : 78 - 85; ISSN 0003-3588

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The present immunocytochemical study identifies unique population of cells containing neuropeptides and neurotransmitters, which are the likely candidates responsible for different aspects of reproduction and spawning in P. canaliculus. Although, the labelling of cells with anti-ELH, anti-APGWamide, anti-5HT and anti-DA does not necessarily confirm any physiological functions at this stage but it does indicate the presence of a preprohormone with ovulation factors and neurotransmitters.

Knowledge Management: Journal