Primary culture and characteristic morphologies of neurons from the cerebral ganglion of the mud crab, Scylla paramamosain

Abstract

Crustacean neurons, obtained from the cerebral ganglion of the mud crab Scylla paramamosain, were successfully cultured in vitro. They maintained typical morphological characteristics and showed better outgrowth in modified Medium 199 (M199) medium than that in Liebowitz's L-15 medium. Fetal bovine serum (FBS), muscle extracts, and hemolymph of the mud crab S. paramamosain were added as supplements. Only 20% FBS could promote neuron outgrowth, while muscle extracts and hemolymph of S. paramamosain did not improve neuron outgrowth. For cell dissociation, both collagenase type I and trypsin worked well as determined by initial cell viability and following cell outgrowth potential. More than six kinds of cells with different morphological characteristics were identified in the neuron outgrowth. They were "small cells", "veilers", "branchers", "multipolar cells", "super-large cell", and "bipolar cells". Among all of the cells, bipolar cells were identified for the first time in crustacean neurons culture and they could live longer than other cells. The neurons could grow for more than a week before retraction and eventual degradation.

Primary Cell Culture and Characteristic Morphologies of Neurons in the Eyestalks of the Kuruma Shrimp, Marsupenaeus Japonicus (Decapoda, Penaeoidea)

“…Furthermore, it had been reported that the haemolymph of Ortosquilla oratoria (De Haan, 1844) was inhibitory in ovary cultures of Fennero penaeus chinensis (Osbeck, 1765) (cf. Tong & Miao, 1996); also, Scylla paramamosain Estampador, 1949 neuron growth was restrained in the presence of muscle extracts (Xu et al, 2010). We conclude that muscle extracts do not always play a positive role in the culture of all types of cells.…”

“…However, the second kind of bipolar cell was very rare in earlier eyestalk neuron cultures. This type of cell was, for example, found in the cerebral ganglion of the mud crab (Xu et al, 2010) and in insect neuron culture (Smith & Howes, 1996;Weigel, 2005). In our studies, they had great vitality and could maintain their cell shape between 8 and 11 days.…”

Culture of neural cells of the eyestalk of a mangrove crab is optimized on poly-l-ornithine substrate

“…Protocols have been developed for nervous system cells of invertebrates, particularly adult and developing insects and adult worms (Beadle 2006;Srivatsan and Peretz 1997). However, for adult decapod crustaceans, we are aware of only a few reports of nerve cell culture: Chun-Lei et al (2003) described the morphology of cultured neurons from the terminal medulla of the Chinese white shrimp Fenneropenaeus chinensis; Stepanyan et al (2004) described a protocol for olfactory neurons from the American lobster Homarus americanus, focusing on olfactory physiology; and Xu et al (2010) studied the morphology of the cells of the cerebral ganglion of the mud crab Scylla paramamosain. None of these reports described a cell-culture protocol that aimed to characterize cell types from the adult decapod crustacean nervous system in detail, as we describe here.…”

“…Further, the characterization of invertebrate neurons and glia either in vivo or in vitro has not been explored as thoroughly as it has in vertebrates (Barres 2008). Culturing invertebrate nervous system cells may allow experimental manipulations under controlled conditions, facilitating studies on their morphology (Xu et al 2010) and functions (Stepanyan et al 2004).…”

“…Decapod crustaceans are excellent models because of their well-organized nervous system, attention-grabbing behavior patterns ranging from reflexes to complex social interactions (Sandeman et al 1992) and ease of handling. To our knowledge, few protocols for nervous-tissue culture have been reported for adult or developing decapod crustaceans (Chun-Lei et al 2003;Mitsuhashi 2002;Stepanyan et al 2004;Toullec 1999;Xu et al 2010).…”