Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identifiedHelix neurons in cell culture

Abstract

Hemolymph of adult Aplysia californica significantly affects neurite outgrowth of identified neurons of the land snail Helix pomatia. The metacerebral giant cell (MGC) and the motoneuron C3 from the cerebral ganglion and the neuron B2 from the buccal ganglion of H. pomatia were isolated by enzymatic and mechanical dissociation and plated onto poly-L-lysine-coated dishes either containing culture medium conditioned by Helix ganglia, or pre-treated with Aplysia hemolymph. To determine the extent of neuronal growth we measured the neurite elongation and the neuritic field of cultured neurons at different time points. Aplysia hemolymph enhances the extent and rate of linear outgrowth and the branching domain of Helix neurons. With the hemolymph treatment the MGC neuron more consistently forms specific chemical synapses with its follower cell B2, and these connections are more effective than those established in the presence of the conditioned medium.

Subconvulsant doses of pentylenetetrazol uncover the epileptic phenotype of cultured synapsin-deficient Helix serotonergic neurons in the absence of excitatory and inhibitory inputs

“…Cell cultures were performed as previously described by Ghirardi et al (1996) and modified according to Brenes et al (2015a). Briefly, snails were anesthetized with MgCl 2 , ganglia were surgically isolated and incubated for enzymatic digestion in protease type XIV (0.3 U/mL) at 34°C for 3-3.5 h. Neurons were individually isolated and transferred to dishes pretreated with 5% bovine serum albumin to prevent cell-substrate adhesion.…”

“…Cultured Helix neurons (Ghirardi et al, 1996) offer great advantages for studying neuronal activity and epileptic-like patterns. They are identifiable, form monosynaptic connections in vitro (Fiumara et al, 2001;Fiumara et al, 2005), and allow a variety of experimental manipulations that are not feasible with mammalian neurons, such as single cell-electrode coupling on microelectrode arrays (MEAs) (Massobrio et al, 2009;Massobrio et al, 2013) and intranuclear DNA injection (Brenes et al, 2015a;Brenes et al, 2015b).…”

Knock-down of synapsin alters cell excitability and action potential waveform by potentiating BK and voltage-gated Ca2+ currents in Helix serotonergic neurons

“…Cell cultures were performed as previously described (Ghirardi et al, 1996). Briefly, the snails were anesthetized by the injection of isotonic MgCl 2 in the foot.…”

“…To better analyze the role of Syn on single cell excitability, we decided to use an invertebrate culture cell preparation that is particularly advantageous since specific identifiable neurons may be isolated from their synaptic inputs and cultured in isolation where they maintain their electrical properties (Ghirardi et al, 1996). In these experiments, serotonergic C1 neurons were isolated from cerebral ganglia of Helix snails and kept in culture as floating somata for 3 days following asRNA injections.…”

“…The aim of the present study is to investigate the effects of Syn silencing on the electrophysiological properties of isolated identified neurons completely deprived of their synaptic inputs. The neurons of the land snail Helix provide several advantages for this study since specific identifiable neurons can be isolated independently and cultured avoiding the effects of surrounding tissue or excitatory and inhibitory synaptic inputs (Ghirardi et al, 1996;Fiumara et al, 2001). These neurons also allow performing complex experimental manipulations such as plasmid intra-nuclear microinjection and provide a convenient genetic organization with a single Syn gene (Fiumara et al, 2007) that may be blocked by antisense RNA (asRNA) (Brenes et al, 2015).…”