Optimization of neuronal cultures from rat superior cervical ganglia for dual patch recording

Julien Amendola (1) , Norah Boumedine (1) , Marion Sangiardi (1) , Oussama El Far (1)

1 UNIS - Inserm U1072 - Unité de Neurobiologie des canaux Ioniques et de la Synapse

Superior cervical ganglion neurons (SCGN) are often used to investigate neurotransmitter release mechanisms. In this study, we optimized the dissociation and culture conditions of rat SCGN cultures for dual patch clamp recordings. Two weeks in vitro are sufficient to achieve a significant CNTF-induced cholinergic switch and to develop mature and healthy neuronal profiles suited for detailed patch clamp analysis. One single pup provides sufficient material to prepare what was formerly obtained from 12 to 15 animals. The suitability of these cultures to study neurotransmitter release mechanisms was validated by presynaptically perturbing the interaction of the v-SNARE VAMP2 with the vesicular V-ATPase V0c subunit. Adult sympathetic rat superior cervical ganglion neurons (SCGN) were one of the first neuronal systems used to investigate synaptic neurotransmission mechanisms 1. These neurons have been a very useful system, as illustrated by an abundant literature, especially to study intracellular pathways of ion channel regulation 2,3 and ion channel signalling mediated via muscarinic receptors 4–6. SCGNs have also been exploited to develop a dissociated neuronal SCG culture model to study synaptic neurotransmission by perturbing presynaptic protein-protein interactions through injection of interfering molecules 7–9. In vivo, SCG neurons are principally adrenergic with a few cells displaying a cholinergic phenotype 10. Depending on culture conditions, SCGN can switch phenotype in vitro and develop cholinergic transmission 10–14. This phenotypic switch has been made use of to follow up membrane currents and voltage changes associated with acetylcholine release using sharp electrode intracellular recording in studying neuro-transmission mechanisms. A detailed characterization of the presynaptic mechanisms SNARE-mediated neurotransmitter release has been performed using SCGNs 7. This was facilitated by the very large size of these neurons, allowing direct injection in the soma of interfering molecules such as peptides, fusion proteins and even intra-nuclear injection of cDNA 15 coding for specific proteins. In this study, we present a protocol to prepare up to six SCGN coverslips from one single ganglion and therefore significantly reduce the number of animals used. In addition, our dissociation and culture conditions allow dual patch-clamp recording and monitoring of cholinergic neurotransmission in as early as two week-old dissociated cultures, rather than the commonly used six to eight week-old cultures 7

Mots clés: SYMPATHETIC NEURONS; SYNAPTIC-TRANSMISSION; CALCIUM-CHANNELS; SYNAPSES; CILIARY; CELLS; DIFFERENTIATION; EXPRESSION; MOTONEURONS; INHIBITION

Domaines: Neurosciences [q-bio.NC]

Julien Amendola, Norah Boumedine, Marion Sangiardi, Oussama El Far. Optimization of neuronal cultures from rat superior cervical ganglia for dual patch recording. Scientific Reports, 2015, 5 (14455), ⟨10.1038/srep14455⟩. ⟨hal-01236867⟩