Visualization of Exocytosis and Endocytosis in Cultured Neurones Using TIRF Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Multiple effects of artemin on sympathetic neurone generation, survival and growth

To define the role of artemin in sympathetic neurone development, we have studied the effect of artemin on the generation, survival and growth of sympathetic neurones in low-density dissociated cultures of mouse cervical and thoracic paravertebral sympathetic ganglia at stages throughout embryonic and postnatal development. Artemin promoted the proliferation of sympathetic neuroblasts and increased the generation of new neurones in cultures established from E12 to E14 ganglia. Artemin also exerted a transient survival-promoting action on newly generated neurones during these early stages of development. Between E16 and P8, artemin exerted no effect on survival, but by P12, as sympathetic neurones begin to acquire neurotrophic factor independent survival, artemin once again enhanced survival, and by P20 it promoted survival as effectively as nerve growth factor (NGF). During this late period of development, artemin also enhanced the growth of neurites from cultured neurones more effectively than NGF. Confirming the physiological relevance of the mitogenic action of artemin on cultured neuroblasts, there was a marked reduction in the rate of neuroblast proliferation in the sympathetic ganglia of mice lacking the GFRα3 subunit of the artemin receptor. These results indicate that artemin exerts several distinct effects on the generation, survival and growth of sympathetic neurones at different stages of development.

K(+) currents in cultured neurones from a polyclad flatworm

Cells from the brain of the polyclad flatworm Notoplana atomata were dispersed and maintained in primary culture for up to 3 weeks. Whole-cell patch-clamp of presumed neurones revealed outwardly directed K(+) currents that comprised, in varying proportions, a rapidly activating (time constant tau =0.94+/−0.79 ms; N=15) and inactivating (tau =26.1+/−1.9 ms; N=22) current and a second current that also activated rapidly (tau =1.1+/−0.2 ms; N=9) (means +/− s.e.m.) but did not inactivate within 100 ms. Both current types activated over similar voltage ranges. Activation and steady-state inactivation overlap and are markedly rightward-shifted compared with most Shaker-like currents (half-activation of 16.9+/−1. 9 mV, N=7, half-inactivation of −35.4+/−3.0 mV, N=5). Recovery from inactivation was rapid (50+/−2.5 ms at −90 mV). Both currents were unaffected by tetraethylammonium (25 mmol l(−1)), whereas 4-aminopyridine (10 mmol l(−1)) selectively blocked the inactivating current. The rapidly inactivating current, like cloned K(+) channels from cnidarians and certain cloned K(+) channels from molluscs and the Kv3 family of vertebrate channels, differed from most A-type K(+) currents reported to date. These findings suggest that K(+) currents in Notoplana atomata play novel roles in shaping excitability properties.