Modeling of Neuronal Growth In Vitro: Comparison of Simulation Tools NETMORPH and CX3D.

We simulate the growth of neuronal networks using the two recently published tools, NETMORPH and CX3D. The goals of the work are (1) to examine and compare the simulation tools, (2) to construct a model of growth of neocortical cultures, and (3) to characterize the changes in network connectivity during growth, using standard graph theoretic methods. Parameters for the neocortical culture are chosen after consulting both the experimental and the computational work presented in the literature. The first (three) weeks in culture are known to be a time of development of extensive dendritic and axonal arbors and establishment of synaptic connections between the neurons. We simulate the growth of networks from day 1 to day 21. It is shown that for the properly selected parameters, the simulators can reproduce the experimentally obtained connectivity. The selected graph theoretic methods can capture the structural changes during growth.

Electrical detection of the contact between a microinjection pipette and cells.

This work presents the basic design and tests of a device designed for detecting the contact between a microinjection pipette and cell membrane. The device facilitates the automation of the microinjection procedure of living adherent cells. The measurement of the contact is based on measuring the resistance of the pipette. Breakage and clogging of the pipette can be detected with the same measurement.

Serotonin induces inward potassium and calcium currents in rat cortical astrocytes.

Ca2+ imaging and patch-clamp techniques were used to study the effects of serotonin (5-HT) on ionic conductances in rat cortical astrocytes. 1 and 10 microM serotonin caused a transient increase in intracellular calcium (Ca(i)) levels in fura-2AM-loaded cultured astrocytes and in astrocytes acutely isolated and then cultured in horse serum-containing medium for over 24 h. However, the acutely isolated (less than 6 h from isolation) astrocytes, as well as acutely isolated astrocytes cultured in serum-free media, failed to respond to 5-HT by changes in Ca(i). Coinciding with the changes in Ca(i) levels, inward currents were activated by 10 microM 5-HT in cultured, but not in acutely isolated astrocytes. Two separate types of serotonin-induced, small-conductance inward single-channel currents were found. First, in both Ca2+-containing and Ca2+-free media serotonin transiently activated a small-conductance apamin-sensitive channel. Apamin is a specific blocker of the small-conductance Ca2+-activated K+ channel (sK(Ca)) When cells were pre-treated with phospholipase C inhibitor U73122 no 5-HT-induced sK(Ca) channel openings were seen, indicating that this channel was activated by Ca2+ released from intracellular stores via IP3. A second type of small inward channel activated later, but only in the presence of external Ca2+. It was inhibited by the L-type Ca2+ channel blockers, nimodipine and nifedipine. Both types of channel activity were inhibited by ketanserin, indicating activation of the 5-HT2A receptor.

Simultaneous detection of action potential current waveforms and single ion channel openings in rat cerebellar granule cells.

Spontaneous Action Potential Current waveforms (APCs) and single ion channel openings were recorded in cultured rat cerebellar granule cells using the cell-attached configuration of the patch-clamp technique. APCs were activated without any externally applied stimuli. Both mono- and biphasic APCs were detected with a typical amplitude (positive waveform component) of 25-60 pA. Correlation between cell APCs and inward and outward single ion channel openings immediately before and after the capacitive transients was analyzed. In a detailed analysis it was found that the opening of 1.5 pA inward channel before the waveform was associated with 16%, and the opening of 1.0 pA outward channel after the waveform with 88% of APCs (of total 327 APCs analyzed). The modulation of APC activity by bath application of the amino acid taurine was found to increase the frequency of APCs, transform the APCs from mixed monophasic/biphasic to completely biphasic, and enhance the activated inward current deflections seemingly connected to the increased firing frequency.