Abstract
Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.
Highlights
Transient increases in intracellular calcium regulate a wide range of cellular processes in developing and mature neurons, from rapid enhancement of neurotransmitter release to longlasting changes in gene expression (Carey and Matsumoto 1999; Emptage et al 2001; Spitzer et al 2004; Syed et al 2004; Yuste et al 1992)
In Drosophila, GAL4 driving expression of the calcium-sensitive luminescent protein, apoaequorin, reveals slow rhythmic oscillations in intracellular calcium levels localized in mushroom bodies, a region of the insect brain required for olfactory associative learning (Rosay et al 2001)
To explore the cellular mechanisms involved in regulation of calcium in neurons from the mushroom body region, intracellular calcium levels were monitored in Kenyon cells grown in primary culture
Summary
Transient increases in intracellular calcium regulate a wide range of cellular processes in developing and mature neurons, from rapid enhancement of neurotransmitter release to longlasting changes in gene expression (Carey and Matsumoto 1999; Emptage et al 2001; Spitzer et al 2004; Syed et al 2004; Yuste et al 1992). Two other calcium sensors that have been transgenically expressed in the fly, have provided increased spatial resolution in analysis of calcium dynamics These have been used to discriminate between odorevoked responses in pre- and postsynaptic regions, or between acetylcholine (ACh)-evoked responses in different lobes, of the mushroom bodies (Fiala et al 2002; Yu et al 2003). O’DOWD erate transients is up regulated during pupal development, transient frequency is similar to the frequency of calcium oscillations in vivo, but spontaneous calcium transients are not specific to Kenyon cells This suggests that, while the transients are likely to represent cellular events that contribute to calcium oscillations in the mushroom bodies, they may play a more general role in adult brain neurons
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