Abstract
Synaptic activity is intimately linked to neuronal structure and function. Stimulation of live cultured primary neurons, coupled with fluorescent indicator imaging, is a powerful technique to assess the impact of synaptic activity on neuronal protein trafficking and function. Current technology for neuronal stimulation in culture include chemical techniques or microelectrode or optogenetic based techniques. While technically powerful, chemical stimulation has limited spatial resolution and microelectrode and optogenetic techniques require specialized equipment and expertise. We report an optimized and improved technique for laser based photoconductive stimulation of live neurons using an inverted confocal microscope that overcomes these limitations. The advantages of this approach include its non-invasive nature and adaptability to temporal and spatial manipulation. We demonstrate that the technique can be manipulated to achieve spatially selective stimulation of live neurons. Coupled with live imaging of fluorescent indicators, this simple and efficient technique should allow for significant advances in neuronal cell biology.
Highlights
Advances in light microscopic techniques and availability of a variety of fluorescent indicators for examining protein trafficking and cellular function in live neurons has led to significant advances in our understanding of the cellular changes that underlie neuronal structure and function (Granger et al, 2013; Karpova et al, 2013)
Stimulation of live cultured primary neurons, coupled with fluorescent indicator imaging, is a powerful technique to assess the impact of synaptic activity on neuronal protein trafficking and function
We report an optimized and improved technique for laser based photoconductive stimulation of live neurons using an inverted confocal microscope that overcomes these limitations
Summary
Advances in light microscopic techniques and availability of a variety of fluorescent indicators for examining protein trafficking and cellular function in live neurons has led to significant advances in our understanding of the cellular changes that underlie neuronal structure and function (Granger et al, 2013; Karpova et al, 2013). Several types of neurons, including cortical, hippocampal (Li et al, 2010; Sylwestrak and Ghosh, 2012) and cerebellar granule neurons (Kim et al, 2009), are highly amenable to being cultured in a primary neuronal cell culture system (Beaudoin et al, 2012), allowing cell biological studies in a neuron type specific manner. These culture systems are well established and have been used by several groups to make fundamental advances in neuronal cell biological studies (Aoto et al, 2013; Siddiqui et al, 2013). The combination of live cell imaging techniques coupled with the ability to stimulate live neurons has great advantages for neuronal cell biological studies (Okada et al, 2009)
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