Kitamura K, Judkewitz B, Kano M, Denk W, Häusser M.
Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo.
Nat Methods. 2008 Jan;5(1):61-7.
PubMed.
Grace Stutzmann Rosalind Franklin University/The Chicago Medical School
Posted:
This article provides some practical alternatives for visualizing and patching neurons in an in vivo preparation using two-photon imaging techniques. Although currently there are relatively few researchers conducting these types of experiments, perhaps these “tricks” can make these techniques more accessible to others, and increase the overall productivity rate in this low-throughput, but high-gain, approach to studying neuronal function in vivo. For somatic patching, shadowpatching will certainly improve productivity compared to blind patching, and for dendritic patching, it is likely a necessity.
Usually, the excess fluorescent dye that is ejected into the extracellular space is considered an undesirable effect in imaging experiments, yet here, it is used to generate a shadow image of neurons—a dark soma against the fluorescent background noise. The contrast generated appears high, but the inherent optical sectioning ability of two-photon imaging requires a comprehensive z-stack of the neuron in order to determine morphology. Ironically, this optical sectioning feature is one of the significant advantages of two-photon imaging, but works against the shadowpatching objectives here. Yet, creating images from stacked z-planes need not be difficult with most imaging software applications and will provide a nice 3D image of the neuron recorded from. Additionally, one could also accelerate the process by focusing up and down to quickly ascertain the neuronal morphology, and verify cell type upon patching and filling the neuron.
The authors also coupled their patching approach with single-cell electroporation of plasmid DNA in vivo. By placing an electrode filled with fluorescent dye and plasmid DNA encoding eGFP close to the soma and administering negative voltage pulses, individual neurons took up the dye and plasmids. Expression of the eGFP was determined at 24 and 48 hours afterwards. Identified single neurons in the cortex were electroporated; however, in more dense regions where neurons are closely packed, electroporation of individual cells may be more problematic.
These helpful tips can certainly facilitate the patching and imaging of individual neurons in vivo, an already challenging and daunting task. I would imagine some practice and fine-tuning would be needed to optimize the amount of positive pressure required to eject the dye into the extracellular space. Likewise, there is likely a fine line between the voltage needed for a successful electroporation and a stimulus that is damaging or lethal to the cell. Once these and other empirical issues are worked out, not seeing your neurons may be the way to go for targeted in vivo imaging.
There may be some useful variants of this approach that could be applied to neurodegenerative disease studies, such as AD. As any fluorescently labeled object can be imaged, in vivo two-photon imaging is not limited to cells. One thought is to fill the patch pipette with thioflavin S or a similar fluorescent plaque indicator and a conventional fluorescent dye, then shadowpatch neurons to measure effects of plaque deposits on neuronal morphology and physiology. Likewise, plaque markers can be injected systemically, and neurons in varying proximity to these labeled plaques can be shadowpatched using a fluorescent dye of a different emission wavelength. These plaque and filled neuron images can be overlaid, or, simultaneously acquired if multiple emission detections systems are in place. Analogous applications may involve apoptotic markers, Lewy body markers, or other pathogenic indicators. As long as it fluoresces, it can likely be detected with two-photon imaging.
Comments
Rosalind Franklin University/The Chicago Medical School
This article provides some practical alternatives for visualizing and patching neurons in an in vivo preparation using two-photon imaging techniques. Although currently there are relatively few researchers conducting these types of experiments, perhaps these “tricks” can make these techniques more accessible to others, and increase the overall productivity rate in this low-throughput, but high-gain, approach to studying neuronal function in vivo. For somatic patching, shadowpatching will certainly improve productivity compared to blind patching, and for dendritic patching, it is likely a necessity.
Usually, the excess fluorescent dye that is ejected into the extracellular space is considered an undesirable effect in imaging experiments, yet here, it is used to generate a shadow image of neurons—a dark soma against the fluorescent background noise. The contrast generated appears high, but the inherent optical sectioning ability of two-photon imaging requires a comprehensive z-stack of the neuron in order to determine morphology. Ironically, this optical sectioning feature is one of the significant advantages of two-photon imaging, but works against the shadowpatching objectives here. Yet, creating images from stacked z-planes need not be difficult with most imaging software applications and will provide a nice 3D image of the neuron recorded from. Additionally, one could also accelerate the process by focusing up and down to quickly ascertain the neuronal morphology, and verify cell type upon patching and filling the neuron.
The authors also coupled their patching approach with single-cell electroporation of plasmid DNA in vivo. By placing an electrode filled with fluorescent dye and plasmid DNA encoding eGFP close to the soma and administering negative voltage pulses, individual neurons took up the dye and plasmids. Expression of the eGFP was determined at 24 and 48 hours afterwards. Identified single neurons in the cortex were electroporated; however, in more dense regions where neurons are closely packed, electroporation of individual cells may be more problematic.
These helpful tips can certainly facilitate the patching and imaging of individual neurons in vivo, an already challenging and daunting task. I would imagine some practice and fine-tuning would be needed to optimize the amount of positive pressure required to eject the dye into the extracellular space. Likewise, there is likely a fine line between the voltage needed for a successful electroporation and a stimulus that is damaging or lethal to the cell. Once these and other empirical issues are worked out, not seeing your neurons may be the way to go for targeted in vivo imaging.
There may be some useful variants of this approach that could be applied to neurodegenerative disease studies, such as AD. As any fluorescently labeled object can be imaged, in vivo two-photon imaging is not limited to cells. One thought is to fill the patch pipette with thioflavin S or a similar fluorescent plaque indicator and a conventional fluorescent dye, then shadowpatch neurons to measure effects of plaque deposits on neuronal morphology and physiology. Likewise, plaque markers can be injected systemically, and neurons in varying proximity to these labeled plaques can be shadowpatched using a fluorescent dye of a different emission wavelength. These plaque and filled neuron images can be overlaid, or, simultaneously acquired if multiple emission detections systems are in place. Analogous applications may involve apoptotic markers, Lewy body markers, or other pathogenic indicators. As long as it fluoresces, it can likely be detected with two-photon imaging.
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