Ca2+ imaging of identifiable neurons labeled by electroporation in insect brains

Fujiwara, Terufumi; Kazawa, Tomoki; Haupt, Stephan Shuichi; Kanzaki, Ryohei

doi:10.1097/wnr.0b013e32832e7d93

Cited by 15 publications

(15 citation statements)

References 18 publications

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“…Electroporation is a widely used technique which has been used both in vitro and in vivo for rapid delivery of large macromolecules in the cell population such as DNA and siRNA (Boudes, Pieraut et al, 2008; Chu, Hayakawa et al, 1987; Haas, Sin et al, 2001; Potter, Weir et al, 1984; Tabata and Nakajima, 2001), fluorescent dyes (Lodovichi, Belluscio et al, 2003; Pinault, 1996), and calcium indicators (Bonnot, Mentis et al, 2005; Fujiwara, Kazawa et al, 2009; Nagayama, Zeng et al, 2007). However, as described previously, they are limited in their ability to target specific populations and require cumbersome and complex apparatuses.…”

Section: Discussion (1045 Words)mentioning

confidence: 99%

A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

Hovis

Padmanabhan

Urban

2010

Journal of Neuroscience Methods

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Since Cajal's early drawings, the characterization of neuronal architecture has been paramount in understanding neuronal function. With the development of electrophysiological techniques that provide unprecedented access to the physiology of these cells, experimental questions of neuronal function have also become more tractable. Fluorescent tracers that can label the anatomy of individual or populations of neurons have opened the door to linking anatomy with physiology. Experimentally however, current techniques for bulk labeling of cells in vitro often affect neuronal function creating a barrier for exploring structure-function questions. Here we describe a new technique for highly localized electroporation within a cell or cell population that enables the introduction of membrane impermeable charged dyes including dextran-conjugated fluorophores, hydrazide tracers, and calcium indicator dyes in vitro. We demonstrate that this technique is highly versatile, allowing for labeling of large or small areas of tissue, allowing for the investigation of both cellular morphology and physiological activity in identified neuronal circuits in acute brain slices. Furthermore, this approach allows subsequent targeted whole-cell patch recording based on well-defined connectivity as well as assessment of physiological activity in targeted circuits on a fast time scale.

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Section: Discussion (1045 Words)mentioning

confidence: 99%

A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

Hovis

Padmanabhan

Urban

2010

Journal of Neuroscience Methods

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“…Under visual control through a stereo microscope, we inserted the micro-electrode superficially into the medulla and applied negative current pulses (8 μA amplitude, 30 ms pulses with 270 ms intervals) for 45 s with a high-gain micro-electrode amplifier (VF-1800, Bio-Logic, Claix, France) to label the neurons by electroporation (method modified after Fujiwara et al, 2009). In some animals, a second and third injection site was stained in the same manner.…”

Section: Methodsmentioning

confidence: 99%

Neuronal representation of visual motion and orientation in the fly medulla

Spalthoff

Gerdes

Kurtz

2012

Front. Neural Circuits

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In insects, the first extraction of motion and direction clues from local brightness modulations is thought to take place in the medulla. However, whether and how these computations are represented in the medulla stills remain widely unknown, because electrical recording of the neurons in the medulla is difficult. As an effort to overcome this difficulty, we employed local electroporation in vivo in the medulla of the blowfly (Calliphora vicina) to stain small ensembles of neurons with a calcium-sensitive dye. We studied the responses of these neuronal ensembles to spatial and temporal brightness modulations and found selectivity for grating orientation. In contrast, the responses to the two opposite directions of motion of a grating with the same orientation were similar in magnitude, indicating that strong directional selectivity is either not present in the types of neurons covered by our data set, or that direction-selective signals are too closely spaced to be distinguished by our calcium imaging. The calcium responses also showed a bell-shaped dependency on the temporal frequency of drifting gratings, with an optimum higher than that observed in one of the subsequent processing stages, i.e., the lobula plate. Medulla responses were elicited by on- as well as off-stimuli with some spatial heterogeneity in the sensitivity for “on” and “off”, and in the polarity of the responses. Medulla neurons thus show similarities to some established principles of motion and edge detection in the vertebrate visual system.

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“…Microruby (Invitrogen©, Life Technologies, Grand Island, NY) fluorescent dye (4% in distilled water) was introduced into the MGC using an electroporation method as described in (Fujiwara et al 2009) but with some modifications. In short, a stimulus isolator (World Precision Instruments, Sarasota, Florida, USA) was used to generate about −80 Volts DC potential, which was applied to patch-type electrodes with 5–20 MΩ resistance.…”

Section: Methodsmentioning

confidence: 99%

Responses of protocerebral neurons in Manduca sexta to sex-pheromone mixtures

2013

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Male Manduca sexta moths are attracted to a mixture of two components of the female's sex pheromone at the natural concentration ratio. Deviation from this ratio results in reduced attraction. Projection neurons innervating prominent male-specific glomeruli in the male's antennal lobe produce maximal synchronized spiking activity in response to synthetic mixtures of the two components centering around the natural ratio, suggesting that behaviorally effective mixture ratios are encoded by synchronous neuronal activity. We investigated the physiological activity and morphology of downstream protocerebral neurons that responded to antennal stimulation with single pheromone components and their mixtures at various concentration ratios. Among the tested neurons, only a few gave stronger responses to the mixture at the natural ratio whereas most did not distinguish among the mixtures that were tested. We also found that the population response distinguished among the two pheromone components and their mixtures, prior to the peak population response. This observation is consistent with our previous finding that synchronous firing of antennal-lobe projection neurons reaches its maximum before the firing rate reaches its peak. Moreover, the response patterns of protocerebral neurons are diverse, suggesting that the representation of olfactory stimuli at the level of protocerebrum is complex.

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Ca2+ imaging of identifiable neurons labeled by electroporation in insect brains

Cited by 15 publications

References 18 publications

A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

Neuronal representation of visual motion and orientation in the fly medulla

Responses of protocerebral neurons in Manduca sexta to sex-pheromone mixtures

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