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RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines.

Fino E, Araya R, Peterka DS, Salierno M, Etchenique R, Yuste R - Front Neural Circuits (2009)

Bottom Line: RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation.Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics.RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biological Sciences, Columbia University New York, NY, USA.

ABSTRACT
We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. It has high quantum efficiency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, two-photon RuBi-Glutamate uncaging can also be used to depolarize and fire pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

No MeSH data available.


Related in: MedlinePlus

Use of RuBi-Glutamate uncaging to optically activate dendritic spines. (A) Layer 2/3 neuron loaded with Alexa-594 to detect dendritic spines. (B) Higher resolution image of a dendritic spine selected for uncaging. Red dots indicate the sites of uncaging. (C) Plot of the XY spatial resolution of uncaging from the spine shown in (B) (Inset, traces corresponded to averages of 15 uncaging potentials at the different locations [1–4; red dots in (B)]. (D) Spatial axial resolution of uncaging. Z-stack of images from the stimulated spine. Traces corresponded to averages of 15 uncaging potentials of its corresponding image stack. Red dots indicate the site of laser beam parking, not the actual size of the beam profile. (E) Plot of the axial resolution at the three different locations showed in (D).
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Figure 4: Use of RuBi-Glutamate uncaging to optically activate dendritic spines. (A) Layer 2/3 neuron loaded with Alexa-594 to detect dendritic spines. (B) Higher resolution image of a dendritic spine selected for uncaging. Red dots indicate the sites of uncaging. (C) Plot of the XY spatial resolution of uncaging from the spine shown in (B) (Inset, traces corresponded to averages of 15 uncaging potentials at the different locations [1–4; red dots in (B)]. (D) Spatial axial resolution of uncaging. Z-stack of images from the stimulated spine. Traces corresponded to averages of 15 uncaging potentials of its corresponding image stack. Red dots indicate the site of laser beam parking, not the actual size of the beam profile. (E) Plot of the axial resolution at the three different locations showed in (D).

Mentions: We then tested the use of two-photon uncaging of RuBi-Glutamate for activating dendritic spines, one of the most useful applications of two-photon glutamate uncaging (Araya et al., 2006; Carter and Sabatini, 2004; Gasparini and Magee, 2006; Matsuzaki et al., 2004; Sobczyk et al., 2005). For these experiments, we performed whole-cell recordings from pyramidal neurons and filled them with Alexa 594 to optimally visualize their dendritic spines. We then bathed the slice in RuBi-Glutamate (300 μM) and directed a pulsed laser at the neuropil adjacent to a chosen spine (Figures 4A,B). With 4 ms laser pulses, we triggered reliable depolarizations in the neuron (see traces in Figures 4C,D). These responses were similar in amplitude to events recorded after two-photon uncaging of MNI-glutamate (see Table 1). However, the RuBi-Glutamate generated neuronal responses had a rate of rise nearly twice as fast as those generated from uncaging MNI-glutamate (p < 0.001, n = 65 RuBi-Glutamate uncaging events from four spines vs. n = 127 MNI-glutamate uncaging events from nine spines). In addition, 10–90% rise times and 37% decay time kinetics were faster then those observed with uncaging of MNI-glutamate (p = 0.001 and p < 0.001, respectively, Table 1), and more closely resemble the kinetics of spontaneous mEPSP (Table 1, see Materials and Methods for detection of mEPSPs).


RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines.

Fino E, Araya R, Peterka DS, Salierno M, Etchenique R, Yuste R - Front Neural Circuits (2009)

Use of RuBi-Glutamate uncaging to optically activate dendritic spines. (A) Layer 2/3 neuron loaded with Alexa-594 to detect dendritic spines. (B) Higher resolution image of a dendritic spine selected for uncaging. Red dots indicate the sites of uncaging. (C) Plot of the XY spatial resolution of uncaging from the spine shown in (B) (Inset, traces corresponded to averages of 15 uncaging potentials at the different locations [1–4; red dots in (B)]. (D) Spatial axial resolution of uncaging. Z-stack of images from the stimulated spine. Traces corresponded to averages of 15 uncaging potentials of its corresponding image stack. Red dots indicate the site of laser beam parking, not the actual size of the beam profile. (E) Plot of the axial resolution at the three different locations showed in (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691658&req=5

Figure 4: Use of RuBi-Glutamate uncaging to optically activate dendritic spines. (A) Layer 2/3 neuron loaded with Alexa-594 to detect dendritic spines. (B) Higher resolution image of a dendritic spine selected for uncaging. Red dots indicate the sites of uncaging. (C) Plot of the XY spatial resolution of uncaging from the spine shown in (B) (Inset, traces corresponded to averages of 15 uncaging potentials at the different locations [1–4; red dots in (B)]. (D) Spatial axial resolution of uncaging. Z-stack of images from the stimulated spine. Traces corresponded to averages of 15 uncaging potentials of its corresponding image stack. Red dots indicate the site of laser beam parking, not the actual size of the beam profile. (E) Plot of the axial resolution at the three different locations showed in (D).
Mentions: We then tested the use of two-photon uncaging of RuBi-Glutamate for activating dendritic spines, one of the most useful applications of two-photon glutamate uncaging (Araya et al., 2006; Carter and Sabatini, 2004; Gasparini and Magee, 2006; Matsuzaki et al., 2004; Sobczyk et al., 2005). For these experiments, we performed whole-cell recordings from pyramidal neurons and filled them with Alexa 594 to optimally visualize their dendritic spines. We then bathed the slice in RuBi-Glutamate (300 μM) and directed a pulsed laser at the neuropil adjacent to a chosen spine (Figures 4A,B). With 4 ms laser pulses, we triggered reliable depolarizations in the neuron (see traces in Figures 4C,D). These responses were similar in amplitude to events recorded after two-photon uncaging of MNI-glutamate (see Table 1). However, the RuBi-Glutamate generated neuronal responses had a rate of rise nearly twice as fast as those generated from uncaging MNI-glutamate (p < 0.001, n = 65 RuBi-Glutamate uncaging events from four spines vs. n = 127 MNI-glutamate uncaging events from nine spines). In addition, 10–90% rise times and 37% decay time kinetics were faster then those observed with uncaging of MNI-glutamate (p = 0.001 and p < 0.001, respectively, Table 1), and more closely resemble the kinetics of spontaneous mEPSP (Table 1, see Materials and Methods for detection of mEPSPs).

Bottom Line: RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation.Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics.RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biological Sciences, Columbia University New York, NY, USA.

ABSTRACT
We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. It has high quantum efficiency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, two-photon RuBi-Glutamate uncaging can also be used to depolarize and fire pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

No MeSH data available.


Related in: MedlinePlus