Optical detection of neuron connectivity by random access two-photon microscopy

Shafeghat, Nasrin; Heidarinejad, Morteza; Motomura, Noboru; Nakamura, Hideki; Inoue, Takafumi

doi:10.1016/j.jneumeth.2016.01.023

Cited by 8 publications

(6 citation statements)

References 61 publications

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“…Analysis revealed both the number of bursts and spikes per bin had Poisson distribution and more than one burst or two spikes per bin were considered significant events, and that SI theta power per bin had lognormal distribution. Therefore, the relationship between fluctuation of SI theta power and spikes or bursts was analyzed using cross-correlation analysis as described previously 59–61 . Briefly,was calculated as previously described 60 .…”

Section: Methodsmentioning

confidence: 99%

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Thalamic Bursts Down-regulate Cortical Theta and Nociceptive Behavior

LeBlanc

Cross

Smith

et al. 2017

Sci Rep

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Section: Methodsmentioning

confidence: 99%

“…Thus, Z value was calculated for each Q ( t ) as follows:whereandZ(t) was calculated for each mouse, and then, the average of Z(t) and 95% confidence interval of Z(t) were calculated (see ref. 61). …”

Section: Methodsmentioning

confidence: 99%

Thalamic Bursts Down-regulate Cortical Theta and Nociceptive Behavior

LeBlanc

Cross

Smith

et al. 2017

Sci Rep

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“…For each optical trace, high-frequency noise was removed by low-pass Butterworth filter, and periods with shaky baseline were discarded. Then, APs were detected as fluorescence spikes in TI Workbench using a Schmitt trigger method 17 and kept exceeding SD for more than 2 ms, this data range was determined as a spike event (fAP) with peak amplitude and peak time as the maximum ΔF value within the period and time of the maximum ΔF, respectively. Noise level was defined as SD / Fbase(i), and SNR was calculated as SNR(i) = ΔF(i) / SD.…”

Section: Discussionmentioning

confidence: 99%

“…A custom-built random-access two-photon microscope was used to perform voltage recording as described 17 . In brief, a femtosecond pulsed Ti:Sapphire laser (output = 600-700 mW, Tsunami, Spectra Physics Japan, Tokyo, Japan) was tuned at 900 nm.…”

Section: Fast Random-access Two-photon Voltage Imaging In Acute Brainmentioning

confidence: 99%

Two-photon voltage imaging of spontaneous activity from multiple neurons reveals network activity in brain tissue

Chavarha

Kobayashi

et al. 2020

Preprint

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Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the structure and function of neuronal circuits. Using fluorescent genetically encoded voltage indicators (GEVI) would be especially desirable, as it would allow cell type-selectivity, longitudinal recordings, and further optical manipulations. By expressing the GEVI ASAP3 via in utero electroporation and rapidly imaging neurons in densely labelled tissues via random-access multi-photon microscopy, we achieve voltage recording of multiple neurons in brain slice with single-trial single-voxel resolution. This approach enables monitoring of subthreshold membrane potential changes and action potentials from multiple locations in soma and dendrites for tens of minutes. By optically recording spontaneous electrical activities in somatosensory cortex neurons, we provide evidence for the development of intralaminar horizontal connections in layer 2/3 with greater sensitivity than calcium imaging. Single-trial optical voltage recordings using ASAP3 thus enables the investigation of network connectivity at cellular resolution.indicators must reside within the plasma membrane to sense changes in transmembrane voltage 6 , the number of responding indicator molecules, and thereby changes in photon flux per voltage transient are significantly limited 7 . In addition to the unfavorable spatial constraint on voltage indicators, the transient nature of voltage also poses a problem. While GECIs can be sampled at slow frame rates of ~30 Hz to detect the long-lasting calcium fluctuations that follow action potentials, sub-millisecond sampling rates are required to detect the transient optical spikes of fast GEVIs in response to action potentials 7 . The higher sampling rate resulting fewer photons collected per frame leads to lower signal-to-noise ratios (SNRs). Excitation power can be increased to boost SNR, but at the price of greater photobleaching and phototoxicity 8 . Given these constraints, to detect fast voltage transients by voltage imaging, GEVIs must have high response amplitudes and fast response kinetics, and be bright and photostable. At the same time, methods to measure GEVI signals must maximize photon flux from the limited area of plasma membrane over the short durations of voltage transients, i.e. be both sensitive and fast.After years of intense engineering efforts, the latest generation of GEVIs now have sufficiently improved performance to be deployed in functional in vivo studies 7,9,10 . For this study we selected a recently reported ASAP3 for its fast kinetics (fast time constant of 0.94 ± 0.06 ms at physiological temperatures) and high responsivity (17.0 ± 0.8% per action potential (AP) in cultured neurons). Most importantly, ASAP3 maintains its responsivity to voltage changes under two-photon excitation, which enabled discrimination of individual spikes in fast-firing neurons in tissue 7 .Although GEVIs have been used to monitor electrical activity in neuronal tissue using widefield singlephoton mic...

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“…Elevations with peaks existing prior to the application of ATP were excluded from the analysis. For other analyses, data were analyzed using a TI workbench [23].…”

Section: -7 Ca 2+ Imagingmentioning

confidence: 99%

Dissection of local Ca2+ signals inside cytosol by ER-targeted Ca2+ indicator

Niwa

Sakuragi

Kobayashi

et al. 2016

Biochemical and Biophysical Research Communications

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Calcium (Ca(2+)) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca(2+) signals, generated by the coordination of Ca(2+) influx from the extracellular space and Ca(2+) release from the intracellular Ca(2+) store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca(2+) signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca(2+) signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca(2+) indicator (GECI) targeted to the outer ER membrane, can monitor Ca(2+) release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca(2+) imaging of C. elegans. We also found that the spontaneous Ca(2+) elevation in cultured astrocytes reported by OER-GCaMP6f showed a distinct spatiotemporal pattern from that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f); less frequent Ca(2+) signal was detected by OER-GCaMP6f, in spite of the fact that Ca(2+) release from the ER plays important roles in astrocytes. These findings suggest that targeting of GECIs to the ER outer membrane enables sensitive detection of Ca(2+) release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca(2+). Our results indicate that Ca(2+) imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca(2+) signals, by enabling dissection of Ca(2+) signals at subcellular resolution.

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Optical detection of neuron connectivity by random access two-photon microscopy

Abstract: This novel method reveals a new view of the functional structure of neuronal networks.

Cited by 8 publications

References 61 publications

Thalamic Bursts Down-regulate Cortical Theta and Nociceptive Behavior

Thalamic Bursts Down-regulate Cortical Theta and Nociceptive Behavior

Two-photon voltage imaging of spontaneous activity from multiple neurons reveals network activity in brain tissue

Dissection of local Ca2+ signals inside cytosol by ER-targeted Ca2+ indicator

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