Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

O’Herron, Philip; Shen, Zhiming; Lu, Zhongyang; Schramm, Adrien E.; Levy, Manuel; Kara, Prakash

doi:10.3791/50025

Cited by 17 publications

(23 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Institutional Animal Care and Use Committee of the Medical University of South Carolina approved all animal experiments. Detailed experimental setup, anesthesia, surgical procedures, and drifting grating visual stimulation protocols are described elsewhere (O'Herron et al, 2012; Shen et al, 2012). Briefly, rats were anesthetized with intraperitoneal injection of a fentanyl cocktail (0.04–0.06 mg kg −1 fentanyl citrate, 3.75–6.25 mg kg −1 midazolam, and 0.19–0.31 mg kg −1 dexmedetomidine).…”

Section: Methodsmentioning

confidence: 99%

“…These RBCs appear as dark bands or streaks in space-time images. In both rats and cats, movements of the brain were minimized by sealing the craniotomy with agarose and a coverglass (Levy et al, 2012; O'Herron et al, 2012). In cats, a lumbar suspension and pneumothorax were needed to further reduce the brain movements of cardiac and respiratory origin.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Improved blood velocity measurements with a hybrid image filtering and iterative Radon transform algorithm

Chhatbar

Kara

2013

Front. Neurosci.

Self Cite

View full text Add to dashboard Cite

Neural activity leads to hemodynamic changes which can be detected by functional magnetic resonance imaging (fMRI). The determination of blood flow changes in individual vessels is an important aspect of understanding these hemodynamic signals. Blood flow can be calculated from the measurements of vessel diameter and blood velocity. When using line-scan imaging, the movement of blood in the vessel leads to streaks in space-time images, where streak angle is a function of the blood velocity. A variety of methods have been proposed to determine blood velocity from such space-time image sequences. Of these, the Radon transform is relatively easy to implement and has fast data processing. However, the precision of the velocity measurements is dependent on the number of Radon transforms performed, which creates a trade-off between the processing speed and measurement precision. In addition, factors like image contrast, imaging depth, image acquisition speed, and movement artifacts especially in large mammals, can potentially lead to data acquisition that results in erroneous velocity measurements. Here we show that pre-processing the data with a Sobel filter and iterative application of Radon transforms address these issues and provide more accurate blood velocity measurements. Improved signal quality of the image as a result of Sobel filtering increases the accuracy and the iterative Radon transform offers both increased precision and an order of magnitude faster implementation of velocity measurements. This algorithm does not use a priori knowledge of angle information and therefore is sensitive to sudden changes in blood flow. It can be applied on any set of space-time images with red blood cell (RBC) streaks, commonly acquired through line-scan imaging or reconstructed from full-frame, time-lapse images of the vasculature.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Improved blood velocity measurements with a hybrid image filtering and iterative Radon transform algorithm

Chhatbar

Kara

2013

Front. Neurosci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…All procedures were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina and were based on those previously published (Kara and Boyd, 2009; O'Herron et al, 2012; Shen et al, 2012). Anesthesia was induced by IM injection of ketamine (20 mg/kg) and acepromazine (0.2 mg/kg) and maintained by isofluorane (1–2% during surgery, 0.5–1% during imaging).…”

Section: Methodsmentioning

confidence: 99%

“…Our procedure was adapted from Stosiek et al (2003) and described in detail elsewhere (Kara and Boyd, 2009; O'Herron et al, 2012; Shen et al, 2012). Briefly, 50 μg of OGB-1 AM (Life Technologies) were dissolved in 4 μl DMSO with 20% pluronic acid F-127 (Sigma).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Strategies for mapping synaptic inputs on dendrites in vivo by combining two-photon microscopy, sharp intracellular recording, and pharmacology

Levy¹,

Schramm²,

Kara³

2012

Front. Neural Circuits

Self Cite

View full text Add to dashboard Cite

Uncovering the functional properties of individual synaptic inputs on single neurons is critical for understanding the computational role of synapses and dendrites. Previous studies combined whole-cell patch recording to load neurons with a fluorescent calcium indicator and two-photon imaging to map subcellular changes in fluorescence upon sensory stimulation. By hyperpolarizing the neuron below spike threshold, the patch electrode ensured that changes in fluorescence associated with synaptic events were isolated from those caused by back-propagating action potentials. This technique holds promise for determining whether the existence of unique cortical feature maps across different species may be associated with distinct wiring diagrams. However, the use of whole-cell patch for mapping inputs on dendrites is challenging in large mammals, due to brain pulsations and the accumulation of fluorescent dye in the extracellular milieu. Alternatively, sharp intracellular electrodes have been used to label neurons with fluorescent dyes, but the current passing capabilities of these high impedance electrodes may be insufficient to prevent spiking. In this study, we tested whether sharp electrode recording is suitable for mapping functional inputs on dendrites in the cat visual cortex. We compared three different strategies for suppressing visually evoked spikes: (1) hyperpolarization by intracellular current injection, (2) pharmacological blockade of voltage-gated sodium channels by intracellular QX-314, and (3) GABA iontophoresis from a perisomatic electrode glued to the intracellular electrode. We found that functional inputs on dendrites could be successfully imaged using all three strategies. However, the best method for preventing spikes was GABA iontophoresis with low currents (5–10 nA), which minimally affected the local circuit. Our methods advance the possibility of determining functional connectivity in preparations where whole-cell patch may be impractical.

show abstract

Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame

Wakao

Oguma

Kushida

et al. 2022

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

Stem cells undergo cytokine-driven differentiation, but this process often takes longer than several weeks to complete. A novel mechanism for somatic stem cell differentiation via phagocytosing ‘model cells’ (apoptotic differentiated cells) was found to require only a short time frame. Pluripotent-like Muse cells, multipotent mesenchymal stem cells (MSCs), and neural stem cells (NSCs) phagocytosed apoptotic differentiated cells via different phagocytic receptor subsets than macrophages. The phagocytosed-differentiated cell-derived contents (e.g., transcription factors) were quickly released into the cytoplasm, translocated into the nucleus, and bound to promoter regions of the stem cell genomes. Within 24 ~ 36 h, the cells expressed lineage-specific markers corresponding to the phagocytosed-differentiated cells, both in vitro and in vivo. At 1 week, the gene expression profiles were similar to those of the authentic differentiated cells and expressed functional markers. Differentiation was limited to the inherent potential of each cell line: triploblastic-, adipogenic-/chondrogenic-, and neural-lineages in Muse cells, MSCs, and NSCs, respectively. Disruption of phagocytosis, either by phagocytic receptor inhibition via small interfering RNA or annexin V treatment, impeded differentiation in vitro and in vivo. Together, our findings uncovered a simple mechanism by which differentiation-directing factors are directly transferred to somatic stem cells by phagocytosing apoptotic differentiated cells to trigger their rapid differentiation into the target cell lineage.

show abstract

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

Cited by 17 publications

References 25 publications

Improved blood velocity measurements with a hybrid image filtering and iterative Radon transform algorithm

Improved blood velocity measurements with a hybrid image filtering and iterative Radon transform algorithm

Strategies for mapping synaptic inputs on dendrites in vivo by combining two-photon microscopy, sharp intracellular recording, and pharmacology

Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame

Contact Info

Product

Resources

About