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1.
Sci Rep ; 9(1): 3917, 2019 03 08.
Article in English | MEDLINE | ID: mdl-30850696

ABSTRACT

We investigated the relationship between whisker mechanoreceptive inputs and the neural responses to optical stimulation in layer 2/upper 3 (L2/U3) of the barrel cortex using optogenetics since, ideally, we should investigate interactions among inputs with spatiotemporal acuity. Sixteen whisker points of a transgenic rat (W-TChR2V4), that expresses channelrhodopsin 2 (ChR2)-Venus conjugate (ChR2V) in the peripheral nerve endings surrounding the whisker follicles, were respectively connected one-by-one with 16 LED-coupled optical fibres, which illuminated the targets according to a certain pattern in order to evaluate interactions among the inputs in L2/U3. We found that the individual L2/U3 neurons frequently received excitatory inputs from multiple whiskers that were arrayed in a row. Although the interactions among major afferent inputs (MAIs) were negligible, negative interactions with the surrounding inputs suggest that the afferent inputs were integrated in the cortical networks to enhance the contrast of an array to its surroundings. With its simplicity, reproducibility and spatiotemporal acuity, the optogenetic approach would provide an alternative way to understand the principles of afferent integration in the cortex and should complement knowledge obtained by experiments using more natural stimulations.


Subject(s)
Optogenetics/methods , Somatosensory Cortex/physiology , Animals , Female , Light , Male , Mechanoreceptors/cytology , Mechanoreceptors/physiology , Neurons, Afferent/cytology , Neurons, Afferent/physiology , Optogenetics/instrumentation , Physical Stimulation , Rats , Rats, Transgenic , Somatosensory Cortex/cytology , Vibrissae/innervation
2.
J Physiol Sci ; 69(1): 65-77, 2019 Jan.
Article in English | MEDLINE | ID: mdl-29761270

ABSTRACT

To investigate how the functional architecture is organized in layer 5 (L5) of the somatosensory cortex of a mouse in vivo, the input-output relationship was investigated using an all-optical approach. The neural activity in L5 was optically recorded using a Ca2+ sensor, R-CaMP2, through a microprism inserted in the cortex under two-photon microscopy, while the L5 was regionally excited using optogenetics. The excitability was spread around the blue-light irradiated region, but the horizontal propagation was limited to within a certain distance (λ < 130 µm from the center of the illumination spot). When two regions were photostimulated with a short interval, the excitability of each cluster was reduced. Therefore, a column-like architecture had functionally emerged with reciprocal inhibition through a minimal number of synaptic relays. This could generate a synchronous output from a region of L5 with simultaneous enhancement of the signal-to-noise ratio by silencing of the neighboring regions.


Subject(s)
Somatosensory Cortex/physiology , Animals , Calcium/metabolism , Female , Mice , Mice, Inbred C57BL , Optogenetics/methods , Somatosensory Cortex/metabolism
3.
Sci Rep ; 8(1): 5435, 2018 04 03.
Article in English | MEDLINE | ID: mdl-29615713

ABSTRACT

Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.


Subject(s)
Opsins/genetics , Optogenetics/methods , Animals , Gene Expression , Genes, Reporter/genetics , Rats , Rats, Transgenic
4.
PLoS One ; 11(5): e0155687, 2016.
Article in English | MEDLINE | ID: mdl-27195805

ABSTRACT

Despite the strength of the Cre/loxP recombination system in animal models, its application in rats trails that in mice because of the lack of relevant reporter strains. Here, we generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato) in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualizes neurons including their projection fibers and spines without any histological enhancement. In addition, a transgenic rat line (FLAME) that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic Cre mRNA into fertilized ova. Our rat reporter system will facilitate connectome studies as well as the visualization of the fine structures of genetically identified cells for long periods both in vivo and ex vivo. Furthermore, FLAME is an ideal model for organ transplantation research owing to improved traceability of cells/tissues.


Subject(s)
Luminescent Proteins/genetics , Rats, Transgenic , Animals , Cell Lineage , Chromosomes, Artificial, Bacterial , Dependovirus , Electroporation , Erythrocytes/cytology , Female , Fertilization , Fibroblasts/metabolism , Genes, Reporter , Hippocampus/metabolism , Imaging, Three-Dimensional , Integrases , Macrophages/metabolism , Male , RNA, Messenger/metabolism , Rats , Transgenes , Red Fluorescent Protein
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