Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin

BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5-10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5-10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state

Light-evoked Somatosensory Perception of Transgenic Rats That Express Channelrhodopsin-2 in Dorsal Root Ganglion Cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain

The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity

Natural anion channelrhodopsins (ACRs) discovered in the cryptophyte alga Guillardia theta generate large hyperpolarizing currents at membrane potentials above the Nernst equilibrium potential for Cl(−) and thus can be used as efficient inhibitory tools for optogenetics. We have identified and characterized new ACR homologs in different cryptophyte species, showing that all of them are anion-selective, and thus expanded this protein family to 20 functionally confirmed members. Sequence comparison of natural ACRs and engineered Cl(−)-conducting mutants of cation channelrhodopsins (CCRs) showed radical differences in their anion selectivity filters. In particular, the Glu90 residue in channelrhodopsin 2, which needed to be mutated to a neutral or alkaline residue to confer anion selectivity to CCRs, is nevertheless conserved in all of the ACRs identified. The new ACRs showed a large variation of the amplitude, kinetics, and spectral sensitivity of their photocurrents. A notable variant, designated “ZipACR”, is particularly promising for inhibitory optogenetics because of its combination of larger current amplitudes than those of previously reported ACRs and an unprecedentedly fast conductance cycle (current half-decay time 2–4 ms depending on voltage). ZipACR expressed in cultured mouse hippocampal neurons enabled precise photoinhibition of individual spikes in trains of up to 50 Hz frequency

Acidification slows algal movement

Ocean acidification is changing the productivity and composition of phytoplankton communities at the base of the aquatic food web. Now a study shows that acidification impairs the swimming ability of flagellated microalgae, suggesting that their capacity to survive is threatened in a high CO2 world