Anterograde monosynaptic transneuronal tracers derived from herpes simplex virus 1 strain H129.

BACKGROUND: Herpes simplex virus type 1 strain 129 (H129) has represented a promising anterograde neuronal circuit tracing tool, which complements the existing retrograde tracers. However, the current H129 derived tracers are multisynaptic, neither bright enough to label the details of neurons nor capable of determining direct projection targets as monosynaptic tracer. METHODS: Based on the bacterial artificial chromosome of H129, we have generated a serial of recombinant viruses for neuronal circuit tracing. Among them, H129-G4 was obtained by inserting binary tandemly connected GFP cassettes into the H129 genome, and H129-ΔTK-tdT was obtained by deleting the thymidine kinase (TK) gene and adding tdTomato coding gene to the H129 genome. Then the obtained viral tracers were tested in vitro and in vivo for the tracing capacity. RESULTS: H129-G4 is capable of transmitting through multiple synapses, labeling the neurons by green florescent protein, and visualizing the morphological details of the labeled neurons. H129-ΔTK-tdT neither replicates nor spreads in neurons alone, but transmits to and labels the postsynaptic neurons with tdTomato in the presence of complementary expressed TK from a helper virus. H129-ΔTK-tdT is also capable to map the direct projectome of the specific neuron type in the given brain regions in Cre transgenic mice. In the tested brain regions where circuits are well known, the H129-ΔTK-tdT tracing patterns are consistent with the previous results. CONCLUSIONS: With the assistance of the helper virus complimentarily expressing TK, H129-ΔTK-tdT replicates in the initially infected neuron, transmits anterogradely through one synapse, and labeled the postsynaptic neurons with tdTomato. The H129-ΔTK-tdT anterograde monosynaptic tracing system offers a useful tool for mapping the direct output in neuronal circuitry. H129-G4 is an anterograde multisynaptic tracer with a labeling signal strong enough to display the details of neuron morphology.

Automated, highly reproducible, wide-field, light-based cortical mapping method using a commercial stereo microscope and its applications.

We introduce a more flexible optogenetics-based mapping system attached on a stereo microscope, which offers automatic light stimulation to individual regions of interest in the cortex that expresses light-activated channelrhodopsin-2 in vivo. Combining simultaneous recording of electromyography from specific forelimb muscles, we demonstrate that this system offers much better efficiency and precision in mapping distinct domains for controlling limb muscles in the mouse motor cortex. Furthermore, the compact and modular design of the system also yields a simple and flexible implementation to different commercial stereo microscopes, and thus could be widely used among laboratories.

[Research report of experimental database establishment of digitized virtual Chinese No.1 female].

OBJECTIVE: To establish digitized virtual Chinese No.1 female (VCH-F1) image database. METHODS: A 19 years old female cadaver was scanned by CT, MRI, and perfused with red filling material through formal artery before freezing and em- bedding. The whole body was cut by JZ1500A vertical milling machine with a 0.2 mm inter-spacing. All the images was produced by Fuji FinePix S2 Pro camera. RESULTS: The body index of VCH-F1 was 94%. We cut 8 556 sections of the whole body, and each image was 17.5 MB in size and the whole database reached 149.7 GB. We have totally 6 versions of the database for different applications. CONCLUSIONS: Compared with other databases, VCH-F1 has good representation of the Chinese body shape, colorful filling material in blood vessels providing enough information for future registration and segmentation. Vertical embedding and cutting helped to retain normal human physiological posture, and the image quality and operation efficiency were improved by using various techniques such as one-time freezing and fixation, double-temperature icehouse, large-diameter milling disc and whole body cutting.

Sustained depolarization-induced propagation of [Ca2+]i oscillations in cultured DRG neurons: the involvement of extracellular ATP and P2Y receptor activation.

Recently emerging evidence implicates a number of neuroactive substances and their receptors in mediating complex cell-to-cell communications in the ganglia. In the present study, we characterized the nonsynaptic chemical coupling mediated by extracellular ATP in dorsal root ganglia (DRG) neuron cultures by using the real time imaging of ATP, whole-cell patch clamping, in conjunction with confocal calcium imaging. Sustained depolarization by electrical stimulation evoked intracellular Ca2+ concentrations ([Ca2+]i) oscillations in individual DRG neurons, and subsequent ATP-dependent propagation [Ca2+]i oscillations to surrounding non-stimulated neighbors. [Ca2+]i oscillations were suppressed by inositol-1,4,5-trisphosphate (IP3) receptor antagonist 2-APB, but not ryanodine. The propagation of [Ca2+]i oscillations was prevented by the presence of the ATP-degrading enzyme, apyrase, and completely abolished by the blockase of G protein-coupled purinergic receptors-PLC-IP3 pathway with suramin, U73122 or 2-APB. In parallel, sustained depolarization elicited robust ATP release and diffusion from the stimulation site. Moreover, exogenous application of ATP to DRG cultures in large concentration elicits the [Ca2+]i oscillations in most neurons. Taken together, this data demonstrates that sustained membrane depolarization elicited ATP release, acting through a highly sensitive P2Y receptors/IP3-mediated signaling pathway to mediate the propagation of intercellular Ca2+ signaling, which suggest a novel signaling pathway for neuronal communication in DRG.

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy.

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.