Next-generation optical technologies for illuminating genetically targeted brain circuits

Abstract

Emerging technologies from optics, genetics, and bioengineering are being combined for studies of intact neural circuits. The rapid progression of such interdisciplinary "optogenetic" approaches has expanded capabilities for optical imaging and genetic targeting of specific cell types. Here we explore key recent advances that unite optical and genetic approaches, focusing on promising techniques that either allow novel studies of neural dynamics and behavior or provide fresh perspectives on classic model systems.

Figure 1.

Chronic and portable fluorescence microendoscopy. A, Photograph of three microendoscope probes, which are 1000, 500, and 350 μm in diameter. Each probe is a compound doublet gradient refractive index lens, comprising an endoscopic objective lens and a longer but weaker relay lens. The objective lenses are oriented to the right, and the relay lenses exhibit a dark coating. Minor ticks on the scale bar are 1 mm apart. Based on Jung et al. (2004). B, Images of CA1 hippocampal pyramidal cell bodies and proximal dendrites expressing YFP, acquired by two-photon microendoscopy in a live mouse. The mouse was of the YFP-H line, which expresses YFP under the control of the Thy1 promotor (Feng et al., 2000). On day 0, a guide tube was surgically implanted so that the microendoscope probe could be inserted repeatedly to the same tissue location just dorsal to CA1. The image acquired on day 239 after the initial surgery (left image, green pseudocolor) closely resembles that acquired on day 241 (middle image, red pseudocolor) as shown by the yellow portions in the merge of the two (right image). This indicates that the same cells have been visualized in each case. Scale bar, 10 μm. Data were taken from Jung et al. (2006). C, Photograph of the inner components of a portable fiber-optic two-photon microendoscopy device that is based on a microelectromechanical systems (MEMS) scanning mirror. A United States dime is shown for size comparison. The scanning mirror is microfabricated in silicon by photolithography methods and deflects light in two angular dimensions with a fast-axis scanning rate of 1.7 kHz (Piyawattanametha et al., 2006). Ultrashort pulses of light from a titanium:sapphire laser are delivered to the instrument by a hollow-core photonic band-gap fiber that virtually eliminates ultrashort pulse distortion (Gobel et al., 2004a). The illumination deflects off the microelectromechanical systems mirror, which scans the light in a raster pattern. The light then enters an optical assembly consisting of a dichroic microprism and four microlenses. Two-photon excited fluorescence is generated in the sample at the focal spot of the objective lens, passes back undeflected through the microprism, and is focused by a collection lens into a multimode optical fiber (not shown) that routes to a photodetector. This portable imaging system was designed by Flusberg et al. (2006). Two-photon imaging using the microelectromechanical systems scanning mirror is described by Piyawattanametha et al. (2006).

Figure 2.

Schematic description of SLICK mice. A, The construct used to generate SLICK transgenic mice consists of two copies of the Thy1 promoter placed back to back. One copy of Thy1 drives expression of a fluorescent protein (YFP), whereas the other expresses an inducible form of Cre recombinase (CreER^T2). B, Gene ablation with SLICK mice. Cre recombinase can delete DNA sequences located between loxP recognition sites (black triangles) and can therefore be used for conditional deletion of genes. C, Induction of transgene expression by SLICK mice. To generate an inducible line of transgenic mice, a transcriptional stop cassette (STOP) is flanked by loxP sites and placed in front of the transgene. When activated, Cre recombinase in SLICK mice can delete the STOP cassette and induce transgene expression. D, Tamoxifen-induced CreER-mediated recombination in SLICK mice. SLICK mice were crossed to a line of Cre reporter mice, Rosa26-loxP-STOP-loxP-LacZ, in which the lacZ expression depends on Cre-mediated excision of transcriptional STOP cassette. When the SLICK/Rosa26-loxP-STOP-loxP-LacZ mice were treated with tamoxifen, which activates CreER, the expression of LacZ is efficiently induced in YFP-labeled neurons (arrowheads).

Figure 3.

Precisely timed, light-driven synaptic transmission within intact mammalian brain tissue. A, Left, ChR2–EYFP expression in an acute hippocampal slice. Data courtesy of Zhang et al. (2006) and Wang et al. (2006). The recording pipette shown is filled with an intracellular solution containing Alexa Fluor 594 (10 μg/ml). Middle, The recorded ChR2–EYFP-negative cell filled with Alexa Fluor 594. Right, Combined image of the recorded cell (red) and mossy fiber projections (green). Scale bars: left, 200 μm; middle and right, 20 μm. B, Excitatory synaptic transmission driven by 5, 10, and 20 Hz repeated light pulses (each blue dash represents one 15 ms light flash). C, The selective glutamatergic transmission blocker CNQX (20 μm) abolishes the synaptic response. Calibration is the same as in B. D, A single postsynaptic event before (filled square in B) and after (open square in C) CNQX application in expanded timescale. E, The 10 Hz synaptic responses evoked via 5, 2, and 1 ms light flashes. All traces were collected without the addition of exogenous all-trans-retinal. This cofactor independence and the demonstrated temporal precision offer considerable utility for other domains of neuroscience and neuroengineering, with potential applications ranging from stem cell engineering (Stroh et al., 2006), to high-throughput optical screening of drugs, to high-speed optics-based neural interfaces for nervous tissue repair (Bi et al., 2006).