research highlights

NEUROSCIENCE All-optical electrophysiology in behaving animals

Three different optical strategies enable and a calcium sensor that can be spectrally the manipulation and imaging of neural separated under two-photon conditions. activity in awake mice. Tank and his colleagues used a different A major goal in neuroscience is the strategy to activate neuronal populations analysis of neural circuits involved in (Rickgauer et al., 2014). They illuminated behavior. The optogenetic manipulation multiple neurons near-simultaneously of defined neural populations and sub- by focusing a two-photon laser to a spot sequent imaging of neural activity using with the size of a neural soma and mov- calcium sensors can help address this goal. ing between neural targets with the help of However, several challenges to perform- fast scanning mirrors. Both groups applied ing all-optical electrophysiology in vivo their methods to awake, head-fixed mice must be overcome. Valentina Emiliani at running on a treadmill. the Université Paris Descartes thinks that “Advantages of these methods compared these challenges are related to the spectral to our method are the possibility of work- overlap of optogenetic actuators and calci- ing [at] a greater depth and a better axial um indicators as well as the requirement for resolution allowed by the use of two-photon simultaneous photostimulation of multiple excitation, …[but they] are not compatible targets with cellular resolution. Moreover, with the use on freely behaving animals, genetic tools to express optogenetic actua- which was the main goal of our work,” says tors are often not specific enough to target Emiliani. The method established by her Peeking into a mouse brain with a fiberscope. defined populations of neurons. and her coworkers takes advantage of com- Figure from Szabo et al., Elsevier. Optical methods to refine the pattern puter-generated holography techniques and

Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature of optogenetic activation may overcome spatial light modulators to establish defined Depending on their experimental needs, the latter problems, whereas new optoge- photostimulation patterns (Szabo et al., researchers interested in all-optical manipu- netic tools, calcium indicators or photo- 2014). These patterns are then delivered to lation and imaging of neural activity in vivo stimulation protocols may address the for- the brain area of interest with a fiberscope, now have multiple options to choose from. npg mer issue. The groups of Emiliani, David which is affixed to the mouse skull and is The two photon–based approaches by the Tank at Princeton University and Michael also used to image calcium signals. Because Tank and Häusser groups permit the analy- Häusser at University College London have they used one-photon illumination as well sis of neural circuits in head-fixed mice, recently independently developed strategies as actuators and sensors with overlapping with good axial resolution and at some to simultaneously activate defined neurons spectral sensitivities, they optimized the depth: advantages that are inherent to two- with high spatial and temporal resolution imaging conditions to minimize optogenetic photon microscopy. Emiliani’s approach, in behaving mice. activation during imaging. However, it may on the other hand, is more suitable to the For targeting individual neurons, the be possible to use red-shifted optogenetic analysis of the neural circuitry underlying illumination of optogenetic actuators has actuators, which could eliminate spectral behaviors in unrestrained animals. to be targeted to defined areas with cellu- overlap and also be helpful in improving Nina Vogt lar resolution. Häusser and his colleagues imaging speed and the signal-to-noise ratio. used a spatial light modulator to split a The fiberscope allows a variety of behav- RESEARCH PAPERS Packer, A.M. et al. Simultaneous all-optical laser beam into several individual beams iors to be analyzed with all-optical elec- manipulation and recording of neural circuit activity and scan the neurons of interest in a spi- trophysiology because the animals do not with cellular resolution in vivo. Nat. Methods 12, ral fashion (Packer et al., 2015). With this need to be restrained. Emiliani thinks that 140–146 (2015). approach, the researchers could activate “performing experiments in freely behav- Rickgauer, J.P. et al. Simultaneous cellular-resolution multiple neurons at the same time while ing animals would not only facilitate the optical perturbation and imaging of place cell firing fields. Nat. Neurosci. 17, 1816–1824 (2014). monitoring the activity of the targeted and experimental procedure … but would also other neurons with wide-field scanning provide a more ‘physiological’, less stressful Szabo, V. et al. Spatially selective holographic photoactivation and functional fluorescence imaging at a different wavelength. This was made environment for the animal (compared to a in freely behaving mice with a fiberscope. Neuron possible by using an optogenetic activator head-fixed situation).” 84, 1157–1169 (2014).

nature methods | VOL.12 NO.2 | FEBRUARY 2015 | 101