Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice

Abstract

Electrical microstimulation can establish causal links between the activity of groups of neurons and perceptual and cognitive functions [1][2][3][4][5][6] . However, the number and identities of neurons microstimulated, as well as the number of action potentials evoked, are difficult to ascertain 7,8 . To address these issues we introduced the light-gated algal channel channelrhodopsin-2 (ChR2) 9 specifically into a small fraction of layer 2/3 neurons of the mouse primary somatosensory cortex. ChR2 photostimulation in vivo reliably generated stimulus-locked action potentials 10-13 at frequencies up to 50 Hz. Naïve mice readily learned to detect brief trains of action potentials (5 light pulses, 1ms, 20 Hz). After training, mice could detect a photostimulus firing a single action potential in approximately 300 neurons. Even fewer neurons (approximately 60) were required for longer stimuli (5 action potentials, 250 ms). Our results show that perceptual decisions and learning can be driven by extremely brief epochs of cortical activity in a sparse subset of supragranular cortical pyramidal neurons.We used in utero electroporation 14 to introduce ChR2 fused to a green fluorescent protein (ChR2-GFP 15 ) together with a red fluorescent cytosolic marker 15 (RFP) into neocortical pyramidal neurons (Fig. 1a, Methods). In the adult brain ChR2-GFP expression was restricted to pyramidal cells in layers 2/3 (> 99.4 %), mainly in the barrel cortex (Fig 1a, 2a). In vivo two-photon imaging and retrospective immunohistology revealed that ChR2-GFP was localized to the neuronal plasma membrane. ChR2-GFP was expressed in about half (48.9 ± 5.3 %, n = 10, 5 mice; see Methods) of red fluorescent layer 2/3 neurons (Supplementary Movie 1). ChR2-GFP invaded the soma, dendrites, and axons (Fig. 1b, 1c). ChR2-GFP expression was stable for at least 8 months and did not seem to perturb neuronal morphology (Fig. 1a-c, Methods).We next characterized the responses of ChR2-GFP-expressing neurons to photostimulation in anesthetized mice. To sample from the entire population of ChR2-GFP-expressing neurons, unbiased by ChR2-GFP expression level, we recorded from red fluorescent neurons using two-photon targeted loose-patch recordings 16 (Fig. 1c, d). Photostimuli consisted of light pulses, produced by a blue miniature light emitting diode (LED; 470 nm), centered on the recording window (Fig. 1d). At maximum light intensities (I max = 11.6 mW/mm 2 at the surface of the brain, centered on the diode; 1-10 ms duration) about half (51 %) of the patched red neurons (n = 39/77, 8 mice) responded reliably to single photostimuli with at AUTOR CONTRIBUTIONS DH and KS designed the experiments. DH performed the behavioral and in vivo physiological experiments. LP, DH and KS performed the brain slice measurements. NG performed histology. SR, TH, ZM, KS provided advice and equipment. DH and KS wrote the paper. All authors discussed the results and commented on the manuscript.

Optimizing the spatial resolution of Channelrhodopsin-2 activation

“…Spatial specificity of light stimulation can be achieved by two methods: Selective expression of ChR2 in a subset of cells (Adamantidis et al, 2007), or restriction of the illumination to a small region of tissue or even to a single cell . Different strategies for introducing ChR2 into neurons have been used: Viral transfection (Adamantidis et al, 2007;Aravanis et al, 2007;Zhang et al, 2008), in utero electroporation (Petreanu et al, 2007;Huber et al, 2008), and the generation of Thy1 transgenic mice (Arenkiel et al, 2007;Wang et al, 2007). Most of these techniques produce a relatively high density of ChR2 expressing cells, and it is not clear whether individual cells can be activated by focused illumination under these conditions.…”

Selective Manipulation of Neural Circuits

“…Although this system made it easy to apply optogenetics to free-moving animals, it is still invasive-wireless headstagemounted systems still need to implant optical fiber into the brain. To reduce invasiveness channels that respond to longer wavelength light have been developed; longer wavelength light penetrates tissue more deeply than short [135][136][137][138][139][140]. Red light-activated opsins such as excitatory red lightactivated channelrhodopsin and inhibitory red-shifted cruxhalorhodopsin (Jaws) can be applied to the surface of the brain or through a thinned skull [119,120].…”

Optogenetics in neuroscience: what we gain from studies in mammals

“…Previous researchers failed to distinguish the precise neuronal groups associated with learning and memory, while optogenetics provided this possibility. Huber et al (2008) successfully calculated the number of [41,42] 426 neurons necessary for perception by using ChR2 in mouse primary somatosensory cortex and found that optogenetic activation of the targeted neurons apparently elicits associative learning [22] . Interestingly, the same clusters of neurons may display different responses with the same micro-stimulation in different temporal patterns.…”