Optogenetic fMRI (opto-fMRI). Neural activation of layer V ChR2-expressing neurons by light stimulation evokes a reliable fMRI response that is equivalent to somatosensory response evoked by vibrissa deflection. A, Opto-fMRI experimental design. Atlas (Paxinos and Franklin, 2001) sagittal (i) and coronal (ii) sections depict the placement of the fiber optic (blue line) over primary somatosensory cortex (SI) barrel field. Monte Carlo simulation of laser intensity contours are plotted (iii) demonstrating that light intensity at 750 μm was 10 %, power sufficient to induce action potentials in layer V pyramidal neurons in vitro. MI: primary motor cortex. B, Wide-field fluorescence depicts ChR2 expression of primarily layer V pyramidal neurons in a transgenic Thy1-ChR2-YFP mouse. Sagittal slice depicts expression throughout the neocortex and an atlas section is superimposed on representative histological and anatomical MRI coronal sections, depicting the site of stimulation for a representative animal. C, A characteristic expression is shown demonstrating fluorescence in layer V pyramidal neurons (top). Maximum intensity projection of confocal laser scanning microscopy demonstrates ChR2 expression (bottom). Denoted by white triangles are apical dendrites approaching the surface, the cell bodies, and axons. D, ‘Localizer’ runs consisted of 16 repetitions of 15 s light-pulses or deflections of the mystacial vibrissae followed by 15 s of no stimulation. A statistical parametric map of a positive BOLD response to light stimulation (p < 0.05 corrected for multiple comparisons) is overlaid on the average fMRI BOLD image of the sequential cross-sections demonstrating the response for the optical drive in a typical animal (‘Optogenetic’). A similar BOLD response was observed to vibrissa deflections (‘Sensory’).

Precisely evoked spike and local field potential activity for 1 s trains of optical stimulation of primary somatosensory cortex. A, Activity of a representative single neuron recorded juxtacellularly is depicted (single-unit activity [SUA]). Raster plot (top) and Peri-stimulus time histogram (PSTH; bottom) show equivalent spike responses for the two trains of 1 s stimulation. Overlaid voltage traces during the first three light pulses (inset) show highly reliable spike times and shape. B, SUA shown for individual neurons, demonstrating that although firing rate was variable between neurons, each neuron showed equivalent firing rate for 1 s train of light pulses in all conditions. C, PSTH of multi-unit activity (MUA) recorded using a laminar electrode is depicted. Responses are averaged from two contacts (depths of 700 μm and 800 μm). D, Similar to SUA, MUA responses were above baseline and equivalent when comparing the 1 s optical stimulation intervals. E, Local field potential (LFP) modulation was observed for each light stimulation train. A representative voltage trace is shown for one trial. The consistency of the response across individual trials is shown by the high overlap of individual traces (inset). F, Similar to SUA and MUA, power modulation was equivalent across the 1 s intervals.

The BOLD response shows linear temporal summation for closely spaced short trains of stimulation. For each animal, time series were extracted from a region of interest (ROI) identified in independent localizer runs. The linear response can be qualitatively observed by subtracting the n from the n+1 conditions (dashed line). A, Optical stimulation evoked equivalent responses across multiple independent 1 s trains. Individual time series (n = 6 animals) of the BOLD fMRI response to one 1 s interval (single) and two trains of 1 s intervals (double) with an inter-stimulus interval of 2 s. Data are expressed as mean ± SEM (individual runs across in each animal). B, Five 1 s (quintuple) and six 1 s trains (sextuple) show an equivalent temporal linearity response (2 animals) to that observed in A. C, D, Sensory stimulation yielded equivalent responses in the single – double (2 animals) and quintuple –sextuple (2 animals) experiments.

BOLD temporal linearity. A, Optical stimulation evoked equivalent responses across multiple independent 1 s trains. Average time series (n = 6 animals) of the BOLD fMRI response to one 1 s interval (single) and two trains of 1 s intervals (double) with an inter-stimulus interval of 2 s. Data are expressed as mean ± SEM (individual runs across all animals). Linear summation of the single trial to a 3 s shifted single trial (dashed line) provides a qualitative estimate of the temporal summation. B, Linear summation similarly holds for sensory stimulation. C, D, The temporal linearity response was quantified by dividing the response for the (n+1) trials condition by the (n) trials condition. E, F, A hemodynamic response function fitted to the single trial condition is plotted against the subtracted responses for the single – double and quintuple – sextuple trial conditions. Optogenetic: optical drive; Sensory: sensory drive; SUA: single unit activity; MUA: multi unit activity; LFP: local field potential.