To combine the line scanning fMRI technique with optogenetics in order to compare sensory and optogenetic stimulation.Line scanning is a novel technique for recording BOLD responses at high temporal resolutions (50 ms)¹. It employs a fast GE sequence without phase encoding, so that instead of images line profiles are acquired. If TR is set to 50 ms the acquired line profiles probe the MR signal at 50 ms intervals. Precise investigation of the temporal evolution of the BOLD response upon stimulation in a defined paradigm becomes possible. Furthermore laminar profiles across cortical layers can be investigated. Here, line scanning was combined with optogenetic methods which enable modulation of defined cellular populations such as excitatory neurons. BOLD responses were detected upon electric forepaw stimulation and upon optogenetic stimulation in the somatosensory (S1FL) cortex of the rat brain, in order to compare the times to half maximum of the BOLD responses.Line scanning was performed on female Fisher rats upon electric forepaw stimulation or optogenetic stimulation of the primary sensorimotor cortex (S1). Rats were locally expressing the opsin C1V1_TT in excitatory neurons after viral transduction into S1FL. Imaging experiments were performed at least 4 weeks after transduction to ensure sufficient opsin expression. For optogenetic stimulation, green light (552 nm) was delivered through a 200 µm diameter optical fiber implanted above the target area. Intensities of 70-105 mW/mm² at the fiber tip lead to reliable optogenetic activations. fMRI experiments used a block design (5 s stimulation: 1 ms electric stimulation at 1.5 mA, 9 Hz or 10 ms optogenetic stimulation at 9 Hz, 25 s rest). All MR imaging experiments were performed at 9.4 T. First, fMRI was performed using a T₂^* weighted GE-EPI sequence (TE = 18 ms, TR = 1 s, spatial resolution 350 x 325 µm², slice thickness 1.2 mm, 9 contiguous slices) to locate the area of BOLD activation and position the line scanning line (Fig. 1). Line scanning was performed using a modified FLASH sequence without phase encoding. Frequency encoding was performed along a line perpendicular to the cortical surface (FA=13°, TE=18 ms, TR=50 ms, 64 frequency encoding steps, FOV 10 x 2.1 x 1.2 mm³, 600 acquisitions including 20 dummy scans, 64 repetitions, scan time 32 min). The signal was analyzed using Matlab. A Fourier transform along the frequency encoding direction produced a line profile across the cortical layers. The edge of the cortex could be determined by the steepest signal rise along the line profile. Averaged time courses were calculated for the whole cortex area and for single cortical layers. The time to half maximum was determined.With line scanning, BOLD responses upon electric forepaw stimulation (n=9) and upon optogenetic stimulation (n=11) (Fig. 2) were reliably detected. Noise levels fluctuated strongly, resulting in baseline SNR ranging from 160-430 for sensory and 190-520 for optogenetic stimulation. The fiber directly in the cortex did not hinder measurements of the BOLD response in cortex with the line scanning method, although such implants regularly caused artefacts in EPI sequences. BOLD responses with high amplitudes (>1.5 %) were analyzed in cortical layers L1 to L6 (Fig. 3). The largest signal change was often found in layer L1, or similar amplitudes were found in L1-L4 (n=10). In 6 cases the highest amplitude was found in L4 or L5. Shorter times to half maximum were found for optogenetic stimulation in cortex (mean: 1.68 s, n=11) vs. sensory stimulation (mean: 1.95 s, n=9) (Mann-Whitney U test, p=0.007) (Fig. 4).High spatial resolution allowed for analysis of layer specific signals. Although the nominal resolution is 0.16 mm/px in the frequency encoding direction, the ability to resolve between cortical layers is difficult to confirm. Partial volume effects, especially due to the thickness of imaging slices (1.2 mm), movement and non-ideal saturation slices certainly spread the MR signal out across layers. Nevertheless BOLD responses can be seen in every cortical layer and signal changes can be observed across layers, so that signals can be assumed to be layer-specific. The largest BOLD amplitude was often found in L1 most likely due to strong BOLD signal from pial veins. Longer onset times for sensory vs. optogenetic stimulation are probably not due to longer neuronal pathways but may point to different hemodynamic responses. Line scanning offers the possibility to analyze BOLD time courses upon different stimulations (varying in kind, strength, duration) for different areas of the brain, without making any model assumptions, at high temporal resolutions and thereby gain insight into variations and dependencies of the hemodynamic response function.