To investigate activation of the visual network by light delivered to the brain for optogenetic stimulation in ofMRI experiments. Such activation may confound the analysis of optogenetically evoked BOLD signal. A control experiment is introduced to distinguish between specific optogenetic stimulation and unspecific activation of the visual pathways.Optogenetics has become a frequently used tool for probing neuronal networks¹. The combination with fMRI has been established by several research groups^2-4. Optogenetic fMRI remains technically challenging, and is prone to light-induced artifacts. While heat-induced artifacts have been studied previously, unspecific activation of the visual pathways has not been addressed to date.For viral transduction of opsins, AAVs encoding either CHR2 or C1V1 were injected into the forelimb region of sensory cortex (S1FL) or into thalamus of female Fisher rats. Optogenetic stimulation experiments were performed via an implanted 200-µm optical fiber in a block paradigm: 10 ms light pulses at 488 nm (ChR2) or 552 nm (C1V1) at 70 – 95 mW / mm², 10 s stimulation at 9 Hz, 20 s rest. The same stimulation was also performed in naïve animals. To specifically stimulate the visual pathway, a second fiber was placed in front of one eye of the animal. Then either pulsed visual stimulation (same paradigm as fiber implanted into brain except for lower light intensity of approx. 0.3 – 3 µW/mm²) or intrabrain illumination during continuous illumination of one eye were applied. Stimulation experiments were performed under medetomidine sedation at 9.4 T with single-shot GE-EPI (TR 1 s, TE 18 ms, 350x325 μm², 1.2 mm slices). fMRI data were smoothed with a 0.5 mm Gaussian kernel and analyzed using a t-test (p < 0.001) with a 2 s time shift to account for the delayed hemodynamic response using ImageJ. After experiments animals were transcardially perfused and brains excised for histological validation of opsin expression.Strong membrane bound expression of opsins was found both at injection sites and at axonal projection targets, but not in visual pathways. Optogenetic stimulation of ChR2 or C1V1 in both S1FL and thalamus evoked a BOLD response in sensory cortex. In addition to these expected clusters (not shown here) a positive BOLD response was regularly observed in subcortical regions (Fig. 1) belonging to the visual pathways, namely the dorsal lateral geniculate nucleus, optic nerve layer of the superior colliculus and the nucleus of the optic tract. Repeating the same experiment in naïve animals confirmed that this activation of the visual pathways was independent of optogenetic stimulation. Pulsed direct stimulation of the rats’ eye resulted in the same activation pattern as the light applied to the brain (Fig. 2). Simultaneous continuous stimulation of one eye abolished activation of the contralateral visual pathway upon intrabrain illumination (Fig. 3). To exclude stray light leaking from the fiber or the fiber-brain interface as stimulation light source (Fig. 4a), the experiment was repeated with blindfolded animals. Direct illumination of blindfolded eyes did not cause activation of the visual pathways. However, intrabrain illumination still resulted in activation of the visual pathways.Application of light to the brain can lead to the activation of visual pathways. This activation is at least partly caused by light scattered diffusely inside the brain (Fig. 4b). It cannot be avoided by blindfolding the animals, but by applying additional continuous illumination. We suggest uncoupling the optogenetic stimulation from stimulation of visual pathways by performing ofMRI routinely with background light inside the scanner. Unspecific activation of the visual network needs to be avoided or recognized as such, as it might confound optogenetic fMRI experiments.