## 3 Results  
### Music-Induced OBBB  
In the first step, we performed a qualitative and quantitative analysis of the BBB disruption using the intravenous injection of EBD (961 Da). The EBD is a commonly used tracer for the estimation of the BBB disruption because it is a non-toxic and not metabolically active dye, which rapidly binds to serum albumines creating the high molecular weight Evans Blue-albumin complex (EBAC, 68.5 kDa). The IBBB is impermeable to EBAC and its extravasation into the brain parenchyma indicates the BBB disruption [23; 24; 34; 40; 41; 42]. We used spectrofluorimetry for the EBAC quantitation 1 h and 4 h after music impact. This time was chosen based on the previous data on mice, in which we showed that the BBB opens 1 h after music effects and the BBB closes 4 h after music listening [23]. Our results clearly show that music caused OBBB for EBAC in mice 1 h after music action that was associated by an accumulation of EBAC in the brain parenchyma vs. intact mice (2.84 \(\pm\) 0.14 \(\upmu\)g/g tissue vs. 0.12 \(\pm\) 0.05 \(\upmu\)g/g tissue, \(p\) < 0.001, \(n\) = 10 in each group). There were no changes in the EBAC levels in the brain parenchyma between the control mice and mice, whose brain were taken 4 h after listening of music (0.14 \(\pm\) 0.10 \(\upmu\)g/g tissue vs. 0.11 \(\pm\) 0.01 \(\upmu\)g/g tissue, \(n\) = 10 in each group). Figure 1a,b illustrates a qualitative analysis of the BBB permeability to FITC-dextran 70 k Da using confocal microscopy. This method also revealed no BBB disruption before music impact (Figure 1a) and distinct distribution of FITC-dextran (Figure 1b) among the cerebral vessels and astrocytes in mice 1 h after music effects. These data are consistent with our previous results demonstrating music-induced OBBB to the high molecular weight substances in 11 brain regions, including frontal, parietal, occipital, temporal, cingulate cortex, hypothalamus, thalamus, hippocampus, inferior and superior colliculi, cerebellum, pons, and medulla [23]. Thus, these results confirm the highly reproducibility of noninvasive method of reversible OBBB by music in mice. Figure 1: **Music-induced OBBB affects the clearance of tracers from the mouse brain and the EEG structure**: (**a,b**) Representative images of IBBB (**a**) and OBBB (**b**). In mice with IBBB, FITC-dextran is present in the cerebral vessels, while in mice with OBBB, the tracer is extravasated from the blood vessels and is distributed among the cerebral microvasculature and astrocytes. (**c,d**) Representative images of real time fluorescence imaging of fast distribution of Faß over the surface of the brain of mice with IBBB (**c**) and OBBB (**d**) immediately after injection of Faß in the right later ventricle. (**e–h**) Representative images of ex vivo fluorescence microscopy of delay distribution of Faß in the lateral and dorsal parts of the brain of mice with IBBB (**e,g**) and OBBB (**f,h**) 1 h after injection of Faß in the right later ventricle. (**i,j**) Representative images of Faß distribution along the cerebral vessels in the pa matter (**i**) and in the meninges (**j**) in mice with OBBB. (**k,l**) Representative images of Faß spreading in the meninges of mice with IBBB (**k**) and OBBB (**l**). (**m,n**) Representative images of presence of Faß in dCLNs of mice with IBBB (**m**) and OBBB (**n**). (**o,p**) Representative images of MRI analysis of Omniscan distribution over the brain 2 h after its injection in the cortex of mice with IBBB (**o**) and OBBB (**p**). (**q,r**) Quantitative analysis of intensity of fluorescent signal from Faß in a.u. on the ventral and dorsal aspects of the brain and in dCLNs in mice with  
IBBB and OBBB. (**o.p**) Quantitative analysis of MRI signal from Omniscan in a.u. in mice with IBBB (**o**) and OBBB (**p**). (**s.t**) Scaling exponents \(\alpha\) and \(\beta\) estimated as local slopes of the dependencies \(F(n)\) and \(dF(n)\) on the double logarithmic plot. Data are shown as mean values \(\pm\) SE (averaging over all available datasets).