The ability of Mn(2+) to follow Ca(2+) pathways upon stimulation transform them into remarkable surrogate markers of neuronal activity using activity-induced manganese-dependent MRI (AIM-MRI). In the present study, a precise follow-up of physiological parameters during MnCl2 and mannitol infusions improved the reproducibility of AIM-MRI allowing in-depth evaluation of the technique. Pixel-by-pixel T1 data were investigated using histogram distributions in the barrel cortex (BC) and the thalamus before and after Mn(2+) infusion, after blood brain barrier opening and after BC activation. Mean BC T1 values dropped significantly upon trigeminal nerve (TGN) stimulation (-38 %, P = 0.02) in accordance with previous literature findings. T1 histogram distributions showed that 34 % of T1s in the range 600-1500 ms after Mn(2+ )+ mannitol infusions shifted to 50-350 ms after TGN stimulation corresponding to a twofold increase of the percentage of pixels with the lowest T1s in BC. Moreover, T1 changes in response to stimulation increased significantly from superficial cortical layers (I-III) to deeper layers (V-VI). Cortical cytoarchitecture detection during a functional paradigm was performed extending the potential of AIM-MRI. Quantitative AIM-MRI could thus offer a means to interpret local neural activity across cortical layers while identification of the role of calcium dynamics in vivo during brain activation could play a key role in resolving neurovascular coupling mechanisms.