The ability of Mn2+ to follow Ca2+ 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 Mn2+ 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 Mn2+ + 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.