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Manganese-enhanced MRI (MEMRI) has been described as a powerful tool to depict the architecture of neuronal circuits. The aim of the present study was to optimize the experimental conditions of MEMRI that permits the study of insult-induced alterations of the somatosensory pathway in a longitudinal way, and to provide functional information on rat corticothalamic connectivity or disturbances thereof. A guidance screw was implanted in the skull of the rats, over the forelimb representation area of the primary somatosensory cortex (S1fl), allowing repetitive injections at the same stereotactic coordinates. MnCl
2 (200 nL, 0.3 M) was injected 1.5 mm below the dura using a calibrated microcapillary. Animals received MnCl
2 injections 3 times at 15 day intervals. Spatiotemporal patterns showed a significant hyperintensity on T1-weighted images induced by manganese transport in structures related to the somatosensory pathway, i.e. globus pallidus, caudate putamen, thalamus and substantia nigra. 7 days after MnCl
2 injection hyperintensity was only evident at some points surrounding the injection site. Complete loss of manganese-induced contrast was achieved after 15 days after injection. Functional MRI (fMRI) experiments were performed under the same conditions, 24 h after MnCl
2 injection. Activation of S1fl was observed showing that fMRI and MEMRI studies are compatible and can be performed in parallel in the same animals. The present study shows, for the first time, a robust and reproducible technique to perform longitudinal MEMRI (L-MEMRI) experiments and to study the time course of alterations of the corticothalamic connections following stroke in the rat.