Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful technique for assessing the functional connectivity of neurons within the central nervous system. Despite the widely held proposition that MEMRI signal is dependent on neuronal activity, few studies have directly tested this implicit hypothesis. In the present series of experiments, MnCl2 was injected into the habenula of urethane-anesthetized rats alone or in combination with drugs known to alter neuronal activity by modulating specific voltage- and/or ligand-gated ion channels. Continuous quantitative T1 mapping was used to measure Mn2+ accumulation in the interpeduncular nucleus, a midline structure in which efferents from the medial habenula terminate. Microinjection of MnCl2 into the habenular complex using a protocol that maintained spontaneous neuronal activity resulted in a time-dependent increase in MEMRI signal intensity in the interpeduncular nucleus consistent with fast axonal transport of Mn2+ between these structures. Co-injection of the excitatory amino-acid agonist AMPA, increased the Mn2+-enhanced signal intensity within the interpeduncular nucleus. AMPA-induced increases in MEMRI signal were attenuated by co-injection of either the sodium channel blocker, TTX, or broad-spectrum Ca2+ channel blocker, Ni2+, and were occluded in the presence of both channel blockers. However, neither Ni2+ nor TTX, alone or in combination, attenuated the increase in signal intensity following injection of Mn2+ into the habenula. These results support the premise that changes in neuronal excitability are reflected by corresponding changes in MEMRI signal intensity. However, they also suggest that basal rates of Mn2+ uptake by neurons in the medial habenula may also occur via activity-independent mechanisms.