%0 Journal Article %A Jennifer J. Siegel %A William Taylor %A Richard Gray %A Brian Kalmbach %A Boris V. Zemelman %A Niraj S. Desai %A Daniel Johnston %A Raymond A. Chitwood %T Trace eyeblink conditioning in mice is dependent upon the dorsal medial prefrontal cortex, cerebellum and amygdala: Behavioral characterization and functional circuitry. %D 2015 %R 10.1523/ENEURO.0051-14.2015 %J eneuro %P ENEURO.0051-14.2015 %X Trace eyeblink conditioning is useful for studying the interaction of multiple brain areas in learning and memory. The goal of the current work was to determine whether trace eyeblink conditioning could be established in a mouse model in the absence of elicited startle responses and the brain circuitry that supports this learning. We show here that mice can acquire trace conditioned responses (tCRs) devoid of startle while head-restrained and permitted to freely run on a wheel. Most mice (75%) could learn with a trace interval of 250 ms. Because tCRs were not contaminated with startle-associated components, we were able to document the development and timing of tCRs in mice, as well as their long-term retention (at 7 and 14 days) and flexible expression (extinction and reacquisition). To identify the circuitry involved we made restricted lesions of the medial prefrontal cortex (mPFC) and found that learning was prevented. Furthermore, inactivation of the cerebellum with muscimol completely abolished tCRs, demonstrating that learned responses were driven by the cerebellum. Finally, inactivation of the mPFC and amygdala in trained animals nearly abolished tCRs. Anatomical data from these critical regions showed that mPFC and amygdala both project to the rostral basilar pons and overlap with eyelid-associated pontocerebellar neurons. The data provide the first report of trace eyeblink conditioning in mice in which tCRs were driven by the cerebellum and required a localized region of mPFC for acquisition. The data further reveal a specific role for the amygdala as providing a CS-associated input to the cerebellum.Significance Statement: The current study characterizes the learned behavior and identifies the brain circuitry necessary for trace eyeblink conditioning in mice. We report that under learning conditions designed to avoid elicited startle responses, mice expressed learned responses that were cerebellar-dependent and required a localized region of the dorsal medial prefrontal cortex. Using functional inactivation paired with anatomical tracers, we further identify the trace eyeblink conditioning circuitry in the mouse model that underlies this basic associative learning task, including a more refined role for how amygdala inputs might support this task in the absence of conditioned stimulus-evoked startle. %U https://www.eneuro.org/content/eneuro/early/2015/07/02/ENEURO.0051-14.2015.full.pdf