Previous studies found that lesions or pharmacological inactivation of the central amygdala (CeA) impairs cerebellum-dependent eyeblink conditioning (Burhans and Schreurs, 2008, Farley et al., 2016, Farley et al., 2018, Freeman et al., 2021, Lee and Kim, 2004, Neufeld and Mintz, 2001). The initial findings were interpreted as supporting a two-process theory of learning, where the amygdala rapidly acquires fear conditioning and this initial learning provides motivational modulation of slower learning within the cerebellum (Lee and Kim, 2004, Neufeld and Mintz, 2001). This process theory is not supported, however, by reversible inactivation studies, which indicate that the CeA also modulates cerebellum-mediated learning after asymptotic performance (Farley et al., 2016, Farley et al., 2018, Freeman et al., 2021). Moreover, blocking CeA consolidation with inhibition of protein synthesis, gene transcription, or NMDA receptors impairs fear conditioning while leaving eyeblink conditioning intact (Steinmetz et al., 2017). These findings indicate that the CeA modulates cerebellar function within the training session and the formation of a fear-based memory is not necessary for its modulation of the cerebellum.

Cerebellar electrophysiological recordings during CeA inactivation showed that the CeA plays a role in acquisition and retention of learning-related plasticity (Farley et al., 2016). This study also found that CeA inactivation impairs learning-specific increases in conditional stimulus (CS)-evoked activity within the cerebellum from the outset of training, suggesting that the CeA plays a role in boosting stimulus input to the cerebellum during learning. Neuroanatomical tracing experiments found no direct projection from the CeA to the cerebellum, but it does project to the basilar pontine nucleus (PN)(Farley et al., 2016), which sends mossy fibers to the cerebellar cortex (Brodal and Jansen, 1946, Burne et al., 1978; J. E. Steinmetz & Sengelaub, 1992). Thus, the CeA may modulate cerebellar learning by facilitating mossy fiber input to the cerebellum. The mossy fiber pathway is the proximal part of the CS pathway necessary for eyeblink conditioning (Halverson & Freeman, 2010, 2010; Hesslow et al., 1999; J. E. Steinmetz et al., 1986, Steinmetz et al., 1987). We hypothesized that the CeA mediates attention to the CS, enhancing input to the cerebellum, and thereby facilitating learning.

Our hypothesis was that the CeA modulates cerebellar function specifically during the CS (the CS facilitation hypothesis), but this temporal specificity could not be verified because the pharmacological inactivation studies inhibited the CeA throughout training. In the current study we used optogenetic inhibition and excitation to determine the time during trials when the CeA modulates cerebellar learning. Optogenetic inhibition (archaerhodopsin) and excitation (channelrhodopsin) of the CeA were given during the CS and just before the CS in separate experiments. We hypothesized that neither pre-CS inhibition nor excitation would affect learning. We confirmed the optogenetic inhibition and excitation of the CeA using single unit optetrode extracellular recordings.