Changes in behavioral state are associated with modulation of sensory responses across visual, auditory and somatosensory cortices. Here we show that locomotor activity independently modulates performance in delay eyeblink conditioning, a cerebellumdependent form of associative learning. Increased locomotor speed in head-fixed mice was associated with earlier onset of learning and trial-by-trial enhancement of learned responses. The influence of locomotion on conditioned responses was dissociable from changes in arousal and was independent of the sensory modality of the conditioned stimulus. Eyelid responses evoked by optogenetic stimulation of mossy fiber terminals within the cerebellar cortex, but not at sites downstream, were also positively modulated by ongoing locomotion. We conclude that locomotor activity modulates delay eyeblink conditioning through mechanisms acting on the mossy fiber pathway within the cerebellar cortex. Taken together, these results suggest a novel role for behavioral state modulation in associative learning and provide a potential mechanism through which engaging in movement can improve an individual's ability to learn.

Attempts to identify cellular mechanisms underlying learning often include knocking out genes involved in candidate forms of synaptic plasticity and assessing subsequent effects on behavior. Within the cerebellum, multiple plasticity mechanisms have been proposed as cellular substrates of learning. For example, type 1-cannabinoid receptors (CB1Rs) mediate several forms of synaptic plasticity in the cerebellar cortex and have been implicated in cerebellum-dependent delay eyeblink conditioning in knockout experiments. However, recent work has shown that eyeblink conditioning is modulated by behavioral state, and global CB1KO mice are known to be hypoactive. We therefore asked to what extent altered locomotor activity vs. impaired CB1-dependent plasticity within the cerebellar cortex contribute to learning impairments in these mice. We find that eyeblink conditioning deficits in global CB1KOs can be fully accounted for by their hypoactivity. Impairments disappear when the level of locomotor activity is taken into account, and externally controlling running speed rescues learning. Moreover, both global and cerebellar granule cell-specific CB1KOs exhibit normal cerebellum-dependent locomotor adaptation. Our results suggest that the apparent effects of CB1R deletion on cerebellar learning are not due to direct effects on CB1-dependent plasticity, but rather, arise as a secondary consequence of hypoactivity. These findings highlight the importance of considering general changes in behavioral state as a powerful means through which individual genes contribute to complex behaviors, particularly in transgenic models.

Supervised learning depends on instructive signals that shape the output of neural circuits to support learned changes in behavior. Climbing fiber inputs to the cerebellar cortex represent one of the strongest candidates in the vertebrate brain for conveying neural instructive signals. However, recent studies have shown that Purkinje cell stimulation can also drive cerebellar learning, and the relative importance of these two neuron types in providing instructive signals for cerebellum-dependent behaviors remains unresolved. Here we used cell-type specific perturbations of climbing fibers, Purkinje cells, and other cerebellar circuit elements to systematically evaluate their contributions to delay eyeblink conditioning. Our findings reveal that while optogenetic stimulation of either climbing fibers or Purkinje cells can substitute for a sensory unconditioned stimulus, subtle reductions in climbing fiber signaling prevent learning entirely. We conclude that climbing fibers and corresponding Purkinje cell complex-spike events provide essential instructive signals for associative cerebellar learning.