Cerebellar-Dependent Expression of Motor Learning during Eyeblink Conditioning in Head-Fixed Mice

Abstract

Eyeblink conditioning in restrained rabbits has served as an excellent model of cerebellar-dependent motor learning for many decades. In mice, the role of the cerebellum in eyeblink conditioning is less clear and remains controversial, partly because learning appears to engage fear-related circuits and lesions of the cerebellum do not abolish the learned behavior completely. Furthermore, experiments in mice are performed using freely moving systems, which lack the stability necessary for mapping out the essential neural circuitry with electrophysiological approaches. We have developed a novel apparatus for eyeblink conditioning in head-fixed mice. Here, we show that the performance of mice in our apparatus is excellent and that the learned behavior displays two hallmark features of cerebellardependent eyeblink conditioning in rabbits: (1) gradual acquisition; and (2) adaptive timing of conditioned movements. Furthermore, we use a combination of pharmacological inactivation, electrical stimulation, single-unit recordings, and targeted microlesions to demonstrate that the learned behavior is completely dependent on the cerebellum and to pinpoint the exact location in the deep cerebellar nuclei that is necessary. Our results pave the way for using eyeblink conditioning in head-fixed mice as a platform for applying next-generation genetic tools to address molecular and circuit-level questions about cerebellar function in health and disease.

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

“…Alpha responses are activity within 50 ms of CS onset that were at least 4SD above the mean baseline activity. Over the training sessions, conditioned and pseudoconditioned mice typically exhibited alpha responses on less than 25% of the trials, with no significant differences between conditioned and pseudoconditioned mice (groups, F (1,15) =2.502, ns). The repeated measures ANOVA did, however, reveal a significant interaction of groups and sessions due to the decrease in pseudoconditioned responses and the maintenance of alpha responses at about 25% for conditioned mice (groups*sessions, F (1,9) =2.074, p<.05).…”

“…Adaptive conditioned responses are considered to have significant activity that is present at least 20 msec before US onset (i.e., at least 4 SD greater than the mean activity present 250 msec before CS onset). Over the training sessions, conditioned mice showed gradual learning of the conditioning paradigm by exhibiting more conditioned adaptive responses with each training session so that conditioned mice exhibited significantly more adaptive conditioned responses than pseudoconditioned mice (groups, F (1,15) =20.62, p<.0005; sessions, F (1,9) =9.987, p<.0001; groups *sessions, F (1,9) = 5.977, p<.0001). Note that the pseudoconditioned group typically exhibited pseudo CRs on less than 20% of the trials.…”

“…(Figure 2A) 1. Construct the cylinder as described by Chettih et al and Heiney et al from a long foam cylinder [14][15] . Cut a 10 cm length of cylinder and drill a hole through the center to fit the axle, a metal rod 12.7 mm (0.5 in.)…”

“…in diameter. Mount the cylinder with its axle onto a metal optical breadboard as described by Heiney et al or another support (e.g., Plexiglas) 15 . 2.…”

“…Head-fixed preparations have been used in previous experiments for optical imaging of the cortex through removable cranial implants, in vivo electrophysiological recordings of the rodent brain with tetrode arrays, two-photon calcium imaging, and also as a platform for eyeblink conditioning in mice [11][12][13][14][15][16] .…”