Granule cells control recovery from classical conditioned fear responses in the zebrafish cerebellum

Abstract

Although previous studies show that the cerebellum is involved in classical fear conditioning, it is not clear which components in the cerebellum control it or how. We addressed this issue using a delayed fear-conditioning paradigm with late-stage zebrafish larvae, with the light extinguishment as the conditioned stimulus (CS) and an electric shock as the unconditioned stimulus (US). The US induced bradycardia in the restrained larvae. After paired-associate conditioning with the CS and US, a substantial population of the larvae displayed CS-evoked bradycardia responses. To investigate the roles of the zebrafish cerebellum in classical fear conditioning, we expressed botulinum toxin or the Ca2+ indicator GCaMP7a in cerebellar neurons. The botulinum-toxin-dependent inhibition of granule-cell transmissions in the corpus cerebelli (CCe, the medial lobe) did not suppress the CS-evoked bradycardia response, but rather prolonged the response. We identified cerebellar neurons with elevated CS-evoked activity after the conditioning. The CS-evoked activity of these neurons was progressively upregulated during the conditioning and was downregulated with repetition of the unpaired CS. Some of these neurons were activated immediately upon the CS presentation, whereas others were activated after a delay. Our findings indicate that granule cells control the recovery from conditioned fear responses in zebrafish.

Imaging Neuronal Activity in the Optic Tectum of Late Stage Larval Zebrafish

“…In the past, three different approaches have been used to extend the period for imaging neuronal activity in zebrafish larvae. First, brighter forms of calcium reporters (e.g., GCaMP5) have been used instead of GCaMP3 (e.g., [ 17 , 18 ]). Second, calcium reporters have been targeted to the nucleus, using a UAS:gal4 system or a different pan-neuronal and/or specific promoter [ 12 , 14 , 16 ].…”

“…Using more recent and brighter forms of GCaMPs (e.g., GCAMP5 and GCaMP6) has allowed for the imaging period to be extended beyond 14 dpf, particularly in the habenula [ 17 , 18 , 19 ]. However, to the best of our knowledge imaging from the optic tectum has not been reported for late stage larvae.…”

Zebrafish use visual cues and geometric relationships to form a spatial memory

“…We uncovered several avoidance strategies, which indicate the presence of flexible neural mechanisms underlying the behavior. Recently developed techniques for embedding of juvenile zebrafish (Bergmann et al, 2018; Matsuda et al, 2017; Vendrell-Llopis & Yaksi, 2016), combined with immersive virtual reality setups, make juvenile zebrafish a promising model organism for future studies of brain activity in a navigating animal.…”

“…There have been reports of learning effects in one-week-old larval zebrafish using classical conditioning paradigms (Aizenberg & Schuman, 2011; Harmon, Magaram, McLean, & Raman, 2017; Lee et al, 2010) and operant conditioning paradigms (Hinz, Aizenberg, Tushev, & Schuman, 2013; Yang, Meng, Li, & Wen, 2019). Robust learning effects were observed in three-week-old juvenile zebrafish (Matsuda, Yoshida, Kawakami, Hibi, & Shimizu, 2017; Valente, Huang, Portugues, & Engert, 2012). While these studies demonstrated the ability of young fish to associate cues with a reward or a punishment, the spatial component of learning has not yet been explored.…”

Integration of Swimming-Related Synaptic Excitation and Inhibition by olig2+Eurydendroid Neurons in Larval Zebrafish Cerebellum

“…Studies of larval zebrafish indicate that cerebellum-dependent behaviors are present as early as 5-6 d post-fertilization (dpf) (Aizenberg and Schuman, 2011;Ahrens et al, 2012;Matsui et al, 2014;Matsuda et al, 2017;Knogler et al, 2019). Electrophysiological recordings of their Purkinje cells reveal synaptic responses from granule cell parallel fibers and inferior olivary climbing fibers and swimming-related increases in firing rate (Matsui et al, 2014;Sengupta and Thirumalai, 2015;Scalise et al, 2016); both synaptic excitation and spiking change gradually during associative motor learning (Harmon et al, 2017).…”