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Roles of zebrafish cerebellar neural circuitry in classical fear conditioning

›ผ“c Œ๕Ži / Koji Matsuda:1@‹g“c ซ”V / Masayuki Yoshida:2@๓์ ˜aG / Kazuhide Asakawa:3@‘ๅ‘q ณ“น / Masamichi Ohkura:4@’†ˆไ ~ˆ๊ / Junichi Nakai:4@์ใ _ˆ๊ / Koichi Kawakami:3@“๚”ไ ณ•F / Masahiko Hibi:1,5@ด… ‹MŽj / Takashi Shimizu:1,5@ 1:–ผŒร‰ฎ‘ๅ‰@—ถ–ฝ—Šw / Div. Biol. Sci., Grad. Sch., Nagoya Univ., Aichi, Japan@2:L“‡‘ๅ‰@ถ•จŒ—‰ศŠw / Grad. Sch. of Bios. Sci., Hiroshima Univ., Hiroshima, Japan@3:‘—งˆโ“`ŒคŒย‘ฬˆโ“`‰Š๚”ญถ / Div. of Mol. and Dev. Biol., NIG., Shizuoka, Japan@4:้‹ส‘ๅ”]‰ศŠw—Z‡Œค‹†ƒZƒ“ƒ^[ / Brain. Sci. Inst., Saitama Univ., Saitama, Japan@5:–ผŒร‰ฎ‘ๅŠwถ•จ‹@”\ŠJ”ญ—˜—pŒค‹†ƒZƒ“ƒ^[ / Biosci. Biotech. Center, Nagoya Univ., Aichi, Japan@

The cerebellar neural circuitry is involved in the classical conditioning in both mammals and teleosts. Cerebellum lesions or drug-mediated silencing of cerebellum in goldfish result in impairments in classical fear conditioning, in which repeating the conditioned stimulus (CS, light) and the unconditioned fear stimulus (US, electric shock) leads to bradycardia and body escape in response to the CS. We adopted delayed conditioning paradigm with a light-off as CS and an electric shock as US in zebrafish. We found that zebrafish older than around 20 days post-fertilization were capable to acquire conditioned bradycardia and body escape responses. However it is not clear which components in the cerebellar neural circuitry are involved in the classical fear conditioning.
To investigate the involvement of the cerebellum in the classical conditioning in zebrafish, we performed the functional inhibition of granule cells. We used the granule cell-specific Gal4 lines and the effector line UAS:BoTx-GFP to express botulinum toxin in the granule cells. In the control larvae, the CS-dependent bradycardia immediately recovered after the conditioning. When the activity of the granule cells in the valvula cerebelli and corpus cerebelli (the rostral and major lobes of the zebrafish cerebellum) was silenced, the resultant larvae were capable to acquire conditioned bradycardia. However they showed no conditioned body escape response but rather displayed a prolonged bradycardia response. The data suggest that the cerebellum is involved in the fear conditioning and at least a portion of the granule cells positively control the conditioned body escape response. The granule cells may function to determine the duration (or timing) of the CS response (or recovery). To examine which cerebellar neurons are activated during the classical fear conditioning, we expressed a Ca indicator GCaMP7a in neurons using a HuC (elavl3) promoter. We found two types of cerebellar neurons that are activated by an electric shock. Type-1 neurons immediately responded to the electric shock and are located in the corpus cerebelli and eminentia granularis (the lateral lobe). Type-2 neurons were activated slightly later than type 1 neurons and were located in the lobus caudalis (the caudal lobe). We shall report our progress in understanding the roles of individual component of the cerebellar neural circuits in the classical fear conditioning.