Research ArticleRole of Olfactorily Responsive Neurons in the Right Dorsal Habenula–Ventral Interpeduncular Nucleus Pathway in Food-Seeking Behaviors of Larval Zebrafish
Introduction
The habenula (Hb) is a highly conserved diencephalic structure with similar afferent inputs as well as efferent targets among vertebrates (Hikosaka, 2010, Okamoto et al., 2012, Proulx et al., 2014). In mammals, the Hb consists of medial and lateral parts (MHb and LHb), and their homologs in zebrafish are dorsal and ventral Hb (dHb and vHb), respectively (Amo et al., 2010). Besides receiving fear- and anxiety-related inputs (Hikosaka et al., 2008, Hong and Hikosaka, 2008, Shabel et al., 2012, Shabel et al., 2014, Yamaguchi et al., 2013), the Hb also receives inputs from multiple sensory modalities, including visual and olfactory pathways (Zhao and Rusak, 2005, Matsumoto and Hikosaka, 2007, Dreosti et al., 2014, Krishnan et al., 2014, Zhang et al., 2017). In zebrafish, visual and olfactory responses are lateralized in the left Hb (L-Hb) and right Hb (R-Hb) (Dreosti et al., 2014), which innervate the dorsal and ventral interpeduncular nucleus (IPN), respectively (Aizawa et al., 2005). The visual response of the left dHb (L-dHb) has recently been demonstrated to play a role in light-preference behavior (Zhang et al., 2017). In terms of the roles of Hb olfactory inputs, it is known that dHb neurons receive inputs from mitral cells of the olfactory bulb (Miyasaka et al., 2009) and respond to a broad range of odor stimuli (Jetti et al., 2014) including nucleotide mixture, amino acid mixture and food odor, suggesting that the Hb may contribute to food seeking-related behaviors. A previous study reported that the right dHb (R-dHb) is involved in gating innate attraction to a bile salt (Krishnan et al., 2014). It is still of interest to examine whether the R-dHb plays a role in food-seeking behavior.
In the present study, through in vivo whole-cell recording and calcium imaging, we first characterized the properties of Hb neurons' olfactory responses and their spatial distribution in the L- and R-Hb neurons. The majority of olfactorily responsive neurons located in the R-dHb. Consistently, the spatial distribution of olfactory responses in the IPN displayed a ventral-to-dorsal asymmetry. We then established a food seeking-related behavior paradigm, in which adding food extract can increase swimming activity of zebrafish larvae. Genetic dysfunction of bilateral Hb, local lesion of the R- but not L-dHb, or local lesion of the ventral IPN (V-IPN) but not dorsal IPN (D-IPN) abolished food-induced increase of swimming activity, indicating the importance of the R-dHb to V-IPN pathway in food-seeking behavior of zebrafish larvae.
Section snippets
Zebrafish preparation
Tg(elavl3:GCaMP5) (Tg(HuC:GCaMP5)), TgBAC(gng8:GAL4FF)c426;Tg(5×UAS:BoTxBLC-GFP)20-39 (TgBAC(gng8:GAL4FF);Tg(UAS:BoTxBLC-GFP)), TgBAC(gng8:GAL4FF)c426;Tg(4 × nrUAS:GFP)c369 (TgBAC(gng8:GAL4FF);Tg(4 × nrUAS:GFP)) and wild type AB adult zebrafish (Danio rerio) were maintained at the National Zebrafish Resources of China (NZRC, Shanghai, China) at 28 °C on a 14/10 light/dark cycle following a standard protocol (Zhang et al., 2017). Embryos were raised in 10% Hank's solution, which consisted of (in
Right-to-left asymmetry of Hb neurons' olfactory responses
To examine the type of Hb neurons' olfactory responses, we first performed in vivo whole-cell recording in 5- to 6-dpf larvae. A brief puffing of food extract either increased (ON-type, 10 out of 34) or decreased the activity (OFF-type, four out of 34) of dHb neurons (Fig. 1A and B). To map the spatial distribution of those responsive neurons, we then performed in vivo calcium imaging in 6-dpf Tg(HuC:GCaMP5) larvae, in which the Ca2+ indicator GCaMP5 was expressed in neurons in the brain. For
Discussion
In zebrafish, neurons in the dHb can respond to a variety of odor stimuli ranging from bile acid mixture, amino acid mixture to food extract (Dreosti et al., 2014, Jetti et al., 2014), and the R-dHb–IPN pathway was found to gate innate attraction to a bile salt (Krishnan et al., 2014). In the present study, we extend the olfactory function of the R-dHb to food-seeking behavior by demonstrating its necessity for increasing locomotion induced by food extract. More importantly, we demonstrated the
Acknowledgments
We thank Dr. Koichi Kawakami for providing the Tg(UAS:BoTxBLC-GFP) line, Dr. Marnie Halpern for providing Tg(gng8:Gal4) and Tg (gng8:GFP) lines, Dr. Qian Hu for image analysis, Dr. Xiaoquan Li for making the Tg(HuC:GCaMP5) line, and Dr. Chen Yin for writing the Matlab program. This work was supported by Young Elite Scientists Sponsorship Program by China Association for Science and Technology, Shanghai Municipal Science and Technology Commission Major Project (18JC1410100, 2018SHZDZX05), Key
Author contributions
B.B.Z. and J.L.D. conceived the research project, designed the experiments, and wrote the manuscript with W.Y.C.'s input. B.B.Z. and W.Y.C. performed the experiments and analyzed the data with help from X.L.P., Q.S.D. and M.J.C.
Competing financial interests
The authors declare no competing financial interests.
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