A Serotonin Circuit Acts as an Environmental Sensor to Mediate Midline Axon Crossing through EphrinB2

Abstract

Modulation of connectivity formation in the developing brain in response to external stimuli is poorly understood. Here, we show that the raphe nucleus and its serotonergic projections regulate pathfinding of commissural axons in zebrafish. We found that the raphe neurons extend projections toward midline-crossing axons and that when serotonergic signaling is blocked by pharmacological inhibition or by raphe neuron ablation, commissural pathfinding is disrupted. We demonstrate that the serotonin receptor htr2a is expressed on these commissural axons and that genetic knock-down of htr2a disrupts crossing. We further show that knock-down of htr2a or ablation of the raphe neurons increases ephrinB2a protein levels in commissural axons. An ephrinB2a mutant can rescue midline crossing when serotonergic signaling is blocked. Furthermore, we found that regulation of serotonin expression in the raphe neurons is modulated in response to the developmental environment. Hypoxia causes the raphe to decrease serotonin levels, leading to a reduction in midline crossing. Increasing serotonin in the setting of hypoxia restored midline crossing. Our findings demonstrate an instructive role for serotonin in axon guidance acting through ephrinB2a and reveal a novel mechanism for developmental interpretation of the environmental milieu in the generation of mature neural circuitry.

Dopaminergic Co-Regulation of Locomotor Development and Motor Neuron Synaptogenesis is Uncoupled by Hypoxia in Zebrafish

“…Transgenic fish lines and alleles used in this paper were the following: Tg(otpb.A:Gal4-VP16 413 À 470 ; myl7:EGFP)) zc57 (referred to as Tg(optb.A:Gal4); Stevenson et al, 2012); Tg (zcUAS:PSD95.FingR-GFP-ZFC(CCR5TC)-KRAB(A)) zc88 (Son et al, 2016); Tg(otpb.A:EGFP-caax) zc49 (Gutnick et al, 2011); and Tg(myl7:EGFP; UAS:TagRFP-caax) zc61 (Xing et al, 2015). The lines Tg(otpb.A:TRFP-caax) zc97 and Tg(Hb9:Gal4VP16 413-470 ) zc98 (referred to as Tg(Hb9: Gal4)) were generated for this project.…”

“…For hypoxia experiments, we followed settings and conditions established in previously published protocols (Stevenson et al, 2012;Xing et al, 2015;Gao et al, 2018). In brief, embryonic zebrafish were placed in a sealed Plexiglas chamber connected via a controller that monitored and adjusted nitrogen gas flow to a desired pO 2 set point (Biospherix Ltd.).…”

“…The DDT is required for locomotor development, and because of our observation of potential synapses between the DDT and motor neurons, we were interested in whether disruption of DDT connectivity would affect motor development. In particular, since hypoxia in developing zebrafish results in pathfinding errors (Stevenson et al, 2012;Xing et al, 2015), and hypoxia has been associated with long-term motor impairment in human infants born prematurely (Martin et al, 2011b;Salmaso et al, 2014), we were interested in whether hypoxia would affect DDT connectivity and lead to resultant swimming deficits. We exposed developing zebrafish larvae to different sublethal hypoxic conditions including 1% pO 2 from 1 to 2 dpf, 3% pO 2 from 2 to 3 dpf, and 5% pO 2 from 3 to 4 dpf.…”

“…We exposed developing zebrafish larvae to different sublethal hypoxic conditions including 1% pO 2 from 1 to 2 dpf, 3% pO 2 from 2 to 3 dpf, and 5% pO 2 from 3 to 4 dpf. The timing and percentage hypoxia was based on previous work, in which these conditions were shown to be non-lethal, and to be non-disruptive to overall development including aspects of CNS development such as cell fate specification and progenitor specification (Stevenson et al, 2012;Xing et al, 2015). Following hypoxia, animals were returned to normoxia, and behavior was then examined at 5 dpf (as the developmental switch in the episodic locomotor (swimming) pattern occurs around 5 dpf; Fig.…”

“…However, the mechanisms linking altered neuronal connectivity, defined as axon pathfinding and synaptic connectivity, to changes in motor function or behavior in vertebrates are still poorly understand. Previous work has shown that developmental hypoxia causes axon pathfinding errors in commissural telencephalic neurons, due to activation of the hypoxia-inducible factor (hif1) pathway in the developing zebrafish brain (Stevenson et al, 2012;Xing et al, 2015). In vivo as well as in vitro studies have demonstrated that hypoxia affects synapse development as well (Curristin et al, 2002;Valdez et al, 2007;Milash et al, 2016;Segura et al, 2016;Son et al, 2016).…”