Optogenetic Localization and Genetic Perturbation of Saccade-Generating Neurons in Zebrafish

Abstract

The optokinetic response (OKR) to a visual stimulus moving at constant velocity consists of a series of two alternating components, a slow phase, during which the eyes follow the stimulus, and a quick phase, which resets the eyes to begin a new response cycle. The quick phases of the OKR resemble the saccades observed during free viewing. It is unclear to what extent the premotor circuitry underlying these two types of jerky, conjugate eye movements is conserved among vertebrates. Zebrafish (Danio rerio) larvae, broadly expressing halorhodopsin (NpHR) or channelrhodopsin-2 (ChR2) in most neurons, were used to map the location of neurons involved in this behavior. By blocking activity in localized groups of NpHR-expressing neurons with an optic fiber positioned above the head of the fish and by systematically varying the site of photostimulation, we discovered that activity in a small hindbrain area in rhombomere 5 was necessary for saccades to occur. Unilateral block of activity at this site affected behavior in a direction-specific manner. Inhibition of the right side suppressed rightward saccades of both eyes, while leaving leftward saccades unaffected, and vice versa. Photostimulation of this area in ChR2-transgenic fish was sufficient to trigger saccades that were precisely locked to the light pulses. These extra saccades could be induced both during free viewing and during the OKR, and were distinct in their kinetics from eye movements elicited by stimulating the abducens motor neurons. Zebrafish double indemnity (didy) mutants were identified in a chemical mutagenesis screen based on a defect in sustaining saccades during OKR. Positional cloning, molecular analysis, and electrophysiology revealed that the didy mutation disrupts the voltage-gated sodium channel Scn1lab (Nav1.lb). ChR2 photostimulation of the putative hindbrain saccade generator was able to fully reconstitute saccades in the didy mutant. Our studies demonstrate that an optogenetic approach is useful for targeted loss-offunction and gain-of-function manipulations of neural circuitry underlying eye movements in zebrafish and that the saccade-generating circuit in this species shares many of its properties with that in mammals.

Functional regionalization of the teleost cerebellum analyzed in vivo

“…This result shows that the caudal region of the cerebellar PC layer controls saccade movements, with the laterality of the movement being predominantly assigned to one hemisphere each. The fact that PC activation neither accelerates nor triggers saccades or swimming movements, regardless of PCs being depolarized or hyperpolarized, is not surprising, because the generators of these behaviors have been mapped to structures outside the cerebellum (42)(43)(44). For proper motor control, PCs need to determine combinations of counteracting muscles (tensor and flexor), for which they have to send well-tuned contraction and relaxation the frame that is three frames before each saccade initiation was used for this graph) and during saccade performance (black; peak signal within five frames after each saccade initiation).…”

Model systems for studying cellular mechanisms ofSCN1A-related epilepsy

“…More recently, a mutant Zebrafish strain that harbors a loss-of-function mutation in the sodium channel gene scn1Lab was discovered in a chemical mutagenesis screen (Schoonheim et al 2010). Although this strain is a homozygous mutant, the scn1Lab zebrafish line is considered analogous to the Scn1a ϩ/Ϫ knockout mouse model of DS given the genome duplication in zebrafish and the presence of an additional Na V 1.1 homolog (scn1Laa) (Baraban et al 2013;Novak et al 2006).…”

Optogenetics: A new enlightenment age for zebrafish neurobiology

“…The best known of these proteins is undoubtedly channelrhodopsin-2 (ChR2), originally isolated from the green algae Chlamydomonas reinhardti (Nagel et al, 2003). ChR2 is a blue light-sensitive cationic channel that Activation of somatosensory and hindbrain neurons (Douglass et al, 2008;Arrenberg et al, 2009;Zhu et al, 2009;Schoonheim et al, 2010) eNpHR Silencing of hindbrain neurons (Arrenberg et al, 2009;Schoonheim et al, 2010) LiGluR Activation of spinal cord neurons Wyart et al, 2009 (Li et al, 2005a,b) GCaMP1.6…”

“…Recent work, using a similar combination of ChR2 and NpHR co-expression, and optic fiber light stimulation, has made it possible to identify in the zebrafish hindbrain the location of the neurons that, when activated, are sufficient for the generation of eye saccades (Schoonheim et al, 2010). The observed latency between ChR2 activation and initial eye movement was 28 ms only.…”