Central projections of RGCs

At the larval stages, the main projection site of RGCs axons is the optic tectum (see section 1.2.2.1), homologous to the mammalian superior colliculus, which constitutes the dor- sal aspect of the zebrafish midbrain (Burrill and Easter, 1994). In addition, by intraocular injection of lipophilic fluorescent dye and tracing the RGC axons, Burrill and Easter iden- tified nine more retinorecipient nuclei, called arborization fields (AFs) (Burrill and Easter, 1994) referred to as AF1 to AF10 (Figure 1.4). Specific RGC types project their axons to one of these AFs or multiple AFs, make connections with target cells to establish a representa- tion of the visual field in the brain.

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Figure 1.4: Retinal projections in the zebrafish. (A)Schematic lateral view of the ten retinorecipi- ent areas (arborization fields) in 6-7 dpf larva, revealed by intraocular injections of DiI.

(B)Camera drawing of DiI labeled ganglion cell axons and their innervation sites. Dor- sal, up; Rostral, left, scale bar represents 50µm. (Figure adapted from Burrill and Easter (1994).)

So far, little is known about the behavioral functions of extratectal AFs, although it is clear that for the execution of a particular visual behavior, one or few of these nuclei should act together (Ullén et al., 1997; Kubo et al., 2014; Semmelhack et al., 2014). It has been shown that AF7 is predominantly innervated by particular bistratified RGCs that are highly selec- tive in their responses to prey-like visual stimuli (Semmelhack et al., 2014; Robles et al.,

1.2 The visual system of zebrafish

2014). Indeed, ablation of the RGC axons innervating in AF7 impairs the prey-capture be- havior in the larval zebrafish. Similarly, neurons surrounding AF9 have been shown to be involved in global optic flow processing (Roeser and Baier, 2003; Kubo et al., 2014).

1.2.2.1 Optic Tectum

The optic tectum, also called AF10, is the biggest retinorecipient area in zebrafish lar- vae and has been intensively studied. 97% of the RGCs send their axons to the tectum and innervate discrete layers (Robles et al., 2013). Similar lamination of retinotectal ax- ons has been observed in many species from birds to mammals (see review (Huberman et al., 2010)). RGCs project their axons into ten distinct laminae that are stacked on top of each other: the stratum opticum (SO), forming the most superficial layer with its two sublaminae, the stratum fibrosum et griseum superficiale (SFGS), which in turn splits into six sublaminae, the stratum griseum centrale (SGC) and finally the deepest layer stratum album centrale (SAC) and the stratum periventriculare (SPV) (Robles et al., 2013) (Figure 1.5). RGCs usually innervate only one layer, although they may form collaterals in other extratectal visual areas (Xiao and Baier, 2007). Recently, application of the Brainbow (Livet et al., 2007) technique in the zebrafish retina revealed the complexity of precise laminar organization of the RGC axons in the tectum (Robles et al., 2013). Moreover, RGCs project their axons in a retinotopic manner, meaning that RGCs from dorsal retina terminate in the ventral tectum and vice versa, and RGCs from temporal retina terminate in the ante- rior tectum and vice versa (Robles et al., 2014). In addition, the tectum receives afferent inputs from other sensory modalities (auditory, somatosensory, lateral line, etc.) which form other sensory maps deeper in the tectum (Nevin et al., 2010).

In zebrafish, each tectal lamina is innervated by several RGC types with different dendritic morphologies (Robles et al., 2013). To investigate how this laminar organization of RGC axons relates to the functional specialization in the tectum, several studies employed two- photonin vivocalcium imaging in the tectum, using genetically encoded calcium indica- tors of the GCaMP family (see section 1.4.2). First, Nikolaouet al. (Nikolaou et al., 2012) imaged the axon terminals of RGCs, expressing SyGCaMP3, in the retinotectal neuropil

Figure 1.5: Retinal ganglion cell projections to the optic tectum.Schematic depiction of ganglion cell axons receiving input from different layers in IPL and via the optic nerve, project to the optic tectum. RGC axons innervate in the tectal neuropil by forming approxi- mately ten distinct layers. Cell bodies of tectal cells extend their axons and dendrites into the neuropil. BM: basement membrane, GCL: ganglion cell layer, PhRL: photore- ceptor layer, SAC: stratum album centrale, SAC/SPV: boundary between SAC and SPV, SFGS: stratum fibrosum et griseum superficiale, SGC: stratum griseum centrale, SM: stratum marginale, SO: stratum opticum, SPV: stratum periventriculare.(Figure taken from Baier (2013) with permission.)

while presenting drifting bars in different directions and orientations in the visual space. By doing so, they discovered that there are three subtypes of direction-selective and two subtypes of orientation-selective retinal inputs to the tectum. Moreover, these two se- lective responses are organized in a reasonably segregated manner within tectal laminae (Figure 1.6). In a follow-up study, Loweet al. (Lowe et al., 2013) explored these parallel direction and orientation maps further and found that visual experience is not necessary for the establishment of direction maps, yet it is required for the formation of orienta- tion maps. Furthermore, they identified two additional orientation-selective subtypes of RGC inputs to the tectum. Second, Gabrielet al. (Gabriel et al., 2012) combined Ca2+ imaging of RGC axons terminating in the tectum with targeted patch clamp recordings of genetically labeled tectal neurons. When they compared the tuning of direction-selective retinal inputs with the tuning of postsynaptic tectal neurons, they discovered two types of direction-selective tectal interneurons: one group received direct input from direction-

1.2 The visual system of zebrafish

selective (DS) RGCs and the other group did not receive direct DS-RGC input, although responding direction-selectively.

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Figure 1.6: Functionally distinct, parallel retinal maps in the tectum. (A)7 dpf transgenic ze- brafish larva Tg(Isl2b:Gal4;UAS:SyGCaMP3), expressing SyGCaMP3 in RGC axons and

(B)magnified view of the boxed region. (Dorsal view)(C)Color coded spatial, com- posite map of direction-selective and(D)orientation-selective RGC axons in the tectal neuropil. A = anterior, L = lateral. Scale bars represent 50µm in(A)and 20µm in(B). (A

andBadapted from Nikolaou et al. (2012),CandDadapted from Dhande et al. (2013).)

Previously, Niell and Smith (Niell and Smith, 2005) described receptive field sizes, topog- raphy, size and direction-selectivity of tectal cell populations and showed that most of these properties are established without a requirement of visual experience. Layer spe- cific tuning of different object sizes was attributed to an inhibitory interneuron type, su- perficial interneurons (SINs) (Del Bene et al., 2010). Depending on their dendritic target layer in the tectal laminae, SINs process small- and large-size-selective signals, filtered by functionally organized RGC inputs (Preuss et al., 2014). Collectively, RGC axon termi- nals positioned in distinct tectal laminae can have tuning properties similar to their target postsynaptic partners. Yet, Hunteret al. (Hunter et al., 2013) showed that direction tun- ing properties of retinal input can be transformed by the tectal cells to generate response

propertiesde novo. Combined, all the abovementioned studies suggest that different RGC types convey information about distinct visual features and converge on a specific lamina, generating parallel maps of feature selectivity.