Morphology of putative direction-selective ganglion cells
traced with DiI in the fish retina.

E.M. Maximova, E.V. Levichkina, I.A. Utina

Laboratory of Sensory Information Processing,
Institute for Information Transmission Problems, Russian Academy of Sciences,
19 Bolshoy Karetny, 127994 Moscow, Russia

This page contains original electronic versions of the figures to the paper by E.M. Maximova et al., as well as some supplementary images of ganglion cells traced with DiI in the fish retinas that were not included in the paper. The paper will be published in Russian in Sensory Systems, 2006, 20 №4.
The page may also be considered as an electronic version of the presentation "Morphology of putative direction-selective ganglion cells traced with DiI in the fish retina as compared with those of mammals" by E. Maximova at the International Biophysics Workshop "Academician Radoslav K. Andjus" on Imaging in Neurosciences and Beyond, Sveti Stefan (Montenegro), September 23-30, 2006.

      Abstract Visual processing starts already in the retina. In the fish there are many types of ganglion cells (GCs) specified in detection of different properties of visual objects such as orientation, size, direction of movement, sign of contrast and so on (Maximova et al., 1971; Maximov et al., 2005). These specific abilities are determined by definite wiring in the inner plexiform layer (IPL) where GCs receive input signals from different bipolar and variety of amacrine cells. Therefore GCs of different physiological functions (detectors of different types) would differ morphologically, in particular, their dendrites could be expected to differ in shape, size, mode of stratification (Rockhill et al., 2002; Mangrum et al., 2002). At the same time physiologically similar GCs of different animals could possess similar morphology (Yang, Masland, 1994; Kittila, Massey, 1997; MacNeil et al., 1999; Weng et al., 2005). There are many methods of visualization of retinal neurons: intracellular dye injection (Yang, Masland, 1994), immunochemistry (Kittila, Massey, 1997), photofilling. Retrograde tracing with the lipophilic dye from the optic nerve is just convenient to visualize GCs (Köbbert et al., 2000).
      In this study GCs in the fish retina were traced by the carbocyanine dye DiI applied to the optic nerve cut in retinas preliminary fixed with 4% paraforme. After 1-2 weeks of incubation in thermostat at 36°C retinal preparations were investigated under UV microscope. This lipofilic dye diffuses in the lipids of cellular membrane. Many axons, cell bodies and completely stained ganglion cell of the pickarel retina traced with DiI are demonstrated in the Fig. 1.
      Six types of GCs (among many other stained) were identified by their clear dendritic morphology. Type 1 and type 2 are GCs of aA and aB types according to the traditional morphological classification with large (1500 µ in dia) sparse flat dendritic arbours stratified in sublaminae a and b of the IPL correspondingly (Fig.2). In mammalian retinas such cells are classified as Y cells of off and on physiological types. Type 3 are GCs with dendritic arborisation situated practically in the same plane as the cell body similar to d cells of the cat retina (Fig.3 Â). Those represent so called "slow" direction-selective (DS) GCs projecting to the accessory optic system. Type 4 are GCs with small dendritic fields of about 100 µ, their dendrites are very thin with varicosities at the endings and in bifurcations (Fig.3 Á). No similar GCs were described in the mammalian retinas. Type 5 and type 6, these two types of the traced GCs according to their peculiar dendritic appearance may be identified as "fast" DS GCs (Fig.4). Their rounded flat "lacy" dendritic arbours are of about 200-300 µ in diameter. Each type of the putative DS GCs in the fish retina is monostratified in one of two sublaminae of the IPL. In mammals such "lacy" dendritic appearance was visualized in the "fast" DS GCs by intracellular dye injection (Yang, Masland, 1994; Weng et al., 2005). Mammalian DS GCs are bistratified corresponding to their on-off physiological properties (Kittila, Massey, 1997). On the contrary, there are two separate (on and off) physiological types of the DS GCs in the fish retina (Maximov et al., 2005). Thus the revealed essentially monostratified morphology of "lacy" dendrites is in agreement with the physiological properties of the DS GCs. Morphological similarities of DS GCs in retinas of different animals imply common cellular mechanisms of the directional selectivity. Two types of monostratified starburst amacrine cells are considered to play a critical role in direction selectivity. However for the fish retina, Djupsund et al. (2003) suggested another candidate for the role of organizer of direction selectivity, namely, a specific type of big fusiform bistratified amacrine cells revealed by different methods exclusively in the fish retina - Fig. 5.

Figures

Fig. 1. A ganglion cell traced with DiI applied on the optic nerve cut in the retina of the pickarel (Spicara smaris).
Á: general view of the cell in the flatmount retina (the image was constructed of five optical sections). The axon of the cell and other axons go to the optic disc. Regularly arranged light spots are axonal terminals of bipolar cells. Â: drawing of the cell on the background of the grid formed by axonal terminals of bipolar cells.

Fig. 2. Type 1 ganglion cells traced with DiI from the optic nerve cut in the retinas of the goldfish Carassius auratus gibelio Bloch - Á and Â, and the eel Anguilla anguilla (Linnaeus) - ×.
Axons of other GCs extending in the direction of the optic disk are seen in Á. A magnified fragment of Á is shown in Â. Dendritic arborizations of these GCs lie in the same optic plane as the cell bodies. Thin short dendritic branches are indicated by arrows. The images were constructed from two optical sections. The sign of contrast was inverted in this figure and in the following ones.

Fig.3. Ganglion cells of two different types in the goldfish retina traced with DiI applied on the nerve cut.
Á: a type 4 GC with small dendritic field, initially thin dendrites and coarse varicosities in the sites of dendritic bifurcations and terminals. The image was made of three optical sections and inverted by sign of contrast. Â: a type 3 large GC of the goldfish retina with flat symmetric evenly branching dendritic tree. The plane of arborization is near the cell body. This GC is similar to ON delta GCs of mammalian retina. The image was made of two optical sections.

Fig. 4. Type 5 and type 6 GCs with flat middle-size rounded dendritic arbours of "lacy" appearance - putative "fast" direction selective ganglion cells:
Á,  and ×: DS GCs of the on-type branching in sublamina b of the IPL (images were constructed from three optical sections in Á, and from two optical sections in  and ×). Ç: two DS GCs of the off-type branching in sublamina a (image was constructed from three optical sections).

Fig. 5. Large fusiform cells (may be amacrine cells) in the flat-mount retina stained with methylene blue in the living goldfish retina.

Supplementary Figures

Fig.S1. Type 4 GCs with small dendritic fields and varicosities.
Two neighboring GCs with overlapping dendritic fields. The image was built from three optical sections.

References

Maximov V, Maximova E, Maximov P (2005) Direction selectivity in the goldfish tectum revisited, Annals of the New York Academy of Sciences 1048: 198-205

Maximova EM, Orlov OYu, Dimentman AM (1971) Investigation of visual system of some marine fishes, Voprocy ichtiologii 11: 893-899 (in Russian)

Kittila CA, Massey SC (1997) Pharmacology of directionally selective ganglion cells in the rabbit retina, J Neurophysiol 77: 675-689

Köbbert CC, Apps R, Bechmann I, Lanciego JL, Mey J, Thanos S (2000) Current concepts in neuroanatomical tracing, Progress in Neurobiology 62: 327-351

MacNeil MA, Heussy JK, Dacheux R, Raviola E, Masland RH (1999) The shapes and numbers of amacrine cells: matching of photofilled with Golgi-stained cells in the rabbit retina and comparison with other mammalian species, J Comp Neurol 413: 305-326

Mangrum WI, Dowling JE, Cohen ED (2002) A morphological classification of ganglion cells in the zebrafish retina, Visual Neurosci 19: 767-779

Rockhill RL, Daly FJ, MacNeil MA, Brown SP, Masland RH (2002) The diversity of ganglion cells in a mammalian retina, J Neurosci 22: 3831-3843

Weng S, Sun W, He S (2005) Identification of ON-OFF direction-selective ganglion cells in the mouse retina, J Physiol 562: 915-923

Yang G, Masland RH (1994) Receptive fields and dendritic structure of directionally selective retinal ganglion cells, J Neurosci 14: 5267-5280

Djupsund K, Furukawa T, Yasui S, Yamada M (2003) Asymmetric temporal properties in the receptive field of retinal transient amacrine cells, J Gen Physiol 122: 445-458

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