- Title
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Encoding of luminance and contrast by linear and nonlinear synapses in the retina
- Authors
- Odermatt, B., Nikolaev, A., and Lagnado, L.
- Source
- Full text @ Neuron
The Zebrafish ribeye a (ctbp2) Promoter Drives Expression in Neurons Containing Ribbon Synapses (A) Imaging synaptic reporters in the retina of live zebrafish using a two-photon microscope. Full-field stimuli were applied through a light guide.(B and C) A stable transgenic fish expressing membrane targeted EGFP (memEGFP) under control of 1.8 kb of the genomic sequence upstream of the ribeye a gene (Tg(-1.8ctbp2:memEGFP)lmb). At 4 dpf, all sensory organs known to express ribbon synapses were labeled, including the retina, the inner ear (white asterisk), the pineal gland (bold arrow), and the neuromasts (arrow heads). (B) Side view, (C) top view of the fish head. Additionally EGFP expression can be seen in the optic nerve and the optic tectum (black asterisk).(D) In a fish at 7 dpf, EGFP expression is driven in hair cells of the inner ear (side view) and maculae (not shown).(E and F) EGFP expression in the pineal gland and a neuromast, respectively (side view; 7 dpf).(G) In the retina, the ribeye a promoter drove expression of memEGFP in photoreceptors and bipolar cells.(H) Labeled photoreceptors in the outer nuclear layer (ONL), their terminals in the outer plexiform layer (OPL), cell bodies of bipolar cells in the inner nuclear layer (INL), and their terminals in the inner plexiform layer (IPL).(I) Expression of sypHy localized to terminals in the OPL and IPL in the stable Tg(-1.8ctbp2:sypHy)lmb line used in this study (see also Figure S1). |
In Vivo Imaging of Synaptic Transmission in the Retina (A) Field of view showing sypHy expression in synaptic terminals of bipolar cells in the IPL of a fish at 10 dpf.(B) ROIs from the same field highlighted in different colors. When viewed at highest resolution, numbers mark ON terminals and red numbers OFF. Nonresponding terminals numbered in white.(C) Difference images highlighting the change in sypHy fluorescence in response to steps of light. Attenuation of the light source is shown in log units (ND 4 to ND 1). Darker areas show OFF terminals; brighter areas are ON.(D) Raster plot showing the relative change in fluorescence (ΔF/F) for each ROIs marked in (B). The intensity of the stimulus was increased in steps of 1 log unit, with a maximum intensity of 5.5 × 105 photons/μm2/s.(E) Responses of five individual ON terminals to light steps increasing in intensity by 0.5 log units. Darker hues indicate more sensitive terminals. The black arrows highlight some examples of switches in response polarity.(F) Responses of five individual OFF terminals (see also Figure S2). |
Calculating the Rate of Vesicle Release from sypHy Signals (A) Electron micrographs indicate that there are about 15,000 vesicles in an average bipolar cell terminal (background). The number of unquenched sypHy molecules on the surface depends on both the rate of exocytosis and the rate of endocytosis (foreground).(B) Estimating the rate of endocytosis in vivo: comparison of the sypHy signal in response to a bright step of light (ND 1) averaged from a population of 95 ON terminals (green) and 272 OFF terminals (red). In OFF terminals, the sypHy signal decayed exponentially with τ ~10 s (black line). In ON terminals, the signal decayed at the same rate when the light step was turned off. In both channels, acceleration of vesicle release generated a sypHy signal that rose at a constant average rate for the first 2 s (black lines).(C) Estimation of the sypHy surface fraction (αmin) by acid quenching. First, responses to a step of bright light (ND 1) were measured in ON and OFF terminals at pH 7.4. Then, sypHy molecules on the surface in darkness were quenched with a solution at pH 3.2. The difference between the minimum fluorescence at pH 7.4 and pH 3.2 reflects quenching of the surface fraction (dashed lines). Traces averaged from 10 fish. αmin averaged 0.8% in ON and OFF terminals (see also Figure S3).(D) Upper traces: average fluorescence response of ON (green) and OFF (red) terminals to a 40 s light step (ND 1). The response and recovery phases could both be described as double-exponential functions (smooth lines). Lower traces: a comparison is shown of the conversion of the fluorescence response to rates of vesicle release, Vexo(t), using the raw sypHy signal (noisy trace) and the fitted traces that minimize noise. Thick black bars in upper graph show the values of Fmin used for this calculation, as described in Experimental Procedures. |
Expression of fluorescent proteins in the zebrafish retina under the ribeye a (ctbp2) promoter (A) Confocal images of a retinal section from a stable transgenic line of fish (Tg(- 1.8ctbp2:memEGFP)lmb) expressing membrane-targetted EGFP under the ribeye a promoter (8 dpf). The image shows a densely packed layer of photoreceptors in the outer nuclear layer (ONL), their terminals in the outer plexiform layer (OPL), cell bodies of bipolar cells in the inner nuclear layer (INL) and their terminals in the inner plexiform layer (IPL, sublamina a and b). Note the complete overlap between memEGFP and ON bipolar cells positive for PKCα staining (red). The neurons stained blue are positive for parvalbumin, a marker of A2 amacrine cells and displaced amacrine cells in the ganglion cell layer (GCL): note that the ribeye a promoter does not drive expression in these (merged image to right). (B) Confocal images of a retinal section from a stable transgenic line of fish (Tg(- 1.8ctbp2:sypHy)lmb) expressing sypHy under the ribeye a promoter (10 dpf). SypHy expression has been marked with an anti-GFP antibody. Expression is driven in photoreceptor terminals in the outer plexiform layer (OPL), and in bipolar cell terminals in the inner plexiform layer (IPL, sublamina a and b). There was complete overlap of sypHy expression and anti-PKCα staining (red, ON-bipolar terminals) in sublamina b of the IPL, while PKCα negative terminals were mainly situated in sublamina a (OFF-bipolar terminals). There is also green auto-fluorescence in photoreceptor outer segments. |