- Top of page
The visual system processes light images by projecting various representations of the visual world to segregated regions in the brain through parallel channels. Retinal bipolar cells constitute the first parallel channels that carry different light response attributes to different parts of the inner plexiform layer (IPL). Here we present a systematic study on detailed axonal morphology and light response characteristics of over 200 bipolar cells in dark-adapted salamander retinal slices by the whole-cell voltage clamp and Lucifer yellow fluorescence (with a confocal microscope) techniques. Four major groups of bipolar cells were identified according to the patterns of axon terminal ramification in the IPL: 36% were narrowly monostratified (whose axon terminals ramified in one of the 10 strata of the IPL), 27% were broadly monostratified, 19% were multistratified, and 18% bore pyramidally branching axons. By analysing the bipolar cells with narrowly monostratified axon terminals in each of the 10 strata of the IPL, we found that several key light response attributes are highly correlated with the strata in which the cells' axon terminals ramify. The 10 IPL strata appear to be the basic building blocks for attributes of light-evoked signal outputs in all bipolar cells, and several general stratum-by-stratum rules were identified by analysing the broadly monostratified, multistratified and pyramidally branching cells. These rules not only uncover mechanisms by which third-order retinal cells integrate and compute bipolar cell signals, but also shed considerable light on how bipolar cells in other vertebrates process visual information and how physiological signals may shape the morphology and projection of output synapses of visual neurones during development.
In addition to topologically mapping two-dimensional images of the outside world onto the two-dimensional array of retinal photoreceptors in each eye, the visual system projects different attributes of light signals through parallel channels to segregated regions in the brain (Enroth-Cugell & Robson, 1966; Hubel & Wiesel, 1977; Hubel & Livingstone, 1987). The first segregated projection region in the visual system is the inner plexiform layer (IPL) of the retina, in which output synapses of ON bipolar cells ramify at the proximal half (sublamina B) and those of the OFF bipolar cells ramify at the distal half of the IPL (sublamina A) (Famiglietti. & Kolb, 1976; Nelson et al. 1978). In mammalian retinas, anatomical studies have shown that rod bipolar cells send their axon terminals to the proximal regions of the IPL whereas cone bipolar cells send axons to more distal regions (Boycott & Wassle, 1991, 1999; Euler & Wassle, 1995; Roska & Werblin, 2001), but physiological characterization of light response attributes in these cells is still fragmentary. Moreover, it has been suggested that the mammalian retina contains more than a dozen types of retinal ganglion cells, each with dendrites ramifying at different strata in the IPL and each carrying a unique representation of the visual world (Roska & Werblin, 2001; Roska & Werblin, 2003). It is not clear, however, how retinal bipolar cells project various attributes of visual signals to different IPL strata for the ganglion cells to generate stratum-specific light responses. A systematic stratum-by-stratum analysis of the bipolar cell light responses is needed.
By comparing light responses and the cell morphology, a recent study has shown that bipolar cells in the salamander retina can be divided into 12 types, each exhibiting a unique set of light response attributes and a different pattern of axon terminal morphology in the IPL (Wu et al. 2000). However, this 12-type classification scheme may be incomplete because of the limited sample size and imprecise visualization of axonal processes by the conventional light microscope. Additionally, since this scheme only includes narrowly monostratified bipolar cells in five of the 10 strata of the IPL, it is difficult to determine complete stratum-by-stratum rules for the entire IPL. In this study, we investigated over 200 bipolar cells in living tiger salamander retinal slices and correlated their light response characteristics with more precise axon terminal morphology by studying Lucifer yellow fluorescence images with a confocal microscope. In addition to the 12 types of bipolar cells reported in the previous study, we found many more morphologically distinguishable types of bipolar cells, including narrowly monostratified cells in all 10 IPL strata. Moreover, we found that the axon terminal morphology of these cells correlates in an orderly fashion with several key light response attributes, which leads to the identification of stratum-by-stratum rules for narrowly monostratified cells and general rules for all bipolar cells. These rules describe how various attributes of light-evoked signals are projected to different regions of the IPL (where synapses with third-order retinal cells are made), and how parallel signalling pathways are organized in the visual system.
- Top of page
Larval tiger salamanders (Ambystoma tigrinum) purchased from Charles D. Sullivan, Co. (Nashville, TN, USA) and KON's Scientific Co. Inc. (Germantown, WI, USA) were used in this study. All animals were handled in accordance with the policies on treatment of laboratory animals of Baylor College of medicine and the National Institutes of Health.
Before each experiment, salamanders were anaesthetized in MS222 until the animal gave no visible response to touch or water vibration. The animals were then quickly decapitated and the eyes were enucleated. The procedures of dissection, retinal slicing and recording were described in previous publications (Werblin, 1978; Wu, 1987b). Dissection and recording were done under infrared illumination with a dual-unit Fine-R-Scope and the Nitemare infrared scopes BE Meyers, Redmond, WA, USA. Oxygenated Ringer solution was introduced continuously to the superfusion chamber, and the control Ringer solution contained 108 mm NaCl, 2.5 mm KCl, 1.2 mm MgCl2, 2 mm CaCl2 and 5 mm Hepes (adjusted at pH 7.7). All chemicals were dissolved in control Ringer solution. A photostimulator was used to deliver light spots (of diameter 600–1200 μm) to the retina via the epi-illuminator of the microscope. The intensity of unattenuated (log I= 0) 500 nm light was 2.05 × 107 photons μm−2 s−1. Since we delivered an un-collimated stimulus light beam through an objective lens with large numerical aperture (Zeiss 40×/0.75 water), the incident light entered the retinal slice in many directions, and thus the effect of photoreceptor self-screening was minor (Field & Rieke, 2002). The peak amplitude of light-evoked current responses was plotted against light stimulus intensity, and data points were fitted by the Hill equation:
where R is the current response amplitude, Rmax is the maximum response amplitude, σ is the light intensity that elicits a half-maximal response, N is the Hill coefficient, tanh is the hyperbolic tangent function and log is the logarithmic function of base 10. In this article, we used the R–log I plot for our analysis (the right-hand term of the above equation), and for such plots the light intensity span (dynamic range, DR: range of intensity that elicits responses between 5 and 95% of Rmax) of a cell equals 2.56/N (Thibos & Werblin, 1978).
Voltage-clamp recordings were made with an Axopatch 200A amplifier (Axon Instruments, Union City, CA, USA) connected to a DigiData 1200 interface and pCLAMP 6.1 software. Patch electrodes of 5 MΩ tip resistance (when filled with an internal solution containing 118 mm caesium methanesulphonate, 12 mm CsCl, 5 mm EGTA, 0.5 mm CaCl2, 4 mm ATP, 0.3 mm GTP, 10 mm Tris, 0.8 mm Lucifer yellow (Sigma, St Louis, MO, USA), and when adjusted to pH 7.2 with CsOH) were made with Narishige or Sutter patch electrode pullers. The chloride equilibrium potential (ECl) with this internal solution was about −60 mV. The equilibrium potential of cation current was determined by the reversal potential of glutamate-induced current in morphologically identified bipolar cells in Ringer solution containing 2 mm Co2+ (Wu & Maple, 1998). Light-elicited photoreceptor and amacrine cell inputs to bipolar cells were studied by recording the light-evoked cation and chloride currents, ΔIC and ΔICl, at holding potentials ECl and EC, respectively. Estimates of the liquid junction potential at the tip of the patch electrode prior to seal formation varied from −9.2 to −9.6 mV. For simplicity, we corrected all holding potentials by 10 mV. Spontaneous and light-evoked current responses were analysed by in-house software and SigmaPlot (Jandel, San Rafael, CA, USA). The average unitary sEPSCs (I1) in HBCs were analysed by the variance/mean method described by Katz & Miledi (1972). In this analysis, we approximated individual sEPSCs as ‘shot’ effects because the rising phase of sEPSCs is very fast (≤ 1 ms), and at least some events appeared to have single-exponential decay. This leads to I1= (variance of the cation current noise)/(2 × difference mean current in darkness and in bright light). If the decay time courses of individual sEPSC events are multiple exponential, our estimate will be off by a factor between 1 and 2 (Katz & Miledi, 1972).
Three-dimensional cell morphology was visualized in living retinal slices (250–300 μm in thickness) through the use of Lucifer yellow fluorescence with a confocal microscope (Zeiss 510). Images were acquired by using a × 40 water immersion objective (n.a. = 0.75), the 458 nm excitation line of an argon laser, and a long pass 505 nm emission filter. Consecutive optical sections were superimposed to form a single image using Zeiss LSM PC software, and these compressed image stacks were further processed in Adobe Photoshop 6.0 to improve the contrast. Since signal intensity values were typically enhanced during processing to improve the visibility of smaller processes, the cell bodies and larger processes of some cells appear saturated due to their larger volume of fluorophore. Although the background images of the retinal slices were acquired simultaneously with the fluorescent cells, they were imaged using transmitted light. The level at which dendritic processes stratified in the IPL was characterized by the distance from the processes to the distal margin of the IPL. We selected cells in the bipolar cell layers with somas situated beneath the surface of the slice and they usually had relatively intact processes (assessed by rotation of the stacked images).