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Cullen Eye Institute, Baylor College of Medicine, Houston, TX 77030, USA
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Abstract |
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IC) and inhibitory chloride current (ICl) of rod and cone depolarizing bipolar cells (DBCRs and DBCCs) and AII amacrine cells (AIIACs) in dark-adapted mouse retinal slices were studied by whole-cell voltage-clamp recording techniques, and the cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. IC of all DBCRs exhibited similar high sensitivity to 500 nm light, but two patterns of ICl were observed in DBCRs with slightly different axon morphology. At least two types of DBCCs were identified: one with axon terminals ramified in 70–85% of the depth of the inner plexiform layer (IPL) and DBCR-like IC sensitivity, whereas the other with axon terminals ramified in 55–75% of IPL depth and much lower IC sensitivity. The relative rod/cone inputs to DBCs and AIIACs were analysed by comparing the IC and ICl thresholds and dynamic ranges with the corresponding values of rods and cones. On average, the sensitivity of a DBCR to the 500 nm light is about 20 times higher than that of a rod. The sensitivity of an AIIAC is more than 1000 times higher than that of a rod, suggesting that AIIAC responses are pooled through a coupled network of about 40 AIIACs. Interactions of rod and cone signals in dark-adapted mouse retina appear asymmetrical: rod signals spread into the cone system more efficiently than cone signals into the rod system. The mouse synaptic circuitry allows small rod signals to be highly amplified, and effectively transmitted to the cone system via rod–cone and AIIAC–DBCC coupling.
(Received 2 April 2004;
accepted after revision 4 June 2004;
first published online 4 June 2004)
Corresponding author S. M. Wu: Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, NC-205, Houston, TX 77030, USA. Email: swu{at}bcm.tmc.edu
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Introduction |
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Anatomical evidence indicates that DBCRs in most mammals do not send synaptic outputs directly to ganglion cells, the output neurones of the retina, but to the AII amacrine cells (AIIACs) which make electrical synapses with DBCCs (which send signals to ‘on’ ganglion cells) and inhibitory chemical synapses with HBCCs and ‘off’ ganglion cells. Therefore, the rod bipolar cell signals are transmitted to ganglion cells by virtue of the AIIACs that ‘piggyback’ on the cone bipolar cell synaptic circuitry (Kolb & Famiglietti, 1974; Strettoi et al. 1990, 1992, 1994; Tsukamoto et al. 2001). For this reason, the AIIACs serve as communication hubs of the rod and cone signals in the inner mammalian retina. It is of great interest to understand how rod and cone bipolar cell signals are transmitted to AIIACs, and how various electrical and chemical synapses contribute to light-evoked responses of these cells.
Despite the vast amount of anatomical data on synaptic connections between rods, cones, rod and cone bipolar cells, and AIIACs, detailed physiological information on the light responses of these cells in the mammalian retina is still fragmentary. For example, although microelectrode recordings from rabbit and cat retina reveal the voltage response and receptive field properties of DBCRs, DBCCs and AIIACs in rabbits and cats (Nelson, 1982; Bloomfield et al. 1997; Bloomfield & Xin, 2000), it is not clear about the relative contributions of the excitatory and inhibitory synaptic currents to these voltage responses. Additionally, it is uncertain how individual electrical and chemical synapses identified by anatomical studies affect the light-evoked signals.
In this study, we used the whole-cell voltage-clamp technique to record light-evoked responses from the DBCRs, DBCCs and AIIACs in the dark-adapted mouse retinal slices. This technique allows us to separate light-evoked excitatory cation current and inhibitory chloride current and to reveal the cell morphology by Lucifer yellow fluorescence. We chose the mouse retina because physiological results obtained in wild-type mice can later be correlated with findings in genetically manipulated mice. Moreover, the cellular and synaptic inputs to these cells have been well characterized at the ultrastructural level (Carter-Dawson & Lavail, 1979; Tsukamoto et al. 2001) and thus physiological findings can be readily correlated with anatomical observations.
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Methods |
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Our study constitutes a systematic voltage-clamp analysis of rod and cone bipolar and amacrine cell light responses in the dark-adapted mouse retina. The major advantage of this approach is that excitatory and inhibitory current responses can be separated by holding the membrane potential near chloride and cation reversal potentials, respectively. Thus the photoreceptor and amacine cell contributions to the bipolar cells' light responses and the bipolar cell and amacrine cell contributions to the amacrine cells' light responses (i.e. cation (photoreceptor or bipolar cell inputs) and chloride currents (amacrine cell inputs), or IC and ICl, respectively) can be differentially recorded. Since the sensitivity of mouse rod pigment to 500 nm light is about 2 log units higher than that of the M-cone pigment, and about 4 log units higher than that of the S-cone pigment (Lyubarsky et al. 1999), we use the response threshold to 500 nm lights to estimate the relative rod/cone contributions to IC and ICl in each ‘on’ bipolar cell and amacrine cell under dark-adapted conditions (the relative sensitivities of rods, and M- and S-cone pigments to 500 nm lights under light-adapted conditions, on the other hand, are different, and thus the present study is limited to rod/cone contributions to ‘on’ bipolar cells and AIIACs under dark-adapted conditions). Another advantage of this approach is that the cell's morphology can be easily revealed by Lucifer yellow filling with the recording electrode (assisted by a confocal microscope). This allows us to characterize the morphology of each recorded cell and to compare it with results of earlier anatomical studies.
Preparations and light stimulation
The mouse strain used in this study was C57Black6J from Jackson Laboratory (Bar Harbour, ME, USA). All animals were handled in accordance with Baylor College of Medicine's policies on the treatment of laboratory animals. Mice were dark-adapted for 1–2 h prior to the experiment. To maintain the retina in the fully dark-adapted state, all further procedures were performed under infrared illumination with dual-unit Nitemare (BE Meyers, Redmond, WA, USA) infrared scopes. Animals were killed by an I.P. injection of ketamine + xylazine + acepromazine (0.1 ml, 100 mg ml–1) and the eyes were immediately enucleated and placed in oxygenated Ames' medium (Sigma, MO, USA) at room temperature. The dissection and preparation of living retinal slices followed essentially the procedures described in previous publications (Werblin, 1978; Wu, 1987). Oxygenated Ames' solution (adjusted to pH 7.3) was introduced continuously to the recording chamber, and the medium was maintained at 35°C by a temperature control unit (TC 324B, Warner Instruments, CT, USA). All pharmacological agents were dissolved in Ames' medium.
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 (logI= 0) 500 nm light was 1.4 x 106 photons µm–2 s–1. The number of photoisomerizations per rod per second (Rh* rod–1 s–1) was estimated by methods described in a previous publication (Pang et al. 2003). Since we delivered an uncollimated stimulus light beam through an objective lens with large numerical aperture (Zeiss, x40, 0.75 NA, water immersion), the incident light enters the retinal slice in many directions, and thus the effect of photoreceptor self-screening is minor (Field & Rieke, 2002a). The peak amplitude of light-evoked current responses was plotted against light stimulus intensity, and data points were fitted by the Hill equation:
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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 use the R–logI plot for our analysis (the right-hand term of the above equation), and for such plots the light intensity span (dynamic range: 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
Voltage-clamp recordings were made with an Axopatch 200A amplifier connected to a DigiData 1200 interface and pCLAMP 6.1 software (Axon Instruments, Foster City, CA, USA). Whole cell voltage-clamp recordings were made with patch electrodes made with Narishige or Sutter patch electrode pullers that were of 5–7 M
tip resistance when filled with 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, adjusted to pH 7.2 with CsOH. The chloride equilibrium potential, ECl, with this internal solution was about –60 mV. 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 (Pang et al. 2002). For simplicity, we corrected all holding potentials by 10 mV. In order to determine the dark membrane potentials of ganglion cells, we measured the zero-current potentials of 12 bipolar cells and 8 amacrine cells (including all cell types described in this paper) with patch electrodes filled with caesium internal solution (above) and with potassium internal solution (Berntson & Taylor, 2000), and found that the zero-current potentials with K+ were consistently 12–17 mV more hyperpolarized that with Cs+. For cells recorded with only Cs+, we corrected the zero-current potential measured in darkness (dark membrane potential) by 15 mV. Since the DBCRs and DBCCs have relatively narrow dendritic and axonal fields, it is likely that a large portion of the cell membrane was space-clamped. In order to maximize good space-clamping, we selected cells with higher input resistance (> 500 M) when whole cell recording was made. To further verify that DBC processes are space-clamped in our study, we examined the effects of GABA and glycine receptor antagonists (picrotoxin and strychnine) on IC (recorded at ECl) of eight DBCRs and nine DBCCs and found that these compounds did not alter the amplitude of IC (but suppressed ICl), suggesting that DBC processes (where inhibitory synaptic inputs are located) were clamped at the command potential by the patch electrodes in the soma. In order to select cells with minimum slicing damage, we recorded from cells that were 2–3 cell layers below the surface of the retinal slice. AIIACs are electrically coupled with one another and it is impossible to space-clamp the coupled network. Since the dark membrane potential of AIIACs is close to ECl (see Results), we studied the light responses of these cell by measuring IC near ECl.
Visualization of cell morphology
Cell morphology was visualized in retinal slices using Lucifer yellow fluorescence with a confocal microscope (Zeiss 510). Images were acquired with a x40 water immersion objective (NA = 1.20), using 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 signal to noise ratio. 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. The level at which dendritic processes stratified in the IPL was characterized in retinal vertical sections by the distance from the processes to the distal margin (0%) of the IPL.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Under infrared visual guidance in dark-adapted mouse retinal slices, we recorded rod ‘on’ bipolar cells (DBCRs) with patch electrodes filled with internal solution and Lucifer yellow in the whole-cell voltage-clamp configuration. DBCRs were identified by their characteristic morphology (with globula (large knob-shaped swellings) axon terminals ramifying between 70 and 100% of the IPL depth; Euler & Wassle, 1995; Hartveit, 1997) revealed by Lucifer yellow fluorescent images in retinal slices and by their inward light-evoked cation current (IC) recorded at ECl. Figure 1A shows the stacked confocal fluorescent image of a DBCR in the retinal slice; it exhibited typical DBCR morphology with the dendritic field extending about 20 µm horizontally in the outer plexiform layer (OPL), a soma located in the upper half of the inner nuclear layer (INL), an axon extending into the IPL and globular axon terminals stratifying in the inner half of the IPL. The morphology of all 18 DBCRs was very similar with the exception that two-thirds (12/18) of them had axon terminal globules distributed in 75–100% of the IPL depth (similar to Fig. 1A, group 1 DBCR) whereas the remaining one-third (6/18) had axon terminal globules distributed more proximally (70–85% of the IPL depth, Fig. 2A, group 2 DBCR). The light-evoked current responses of these two groups of DBCRs to a 0.5 s 500 nm light step recorded under dark-adapted conditions at various holding potentials are shown in Figs 1B and 2B, and the current–voltage relations in Figs 1C and 2C. The light-evoked current responses of the two groups of DBCRs are very similar, with apparent reversal potentials near –40 mV. Both groups exhibited inward IC (at ECl) and outward ICl (at EC). The mean zero-current potential in darkness of these two groups of cells was –59 ± 5 mV and –57 ± 6 mV, respectively (see Methods). Since DBCRs in the mammalian retina receive glutamatergic inputs from rods (Qin & Pourcho, 1996; Brandstatter et al. 1998) and GABAergic/glycinergic inputs from the reciprocal rod amacrine cells (Freed et al. 1987; Sandell et al. 1989; Fletcher & Wassle, 1999; Zhang et al. 2002), IC is likely to be mediated by rod inputs that gate a cation conductance and the ICl is probably mediated by reciprocal rod amacrine cell inputs that gate a chloride conductance (Cohen & Miller, 1994). To verify that IC and ICl in DBCRs are mediated by different synaptic inputs, we examined the effects of GABA and glycine receptor antagonists (picrotoxin (PTX) and strychnine (STR)) on five group 1 DBCRs and three group 2 DBCRs and found that these compounds did not significantly alter the amplitude of IC (peak IC in 100 µM PTX + 1 µM STR was 1.11 ± 0.23 (n= 6) of peak IC in control solution, and peak IC in 100 µM PTX + 1 µM STR was 0.92 ± 0.18 (n= 2) of peak IC in control solution). This suggests that the rod inputs to DBCRs are not significantly (P= 0.68, paired t test) influenced by GABAergic and glycinergic interneurones in the mouse retina.
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IC and ICl elicited by 500 nm light (as discussed in Methods, 500 nm light allows us to separate the rod, M-cone and S-cone contributions to IC and ICl) of various intensities (Figs 1D and E, and 2D and E). In both groups of DBCRs, the –7 light step elicited a small IC, whereas the –6 (0.7 Rh* rod–1 s–1) light step gave rise to an inward IC of about 25 pA. As the light step became brighter, the inward IC became larger. We measured the light sensitivity of IC to 500 nm light in all 12 group 1 DBCRs and 6 group 2 DBCRs, and the mean (±S.D.) response–intensity (IC–logI) relations are plotted in Figs 1F and 2F. The continuous curve was fitted by the Hill equation (see Methods). The mean thresholds (defined as eliciting 5% of the maximum response) were the same for the two groups of DBCRs (–6.8 = 0.1 Rh* rod–1 s–1), and the dynamic ranges were 3.1 and 3.4 log units, about 1.1–1.4 log units wider (extended to the left along the intensity axis) than that of the rod photocurrent (Field & Rieke, 2002b). Possible mechanisms underlying the dynamic range widening will be discussed later.
Our results show that all DBCRs exhibited light-evoked chloride current (ICl) at EC, which was not observed by Berntson & Taylor (2000). The mean threshold of light-evoked inhibitory current ICl of the 12 group 1 DBCRs was –7.2 (0.044 Rh* rod–1 s–1) and that of the 6 group 2 DBCRs was –7.6 (0.018 Rh*rod–1 s–1), suggesting that the amacrine cells mediating ICl of DBCRs have rod-like threshold. The mean (±S.D.) response–intensity (ICl–logI) relations (shown in Figs 1F and 2F) had mean dynamic ranges of 2.3 and 1.2 log units, suggesting that the amacrine cell inputs to the two groups of cells are different (in the 8 DBCRs in which we examined the effects of GABA and glycine receptor antagonists (see above), we found that ICl of group 1 and group 2 DBCRs exhibited different sensitivities to strychnine and picrotoxin, suggesting that these two groups of cells receive different GABAergic and glycinergic amacrine cell inputs; data not shown, J.-J. Pang, F. Gao & S. M. Wu, manuscript in preparation). Additionally, there are more spontaneous inhibitory postsynaptic currents (sIPSCs) in group 2 DBCRs (Figs 1E and 2E). Mean response thresholds, dynamic range saturation intensities as well as the dark membrane potentials of the two groups of DBCRs are listed in Table 1.
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Anatomical evidence has suggested DBCRs in the mammalian retina do not make output synapses on ganglion cells, but primarily on the AII amacrine cells (AIIACs) (Kolb & Famiglietti, 1974; Freed et al. 1987; Veruki & Hartveit, 2002a,b). In order to study the output of the rod bipolar cells, we examined the light response characteristics of morphologically identified AIIACs. We selected cells with the soma located near the inner half the INL and made whole cell voltage-clamp recordings with patch electrodes filled with internal solution and Lucifer yellow. AIIACs were identified by their characteristic morphology (thick globular dendrites in the distal half and pyramidally branching dendrites in the proximal half of the IPL, and with dendritic width less than 30 µm; Famiglietti & Kolb, 1975; Strettoi et al. 1992) revealed by Lucifer yellow fluorescent images in retinal slices and the inward light-evoked cation current (IC) recorded at ECl. Figure 3A shows the stacked confocal fluorescent image of an AIIAC in the retinal slice with the typical AIIAC morphology. We recorded from a total of 15 cells with very similar morphology in retinal slices, and the dendritic width varied between 20 and 35 µm. The light-evoked current responses of an AIIAC to a 2.5 s 500 nm light step at various holding potentials are shown in Fig. 3B, and the current–voltage (I–V) relations in Fig. 3C. We found that all 15 cells with AIIAC morphology had very similar I–V relations, and that the light-evoked current response is inward at all holding potentials (i.e. it did not have a reversal potential at least between –100 mV and +40 mV). This may be explained by the anatomical findings that AIIACs are extensively coupled with adjacent AIIACs and cone ‘on’ bipolar cells (Bloomfield & Xin, 2000; Veruki & Hartveit, 2002a), and thus light-evoked inward current from unclamped neighbouring cells may dominate the cells' response at all potentials. The mean zero-current potential in darkness of the 15AII ACs was –65 ± 5 mV.
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IC, Figs 1F and 2F). This makes the AIIAC response threshold very low (near –9.0, or 0.0007 Rh* rod–1 s–1), about 100 times lower than that of the DBCRs, suggesting a large signal gain at the DBCR–AIIAC synapses.
Table 2 lists the mean peak IC responses of 10 DBCRs and 11 AIIACs to the 9, –8, –7 and –6 (500 nm) lights. The mean peak IC responses of DBCRs to –6 and AIIACs to –8, –7 and –6 were obtained from original response records; responses of DBCRs to –7 and AIIACs to –9 were obtained from signal-averaged records (10 repeated responses each for increasing signal/noise ratio). DBCR responses to –8 and –9 were extrapolated from the mean response–intensity relation because they were too small (note: the small DBCRIC in Table 2 (0.065 pA) is the theoretical mean response that represents a threshold response of about 9 pA in one out of 143 DBCRs that converge to an AIIAC; see Discussion below). By using this method, we estimated the current gain, defined as the ratio [peak IC(AIIAC)]/[peak IC(DBCR)], elicited by a given light, of the DBCR–AIIAC synapse, and the values are listed in Table 2. It is evident that the current gain is higher at smaller IC values (elicited by the –9 and –8 lights), and became increasingly lower when IC were larger (elicited by the –7 and –6 lights). Since the coupled AIIAC network receives synaptic inputs from many DBCRs, our data suggest that signal convergence from DBCRs to AIIACs is amplitude dependent. Possible mechanisms underlying such non-linearity will be discussed later.
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Cone ‘on’ bipolar cells (DBCCs) were identified by their axonal morphology (ramified in the proximal half of the IPL, with relatively fine axon terminal branches, wider lateral extension and small vesicular expansions along the terminal branches, instead of the globular, large knob-shaped terminals characteristic of the DBCRs (Boycott & Wassle, 1999) revealed by Lucifer yellow fluorescent images in retinal slices and the inward light-evoked cation current (IC) recorded at ECl. Anatomical evidence shows that the morphology of DBCCs is more heterogeneous than the DBCRs, and the axon terminals of different types of DBCCs ramified at different depths in the IPL (Euler & Wassle, 1995). In addition, there are two types of cone pigments in the mouse retina, the M and S (or UV) pigments, and different cones contain M and S pigments of various proportions (Applebury et al. 2000). Furthermore, cones are electrical coupled with rods (Tsukamoto et al. 2001), and thus the light response of some cones may be influenced by the rod pigment. These results suggest that the light response of DBCCs may be more heterogeneous than that of the DBCRs.
Figure 4A shows the stacked confocal fluorescent image of a DBCC in the retinal slice, and it exhibited typical DBCC morphology with branching axon terminals stratifying between 70% and 85% of the IPL depth, and Fig. 5A shows the stacked confocal fluorescent image of another DBCC with branching axon terminals stratifying between 55% and 75% of the IPL depth. These two types of DBCC had similar current–voltage relations (Figs 4B and C, and 5B and C), the same mean dark membrane potential (–56 ± 4 mV), but very different response thresholds and dynamic ranges for IC and ICl elicited by the 500 nm light (Figs 4D, E and F, and 5D, E and F). IC in Fig. 4 exhibited a threshold near –6.9 (0.088 Rh* rod–1 s–1) and a dynamic range of 4.6, and IC in Fig. 5 showed a threshold near –4.9 (8.8 Rh* rod–1 s–1) and a dynamic range of 2.1. Therefore the first types of DBCC (named DBCC1) receive substantial input from rods in addition to the cones (judged by the dynamic range span for the 500 nm light), and the second type of DBCC (DBCC2) receive input predominantly from the cones with much weaker rod inputs (thus lower sensitivity to 500 nm light and narrower dynamic range).
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IC and ICl in DBCCs are mediated by different synaptic inputs by examining the effects of GABA and glycine receptor antagonists on five DBCC1s and four DBCC2s and found that these compounds did not significantly alter the amplitude of IC (peak IC in 100 µM PTX + 1 µM STR was 1.15 ± 0.19 (n= 9) of peak IC in control solution). This suggests that GABAergic and glycinergic interneurones exert little influence on photoreceptor inputs to DBCCs in the mouse retina (the small increase in peak IC in PTX and STR may suggest, however, GABAergic/glycinergic feedback from horizontal cells to cone photoreceptors; detailed analysis will be presented in a future publication, J.-J. Pang, F. Gao & S. M. Wu, in preparation).
ICl in Fig. 4 exhibited a threshold near –6.9 (0.088 Rh* rod–1 s–1) and a dynamic range of 1.8, and that in Fig. 5 had a threshold of 5.3 and a dynamic range of 2.10. Similar to the two groups of DBCRs, DBCC1 and DBCC2 also differed in spontaneous inhibitory postsynaptic currents (sIPSCs). sIPSCs in DBCC2 were larger and of higher frequency. These results suggest that DBCC1s and DBCC2s receive inhibitory inputs from different populations of amacrine cells (in the 5 DBCC1s and 4 DBCC2s in which we examined the effects of GABA and glycine receptor antagonists, we found that ICl of DBCC1s and DBCC2s exhibited different sensitivities to strychnine and picrotoxin, suggesting that these two types of DBCCs receive different GABAergic and glycinergic amacrine cell inputs (data not shown, J.-J. Pang, F. Gao & S. M. Wu, in preparation)). The mean response thresholds and dynamic ranges as well as the dark membrane potentials of the two groups of DBCCs are listed in Table 1.
Among 22 DBCCs, 12 had DBCC1-like morphology and light responses, 4 had DBCC2-like morphology and light responses, and the remaining 6 had axon terminals branching within 60–90% of the IPL depth, and exhibited response thresholds and dynamic ranges between the corresponding values of the DBCC1s and DBCC2s. These results suggest that DBCCs in the mouse retina are heterogeneous, 55% of them are DBCC1-like, 18% are DBCC2-like, and the remainder have intermediate responses.
Mean response–intensity spans of depolarizing bipolar cells and AII amacrine cells in dark-adapted mouse retina
In order to determine the relative contributions of rod and cone inputs to various types of DBCs and AIIACs, we plot in Fig. 6 the intensity spans of various cells in response to 500 nm light stimuli. Each horizontal bar marks the mean dynamic range (light intensity from threshold (5% of the maximum response) to saturation (95% of the maximum response) of a cell type. It is evident that the dynamic range of rod-mediated responses (IC recorded at ECl) in both groups of DBCRs (from –7.0 to –3.88, and from –7.0 to –3.90) is about 1.1–1.4 log units wider than that of the rod photocurrent (from –5.57 to –3.57) (Howes et al. 2002; Field & Rieke, 2002b). The extension of the dynamic range is at the low intensity side (towards left along the intensity axis), while the saturation intensity is close to the saturation intensity for the rod photocurrent.Similarly, the dynamic range of the AIIACs (from –9.0 to –4.08) is about 1.8 log units wider that that of the DBCRs, the widening is at the low intensity side, and the saturation intensity is close to the saturation intensity for the rod and DBCR responses. These plots clearly suggest amplification of small rod signals by the rod–DBCR synapses and small DBCR signals by the DBCR–AIIAC synapses. The observation that both DBCR and AIIAC responses saturate near the saturation intensity for the rod photocurrent (but not extending into the cone response dynamic range) indicate that cone inputs to DBCRs and AIIACs under dark-adapted conditions are relatively weak.
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IC is 4.7 log units (ranging from –6.9 to –2.2), and that of DBCC2IC is 2.68 log units (ranging from –4.9 to –2.22). Both types of DBCCs saturate near the saturation intensity of the M-pigment-dominated cones, but the thresholds to the 500 nm lights are very different. The mean threshold of DBCC1s is very close to that of the DBCRs, suggesting that these cells receive strong rod inputs in their IC. The mean threshold of DBCC2s is two log units higher, indicative of much weaker rod inputs in their IC.
We also plot in Fig. 6 the mean dynamic ranges of ICl of DBCRs and DBCCs. It is evident that ICl of different types of DBCs are very different, suggesting that the amacrine cell inputs to these cells are very complex. In order to compare the DBC and AIIAC responses with their output neurones, we added the mean dynamic ranges of IC and ICl of the mouse 'on' alpha ganglion cells (ON
GCs) derived from a previous publication (Pang et al. 2003) at the bottom of Fig. 6. The mean dynamic range of the ONGC IC is very close to that of the DBCC1 and overlaps with the dynamic range of DBCC2IC, consistent with the notion that ONGC IC is primarily mediated by the DBCC output synapses. The dynamic range of ONGC ICl is wider than the ICl ranges of the DBCRs and DBCCs, and it is located at an intensity range two log units less sensitive than the dynamic range of the AIIAC IC, suggesting that the inhibitory inputs to ONGCs (black arrow 5 in Fig. 7) may involve more than one type of amacrine cell. It is unlikely that the AIIACs make a significant contribution to the ONGC ICl (they contribute ONGC IC through DBCC1s via gap junctions) because the ONGC ICl threshold is much lower than the AIIAC responses. AIIACs may contribute more significantly to the light responses of other ganglion cells, such as the sustained ‘off’GCs described in a previous report (Pang et al. 2003).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Based on our analysis of light-evoked cation and chloride currents (IC and ICl), we propose a functional synaptic circuitry diagram of the rod, cone and AC inputs to DBCRs, DBCCs and AIIACs in the mouse retina (Fig. 7). The outlines of this diagram are consistent with the general plan set forth by anatomical data, but with several new and more detailed findings revealing how synapses in the mouse retina function.
We found that the light-evoked cation current, IC, of DBCRs are homogeneous, although two groups of cells can be distinguished morphologically by the slight difference in axon terminal stratification in the IPL, and more importantly, by their inhibitory inputs. All DBCRs are highly sensitive to 500 nm lights, consistent with anatomical data, suggesting that bipolar cells with globular axon terminals at 70–100% of the IPL depth contact only rods (Euler & Wassle, 1995; Hartveit, 1997; Tsukamoto et al. 2001). The light-evoked chloride current (ICl) of DBCRs are mediated by amacrine cells with rod-like threshold, consistent with anatomical observations that DBCRs receive reciprocal feedback synapses from DBCR-driven amacrine cells (Nelson & Kolb, 1985; Freed et al. 1987; Sandell et al. 1989; Hartveit, 1999). We propose that ICl in the mouse DBCRs are mediated by amacrine cells in the inner retina because physiological evidence has shown that chloride currents were observed when inhibitory neurotransmitters were applied to the axon terminal regions in the IPL (McCall et al. 2002). The dynamic ranges of ICl in DBCRs are substantially narrower than those of IC, indicating that synaptic clipping (Attwell et al. 1987) may occur in the DBCR
reciprocal rod ACDBCR feedback loop. Our observation that the two groups of DBCRs differ in ICl dynamic range and sIPSCs suggests that the reciprocal rod AC inputs to DBCRs with axon terminals at two levels of IPL may not be identical. However, since the dark membrane potential of all DBCRs are very close to ECl (Table 1), light-evoked voltage responses of the two groups of DBCRs under dark-adapted conditions may be similar (the different ICl may contribute small differences in the voltage responses by shunting). For this reason, we do not feel it is justified to state that there are two types of DBCR, but merely propose that DBCRs receive reciprocal inputs from different amacrine cells at two strata of the IPL.
In all 15 AIIACs, we were unable to reverse the light-evoked current. This is consistent with anatomical findings that these cells are strongly coupled with one another, and with DBCCs (Famiglietti & Kolb, 1975; Kolb & Nelson, 1993; Vaney, 2002; Veruki & Hartveit, 2002a). As the light-evoked current in a given AIIAC is pooled from a network of cells, it was impossible to depolarize all coupled neurones beyond their reversal potentials.
We show that there are at least two types of DBCC in the mouse retina with distinguishable morphology and light responses. DBCC1s have axon terminals ramified in 70–85% of IPL depth, a IC threshold near that of the DBCRs and a saturation intensity near that of the M-pigment-dominated cones. It is likely that these cells receive strong rod signals through gap junctions between rods and cones, and/or between AIIACs and their own axon terminals (Deans et al. 2002; Veruki & Hartveit, 2002a) (Fig. 7). DBCC2s have axon terminals ramified in 55–75% of the IPL depth and a much less sensitive IC, indicating that their rod-mediated inputs (rod–cone and AIIAC coupling) are much weaker. The ICl threshold of DBCC1s is about 1.5 log units lower than that of the DBCC2s, suggesting that amacrine cells making reciprocal inhibitory synapses on the former DBCs are more rod-dominated than the amacrine cells synapsing on the latter. In addition to DBCC1s and DBCC2s, we also recorded six DBCCs with mixed DBCC1/DBCC2 light response and morphological characteristics. Although axon terminals of mouse DBCs are rarely as narrowly monostratified in the IPL as the salamander bipolar cells (Wu et al. 2000), the general rod/cone dominance rule set forth by the salamander bipolar cells is obeyed: axon terminals of DBCs with stronger rod inputs ramified closer to the ganglion cell layer and those with stronger cone inputs ramified closer to the centre of the IPL.
It is important to note that we may not have included all morphological types of depolarizing bipolar cells in this study. For example, immunocytochemical evidence has shown that there are four types of ‘on’ cone bipolar cells in the rat retina (Euler & Wassle, 1995). It is possible that some of these cells are less accessible to our patch electrodes and/or they exhibit smaller light responses than DBCC1 and DBCC2 in dark-adapted retinal slices.
Signal convergence and amplification of the DBCR–AIIAC synapse
Anatomical studies show that 22 rods converge to a DBCR in the mouse retina (Tsukamoto et al. 2001), and our data reveal that the threshold of the DBCRs is about 19 ((1.9 Rh* rod–1 s–1)/(0.1 Rh* rod–1 s–1), Table 1) times lower than that of the rods. This suggests that signal convergence plays a major role in sensitivity amplification at the rod–DBCR synapse: a threshold signal (5% of maximum response) can be elicited in a DBCR by an incident light that is just bright enough to generate a threshold signal in any one of its presynaptic rods. We named this type of signal convergence the one-to-one threshold scheme (threshold signal in one presynaptic cell is adequate for generating a threshold response in the postsynaptic cell). As the incident light becomes brighter, threshold signals are elicited in more rods and the DBCR response is larger, and when the light reaches the intensity about 20 times brighter than that for the DBCR threshold, threshold signals are elicited in every rod (defined as rod threshold) and the DBCR sums signals from all presynaptic rods.
Anatomical data revealed that an AIIAC in the mouse retina collects synaptic inputs from several (3–4) DBCRs (Tsukamoto et al. 2001), and they are electrically coupled to one another (Famiglietti & Kolb, 1975; Deans et al. 2002; Veruki & Hartveit, 2002a). It is not clear how strongly AIIACs are coupled together in the mouse retina, and how many DBCRs contribute to the light response of a single AIIAC through the coupled AIIAC network. Our data show that the mean response threshold of the AIIACs to 500 nm light is about 143 ((0.1 Rh* rod–1 s–1)/(0.0007 Rh* rod–1 s–1)) times lower than that of the DBCRs. If the one-to-one threshold scheme also applies to the DBCR–AIIAC synapses, then about 143 DBCRs should contribute to the light response of a single AIIAC, or the light response of an AIIAC is pooled through a coupled network consisting of about 40 (143/(3–4)) AIIACs. This estimate is of the same order of magnitude as the number of coupled AIIACs in the cat retina (Smith & Vardi, 1995). On the other hand, if the one-to-one threshold scheme does not hold for the DBCR–AIIAC synapse (e.g. threshold signals in more than one presynaptic cell are needed to elicit a threshold response in the postsynaptic cell), then the number of AIIACs that share signals would be lower, or less than 40 AIIACs are effectively coupled. We plan to examine the effect of AIIAC coupling on AIIAC light sensitivity by studying light responses of DBCRs and AIIACs in the connexin-36 knockout mice in the future.
The data listed in Table 2 suggest that the current gain of the DBCR–AIIAC synapse is higher for smaller IC values and became progressively lower for larger IC. Similar non-linear input–output relations have been observed in other glutamatergic synapses in the retina (e.g. the rod–horizontal cell synapses in amphibian retinas; Attwell et al. 1987). It has been proposed that such non-linearity is caused by voltage dependence of presynaptic calcium current and glutamate receptor saturation (Falk, 1988; Attwell, 1990). Further investigation is needed to clarify this issue in the mouse retina.
Asymmetric interaction of rod and cone signals in dark-adapted mouse DBCs and AIIACs
In this study, we analysed the dynamic ranges of IC generated by 500 nm light in DBCRs, AIIACs and DBCCs, and compared them with the dynamic ranges of rods and cones (Fig. 6). Our data indicate that under dark-adapted conditions, the cone's influence on the rod system (DBCRs and AIIACs) is weaker than the rod's influence on the cone system (DBCCs). For example, IC in DBCRs and AIIACs saturate near the saturation intensity of the rods (–3.5), and do not extend substantially into the cone dynamic range. On the other hand, IC in DBCC1s saturate near the saturation intensity of the M cones (as they receive inputs exclusively from cones; Tsukamoto et al. 2001), but their dynamic range extends leftward into the DBCR response range (threshold near –7.0, Fig. 6). This nearly 3 log unit leftward extension of DBCC1 dynamic range is unlikely to be mediated by signal convergence (as in rod–DBCR and DBCR–AIIAC convergence discussed above), because on average each cone DBC only contacts four cones in the mouse (Tsukamoto et al. 2001). Therefore DBCC1s must receive strong inputs from the rods.
There are at least two possible explanations for the asymmetric rod–cone signal mixing in DBCs. First, it has been shown that rod–cone contacts in the mouse retina are highly asymmetrical: on average 32 rods contact one cone and only 1.2 cones contact one rod (Tsukamoto et al. 2001). Therefore, rods influence cones much more than cones influence rods. The second reason is that postsynaptic receptors in DBCRs and AIIACs may approach saturation when stimulated by 500 nm light near –4.0 (Xin & Bloomfield, 1999). At higher light intensities when cone inputs join in, IC in DBCRs and AIIACs cannot grow larger.
In summary, by analysing light response thresholds and dynamic ranges of dark-adapted mouse DBCs and AIIACs, we found that small rod signals are greatly amplified by the rod–DBCR–AIIAC synaptic pathway, and they are effectively transmitted to the cone system (DBCCs) via gap junctions between rods and cones and between AIIACs and DBCCs. This allows ‘on’ ganglion cells, which receive synaptic inputs exclusively from DBCCs, to reliably collect information from both the rod and cone signalling systems. Although there is no evidence that the rod–cone and AIIAC–DBCC gap junctions are rectified, these electrical synapses are functionally asymmetrical under dark-adapted conditions: cone-mediated signals contribute much less to the light responses of cells in the rod system (DBCRs and AIIACs) than the rod-mediated signals in cells in the cone system (DBCCs and ‘on’ ganglion cells). Under different physiological conditions, such as in the presence of background light, signal interaction between the rod and cone systems can be very different, and cone-mediated responses may dominate all retinal neurones.
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