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1Discipline of Physiology, School of Medical Sciences and Institute for Biomedical Research, The University of Sydney, Australia; 2Department of Neuroanatomy, Max Planck Institute for Brain Research, Frankfurt am Main, Germany; and 3Ophthalmology and Visual Science, University of Texas Medical School at Houston, Houston, Texas
Submitted 16 August 2004; accepted in final form 7 December 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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RB cell signaling. Consistent with this result, strychnine, a glycine receptor antagonist, was unable to abolish these OFF responses. In addition, we were able to show that some OFF cone bipolar dendrites terminate at rod spherules and make potential contacts. In the mouse retina, however, there seems to be a very low proportion of OFF signals carried by an APB-resistant pathway. No ganglion cells in the rat retina displayed APB- and strychnine-resistant responses. Our data support signaling through flat contacts between rods and OFF CB cells as the alternative route, but suggest that the significance of this pathway differs between species. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Daw et al. 1990). In the mammalian retina, rods synapse onto a unique type of bipolar cell, the ON-depolarizing rod bipolar (RB) cell. These cells do not make direct contact with ganglion cells (GCs). Instead they contact a bistratified narrow field amacrine cell, the AII amacrine cell, which splits the incoming signal to feed the ON and OFF channels of the visual system. AII cells make electrical synapses with ON cone bipolar (CB) cells via gap junctions and inhibitory chemical synapses with OFF CB cells, using glycine as their neurotransmitter (see Fig. 1). The axon terminals of ON and OFF CB cells stratify in sublamina b and a, respectively, of the inner plexiform layer to synapse onto their appropriate ganglion cells.
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). Müller et al. (1988) have shown in the intact cat retina that APB abolishes light responses in scotopic conditions from all ON and OFF brisk sustained (X) and brisk transient (Y) GCs by acting at the rodRB cell synapse and thereby blocking the signal transmission. Correspondingly, it was also found that strychnine, a glycine receptor antagonist, abolished scotopic light responses of OFF GCs by blocking AIIOFF CB cell synapses without substantially affecting the activity of ON GCs.
More recently, two alternative pathways for rod signals have been proposed. First, DeVries and Baylor (1995) showed that, in scotopic conditions, two types of GCs (OFF sluggish and ON-OFF direction-selective) in the rabbit retina were still responsive to light stimulation in the presence of APB. On the basis of anatomical (Raviola and Gilula 1973) and physiological evidence (Nelson 1977; Schneeweis and Schnapf 1995), DeVries and Baylor proposed that rod signals could flow to the cone pedicles via gap junctions and subsequently to the GCs through OFF CB cells (Fig. 1). Additionally, in connexin 36 knockout mice (Cx36 KO mice), extracellularly recorded scotopic ON responses of retinal GCs were suppressed, most likely due to lack of expression of functional gap junctions between rods and cones and between AIIs and CB cells, further supporting the existence of multiple rod pathways for night vision (Deans et al. 2002).
Second, rod-driven signals have been recorded in the presence of APB from OFF and ON-OFF GCs of normal and transgenically derived coneless mouse retinas (Soucy et al. 1998). Based on these results, Soucy et al. suggested a second alternative circuit independent of cone photoreceptors, which implies a direct contact between rods and OFF CB cells (Fig. 1). Further support for this theory came from the localization of the ionotropic glutamate receptors GluR1 and GluR2 to flat contacts between rods and putative OFF CB cells in the normal rodent retina (Hack et al. 1999), as well as from the recent demonstration of such contacts between a specific type of OFF cone bipolar cell, the CB2, and rod spherules in the mouse retina (Tsukamoto et al. 2001). In addition, GluR2 receptors were recently shown to be localized to dendrites of OFF CB cells contacting rod spherules in a large percentage of cells in the rabbit retina (Li et al. 2004).
To investigate whether these alternative pathways transmit rod signals through the mammalian retina, we examined the light responses of GCs in the rabbit, rat, and mouse retina under different light conditions and with specific pharmacological tools. We observed APB-resistant signals frequently in the rabbit, rarely in the mouse, and never in the rat retina. The high proportion of APB-resistant OFF signals in rabbit retina is supported by the high number of rodOFF CB cell contacts observed anatomically. Our results favor the alternative circuit in which rod photoreceptors directly contact OFF CB cells, and they support the existence of this alternative pathway in rabbit as well as rodent retina.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Electrophysiological recordings were performed from retinal GCs in albino and pigmented rabbits (2–3.5 kg), mice from the strain C57Bl/6J (5–7 wk old) and Swiss rats (5–7 wk old). Light responses in the rabbit retina were recorded from both the whole mount preparation and slices, whereas in the case of mouse and rat retinas, only slices were used. For whole mount experiments, rabbits were dark-adapted for 
5 h before they were killed by an overdose of pentobarbital sodium. The right eye was removed, and the retina was isolated under infrared illumination (>900 nm). A small piece (
3 x 4 mm) of the central part of the retina was placed photoreceptor side down in the recording chamber. Retinal vertical slices were prepared from rabbit, mouse, and rat retinas following previously described procedures (Protti et al. 1997). Rabbits were prepared as described above, whereas mice and rats were dark-adapted for 6–7 h and subsequently anesthetized with halothane and decapitated. All procedures were performed under infrared light and in accordance with the guidelines for animal experiments issued by the Federal Republic of Germany (Tierschutzgesetz). The eyes were rapidly enucleated and transferred to a dish containing Ames medium. The cornea was cut along the ora serrata, the lens and vitreous were removed, and the retina was separated from the sclera. The retina was cut into four pieces, one of which was embedded in 2% agar dissolved in Ames medium (kept at 38°C), and the resulting block was rapidly cooled. The block was transferred to a microslicer (Dosaka EM), and 200 µm-thick slices were cut. Slices were kept in Ames equilibrated with a mixture of 95% O2-5% CO2 for 30 min before electrophysiological recording. The tissue could be maintained for 
8 h in oxygenated Ames medium.
Electrophysiological recordings
During recordings, the tissue was mounted in a recording chamber and continuously perfused with oxygenated Ames medium (35°C, pH = 7.4). Cells were viewed on a video monitor coupled to a CCD camera mounted on a Zeiss microscope (ACM, Zeiss, Oberkochen, Germany) equipped with a water immersion objective (x40/0.75 W). The Nomarski optics of the microscope were modified for infrared illumination (900 nm). GCs were identified by their electrophysiological properties and their morphology, namely large sodium currents (>1 nA) and a large soma. Patch-clamp recordings of cells in the GC layer were performed using an EPC-9 patch-clamp amplifier (Heka Electronic, Landau, Germany). The pipette solution contained Cs+ as the main cation and gluconate as the main anion. The intracellular solution consisted of (in mM) 100 CsGlu, 0.5 MgCl2, 10 Na-HEPES, 5 EGTA, 0.5 CaCl2, 3 Mg-ATP, 0.5 Na GTP, 5 tetrabutylammonium chloride (TBA), and 5 QX-314. TBA and QX-314 blocked voltage-gated potassium and sodium conductances, respectively, thus allowing the recording of light-evoked synaptic currents. The chloride concentration yielded an ECl– 
–55 mV. Series resistances, which ranged from 10 to 30 M
, were left uncompensated. Drugs were diluted in Ames medium and perfused at 3 ml/min. Ames medium, strychnine, and all other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany). To block scotopic OFF responses in GCs, a racemic mixture of both D-and L forms of APB (Tocris, Cookson, Bristol, UK) at a concentration of 100 µM was used (see also Jin and Brunken 1996; Soucy et al. 1998).
Visual stimuli
All visual stimuli were achromatic and presented on the 13-in RGB color monitor of a Macintosh IIci computer. Images were generated by software kindly provided by Dr. Markus Meister (Harvard University, Boston, MA) and modified by Dr. Rowland Taylor (Neurological Sciences Institute, Oregon Health and Sciences University, Portland, OR). In both scotopic and mesopic/photopic conditions, two main stimulation patterns were used: 1) bright stimuli on a dark background (ON stimulus) and 2) dark stimuli on an illuminated background (OFF stimulus). Stimuli were focused onto the photoreceptors, and their luminance was adjusted from the software and by neutral density filters and ranged from 6.5 x 10–8 to 0.65 cd/m2. For whole mount experiments, we used optimal spots (50–200 µm diam) to elicit centered responses. For the slice experiments, full-field stimuli were employed.
Analysis
Peak current amplitude and latency of the stimulus-evoked responses were calculated using homemade routines (Igor Pro, WaveMetrics, Lake Oswego, OR). For the intensity-response function, peak amplitude of the currents were normalized and plotted versus the logarithm of the stimulus luminance. The values of I/Imax were fitted with the Hill function: I/Imax = Lh/(Lh + L50h), where Imax represents the maximal current amplitude, L corresponds to the luminance (in cd/m2), h represents the slope of the curve (i.e., the Hill coefficient), and L50 is the luminance evoking a half-maximal response (in analogy to the equations describing the intensity-response function of photoreceptors membrane potential; Normann and Werblin 1974).
Morphology and immunocytochemistry, rabbit retina
Bipolar cells were recognized in whole mount retina pieces prelabeled with 5 µM 4, 6-diamino-2-pheylindole (DAPI, Molecular Probes, Eugene, OR) and impaled under visual control using pipettes tip filled with 4% Neurobiotin (Vector Laboratories, Burlingame, CA) and 1% Lucifer yellow-CH (Molecular Probes) in ddH2O and backfilled with 3 M LiCl. The cells were injected with a biphasic current (+1.0 nA for 100 ms and –1.0 for 100 ms) for 1–2 min. After the last injection, the retina pieces were fixed in 4% paraformaldehyde for 20 min. The tissues were washed extensively with 0.1 M phosphate buffer (PB, pH 7.4) and blocked with 3% donkey serum in 0.1 M PB with 0.5% Triton-X 100 and 0.1% NaN3 over night. The tissues were then incubated in an antibody to mGluR6 in the presence of 1% donkey serum/PBS/0.5% Triton X-100/0.1% sodium azide for 1 wk. After washing, the tissues were incubated in secondary antibody: donkey anti-rabbit Cy3 (1:200; Jackson Immunochemicals, West Grove, PA) and streptavidin 488 (1:200; Molecular Probes) diluted in 0.1 M PB/0.5% Triton X-100/0.1% NaN3 overnight. The tissues were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and observed with an Olympus Vanox photomicroscope (Olympus) and/or a confocal microscope (LSM 410, Zeiss, Thornwood, NY). Confocal sections were imaged in 0.5-µm steps. Images shown are the result of merging short stacks of four to six single confocal sections x 0.5 µm (total thickness, 2–3 µm) to flatten out ripples in the tissue. Brightness and contrast of the final images were adjusted using Photoshop 5.5 (Adobe Systems, San Jose, CA); no filtering was applied to the images.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Whole cell patch-clamp recordings were performed from GCs in whole mount preparations and slices. The GCs were from the lower region of the visual streak and were morphologically identified by Neurobiotin staining. Another parameter used to identify GCs was the presence of robust voltage-dependent Na+ currents (>1 nA), which could only be observed immediately after breaking into the cells, before they were blocked by intracellular dialysis with QX-314. Recordings were obtained from ON, OFF, and ON-OFF GCs. Our observations regarding the pharmacological profile of the light responses proved to be equally valid for transient as well as for sustained responses; therefore no further classification has been done. GCs were held at a voltage of –75 mV. Since light-evoked currents consist of a mixture of cationic (Na+/K+) and anionic (Cl–) currents, at this holding potential, the light-driven synaptic currents are inward. Light-evoked currents were elicited by using spots of different light intensities. Under very low light conditions (scotopic), the lowest intensities we tested occasionally failed to evoke light responses. Above a threshold of 6.5 x 10–7 to 3.25 x 10–6 cd/m2, light-evoked currents were consistently elicited, and intensity-response functions were measured. Under these conditions, light-evoked currents elicited by small spot diameters (50–200 µm) consisted of inward currents and represented responses of the receptive field center. The reversal potential of light-evoked currents was measured by setting the membrane potential to different values between –105 and 45 mV and presenting a spot of optimal size. We found that stimulus-evoked currents reversed at approximately –20 mV (data not shown), suggesting a predominance of excitatory input (as shown in detail in Flores-Herr et al. 2001).
We first tested the ability of 100 µM APB to abolish ON light responses in scotopic and mesopic conditions. APB is an agonist at the metabotropic glutamate receptor type 6, which blocks the light-evoked responses of all ON-depolarizing bipolar cells, including RB cells. As expected, ON responses of ON and ON-OFF cells were completely blocked in all cases (data not shown). This blockade persisted over a wide range of light intensities. After removal of APB from the bath, light-responses were fully recovered (data not shown).
The effect of abolishing synaptic transmission between rods and RB cells using APB was also studied in OFF responses originating from OFF and ON-OFF GCs. OFF responses were elicited either at the cessation of a square light pulse (ON stimulus) or at the presentation of a dark spot on an illuminated background (OFF stimulus). Scotopic OFF stimuli are shown by a black rectangle on a dark gray background, and mesopic/photopic stimuli by a black rectangle on a light gray background. The top trace in Fig. 2A shows a response elicited by an OFF stimulus recorded from an ON-OFF GC under scotopic conditions. Inward currents followed both transitions in light intensity. Due to increased latency and slow response kinetics in scotopic conditions, the ON and OFF part of the light response cannot always be clearly separated when using 400-ms stimuli. APB (100 µM) reversibly abolished the OFF component in 9 (7 OFF and 2 ON-OFF) of 21 cells that presented OFF responses. When using an intensity of 1.625 x 10–5 cd/m2, stimulus-evoked currents were –51.4 ± 15 (SD) pA (n = 7 cells). In four of these cells, currents in control conditions were –51 ± 17 pA and were reduced by 96% in the presence of 100 µM APB, as shown in Fig. 2A (middle). However, in the presence of 100 µM APB, stimulus-evoked OFF currents were restored by increasing light intensity toward the cone activation levels, either by illuminating with brighter spots or by presenting a dark spot on a brighter background (Fig. 2A, bottom). This response under bright conventional conditions indicates direct coneOFF CBOFF GC cell signaling, which is expected to be APB-resistant.
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However, in the remaining 12 cells (4 OFF and 8 ON-OFF), OFF responses, although partly reduced in amplitude, always persisted in the presence of 100 µM APB. Figure 2C represents a typical APB-resistant light-evoked current recorded in scotopic conditions and shows that, although the amplitude of the light-evoked currents was reduced by application of APB, a substantial inward current still persisted. Slower kinetics of the light response after application of 100 µM APB (as in Fig. 2C) was not seen in all cells tested. In the remaining 3 of 7 cells for which currents were measured in response to the same stimulus intensity, the mean amplitude of light-evoked currents in control conditions was –52 ± 16 pA (n = 3) and it was only reduced by 7% by 100 µM APB. This strengthens the idea that 100 µM APB blocks the light responses of ON CB and RB cells without substantially affecting OFF CB cell signaling. Consistent with this result, in this group of cells, 2 µM strychnine exhibited only partial blocking effects in scotopic conditions with stimulus-evoked currents being reduced to 58% of their control amplitude (3 cells), implying that these responses might be independent of the RB cell-AII amacrine cell circuit. A representative recording of the strychnine effect on OFF responses is shown in Fig. 2D, in which presentation of an OFF stimulus elicited an inward current (top), and application of strychnine partly, but not fully, blocked this response (bottom). In summary, these experiments indicate the presence of rod input to OFF GCs, independent of RB cells.
Rod contacts of OFF cone bipolar cells
We set out to investigate the anatomical substrate of the alternative rod pathway in the rabbit retina, as previously described in the rodent retina (Hack et al. 1999; Tsukamoto et al. 2001). This was accomplished by dye-injecting single OFF CB cells with Neurobiotin and Lucifer yellow and double labeling the tissue with an antibody against mGluR6. Each whole mount retina was examined with a confocal microscope. The mGluR6 antibody labeled the dendritic tips of RB cells and ON CB cells, as previously reported (Vardi et al. 2000). Each rod spherule was identified as two bright dots, one for each of the two rod bipolar dendrites forming the central pair in a tetrad of processes that invaginate the rod terminal (Rao-Mirotznik et al. 1995). Cones were identified by multiple ON CB cells contacting one cone pedicle forming a lightly labeled cluster of fine dendritic tips. This pattern of mGluR6 labeling provides a map for the locations of rods and cones.
When viewed in whole mount, the dendritic tree of each OFF CB cell formed a typical pattern of branching dendrites with terminal clusters. As shown in Fig. 3, most of the dendritic tips contact cone pedicles, and almost every cone within the dendritic field is contacted (Fig. 3). Occasionally, the terminals of two separate dendrites meet at a single cone pedicle. As might be expected for OFF CB cells, the dendritic tips were not double labeled for mGluR6.
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Light responses in the mouse retina
Light-evoked currents were recorded from cells in the GC layer in mouse retinal slices. Cells were identified as GCs using the same criteria as described for rabbit GCs. The light stimulus consisted of a full-field pulse (ganzfeld). Typical light responses at a holding potential of –75 mV consisted of inward currents that reversed between –55 and –35 mV, suggesting that there was also a strong inhibitory input presumably originating from the surround illumination.
As in the rabbit retina, APB reversibly abolished ON responses at all intensities tested. We studied light responses from 18 ganglion cells (10 OFF and 8 ON-OFF) that presented OFF responses in scotopic conditions. In 17 of them, 100 µM APB completely abolished OFF responses at low light intensities. Representative responses are shown in Fig. 4. In ON-OFF GCs, inward currents were elicited at both onset and offset of the stimulus (Fig. 4A, top). These responses were completely blocked after bath application of 100 µM APB (Fig. 4A, middle). An increase of stimulus intensity (2–3 log units brighter) rescued OFF responses still in the presence of APB, whereas ON responses remained hindered due to blockade of signal transfer at the photoreceptorON-depolarizing bipolar cell synapse. OFF GCs (Fig. 4B) exhibited a similar behavior: responses to an OFF stimulus were abolished by 100 µM APB (middle) but were recovered by using a brighter background (bottom). The effect of 2 µM strychnine on OFF responses recorded from ON-OFF and OFF GCs was also tested. In ON-OFF cells, strychnine abolished the OFF component of light-evoked currents in response to scotopic stimulation without affecting the ON responses (Fig. 4C, top and middle). Using a brighter uniform illumination as background in the presence of strychnine, application of an OFF stimulus resulted in recovery of the OFF response and an increase in the amplitude of the ON response (bottom). Figure 4D shows the effect of strychnine on light-evoked responses of OFF GCs. OFF responses were evoked by presentation of an OFF stimulus (top) and were abolished by 2 µM strychnine (middle). Using brighter background intensity during perfusion with strychnine rescued OFF responses (bottom). The ON response is most likely inhibitory chloride input to the cell that was not present or masked by spontaneous membrane fluctuations at lower light intensities. Strychnine fully abolished the OFF responses in seven of seven cells (5 ON-OFF and 2 OFF).
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Light-evoked currents were also recorded from cells in the GC layer of rat retinal slices. Cells were identified according to the criteria used for rabbit and mouse GCs. As in the previous sections, light responses were inward currents and reversed between –55 and –35 mV.
As previously described for the rabbit and mouse retinas, 100 µM APB completely and reversibly abolished light-evoked ON responses recorded from all GCs at all intensity levels, and this blockade was reversible. Light-evoked OFF responses were recorded from a total of 20 (11 OFF and 9 ON-OFF) GCs in scotopic conditions. In all cases, stimulus-evoked OFF currents were fully blocked by 100 µM APB, an effect that was reversible. Figure 7A shows the response of an OFF GC in scotopic conditions (top) that is completely blocked by 100 µM APB (middle). However, using brighter background illumination in the presence of APB allowed OFF responses to recover (bottom). This indicates input mediated by OFF CB cells in the photopic range.
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Analysis of the intensity-response relationship confirms the blocking action of APB for OFF responses at low intensities. When higher stimulus intensities were applied during the application of APB, light responses could be generated via direct input from OFF CB cells. Further increases in light intensity produced larger responses (Fig. 8). These results suggest that, in the rat retina, the majority of rod level signals are processed via the RB pathway, which is sensitive to APB. We were unable to detect alternative rod-driven pathways in this sample of GCs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Ramon y Cajal 1972; Wässle and Boycott 1991), recent results have modified our views of signaling in the rod pathway. The simplest rod circuit is via RB cells (Bloomfield and Dacheux 2001; Daw et al. 1990; Famiglietti and Kolb 1975; Kolb and Famiglietti 1974; Müller et al. 1988). In addition to this well-documented circuit, recent evidence suggests the existence of alternative parallel pathways that can also carry rod signals (Deans et al. 2002; DeVries and Baylor 1995; Hack et al. 1999; Soucy et al. 1998; Tsukamoto et al. 2001). We have conducted physiological studies to investigate the presence of these alternative routes in three mammalian species of interest for retinal studies. Additionally, we have explored the morphological basis for one alternative rod pathway in the rabbit retina.
In rabbit and mouse retina, we observed that light signals are able to flow through a circuit independent of the RB and AII amacrine cell interneurons. Our conclusions, which support the existence of an alternative pathway, are based on the observation that some OFF light responses persisted in the presence of APB and strychnine. These data imply that both rodRB cell and AII amacrine cellOFF CB cell synapses were not required. The APB- and strychnine-resistant light-evoked responses were observed in a high proportion of OFF and ON-OFF GCs in the rabbit retina in a small proportion of cells in the mouse retina and in no cells in the rat retina from our sample. The APB- and strychnine-resistant responses cannot be attributed to the concentrations used here, because these doses were effective in other studies (Jin and Brunken 1996; Soucy et al. 1998). Anatomically, we have observed that some OFF CB cells in the whole mount rabbit retina establish not only contacts with cone pedicles but also some terminal contacts with rod spherules, as previously reported for mouse and rat retina (Hack et al. 1999; Tsukamoto et al. 2001). These terminal contacts cannot be confused with dendrites merely passing close to rod spherules on the way to a cone pedicle. Rather, they suggest the presence of direct basolateral contacts between OFF CB cell and rod spherules, as in the study by Li et al. (2004), in which these contacts were confirmed by colocalization with the glutamate receptor GluR2. Nevertheless, this type of contact could be the locus of direct synaptic transmission from rods to OFF CB cells in the rabbit retina.
Our electrophysiological results in the rabbit retina are very similar to the observations made by DeVries and Baylor (1995). However, while they suggested that APB-resistant signaling could occur via rodcone gap junctions, we interpret these results in a different manner. Because it is very likely that single cones contact all types of CB cells (Boycott and Wässle 1991, 1999; Grünert et al. 1994), if rod signals were to spread to the cones and were subsequently transmitted to the CB cells, theoretically all OFF and ON-OFF GCs should exhibit APB-resistant OFF responses. However, this was not the case (DeVries and Baylor 1995; this study), and therefore we propose that these signals do not arise from rod signals spreading into cones but from direct rodOFF CB cell contacts. Such contacts, at least in mouse retina, have been shown to involve only one type of OFF CB cell, and hence, it is highly likely that their signals reach only a subset of GCs. In rabbit retina, all three OFF CB cells establish dendritic contacts with rod spherules in a relatively high proportion (Li et al. 2004). These results are in agreement with Deans et al. (2002), who showed that, in scotopic conditions, GC ON responses from connexin 36 knockout mice, but not OFF responses, were suppressed. Because Cx36 KO mice most likely lack functional gap junctions between rods and cones, the persistence of light responses in OFF GCs suggests that rodcone signaling does not play a major role for the OFF pathway at scotopic intensities.
Soucy et al. (1998) have also described an alternative pathway for scotopic vision in both normal and coneless mouse retina, which carries signals to all OFF and ON-OFF GCs. The anatomical substrate suggested to transmit these signals are flat contacts established by a particular type of OFF CB2 with rod spherules (Tsukamoto et al. 2001), and where ionotropic glutamate receptors have been localized (Hack et al. 1999). In the mouse retina, however, we only observed APB-resistant OFF responses in 1 of 18 cells. What possible explanations could account for this difference? First, we recorded synaptic currents by doing patch-clamp recordings in the slice preparation, whereas Soucy et al. (1998) recorded spike trains with a multielectrode array in the whole mount retina. If when doing multielectrode recordings, there were a bias toward recording from particular GC types that happened to be those cells receiving input from an APB-resistant pathway, the role of this pathway might have been overestimated. Along a similar line of reasoning, if GCs that receive input from CB2 have a particular morphology that made them unsuitable for our recordings, or if either CB2 or their postsynaptic GCs do not survive the slicing procedure, we may have possibly missed recording some APB-resistant OFF responses. Nevertheless, in the rabbit retina, we have obtained similar results recording from slices and from whole mount retina, suggesting that the functionality of the alternative pathway is independent of the preparation. Second, we dark-adapted the animals for a substantially longer time period than Soucy et al. (6 vs. 1 h), since in preliminary experiments, we observed that after short dark-adaptation periods the retina was unresponsive to low intensity stimulation. Therefore a difference in photoreceptor sensitivity between the two studies cannot be excluded. Finally, according to Soucy et al. (1998), the appearance of APB-resistant OFF responses was dependent on stimulus contrast, being most prevalent at high contrast. To detect APB-resistant OFF responses at all intensities, we used the maximum possible contrast (100%), thus ruling out a difference between the stimulus employed to evoke responses in their experiments and in this study
Although no more than 20% of rods were found to make contacts with the CB2, it was proposed that rodrod gap junctions in the mouse retina may be the route by which all rod signals could be fed into CB2 cells and their corresponding GCs (Tsukamoto et al. 2001). It is well established, however, that GCs stratify at different strata in the inner plexiform layer, where each particular type receives input from only a few bipolar cell types (Boycott and Wässle 1999; DeVries 2000; Roska and Werblin 2001). Consequently, CB2 bipolar cells are expected to synapse onto some but not all GC types. Additionally, only an estimated 5% of rods were shown to possess flat contact synapses with glutamate receptors (Hack et al. 1999). Taking into account these considerations and consistent with our results, signaling through an APB-resistant pathway is expected to be transferred to only a subset of OFF and ON-OFF GCs. Our data suggest that, in rodents, the percentage of OFF GCs might be considerably lower than in rabbit.
Soucy et al. (1998) also observed APB-resistant light responses in coneless mice, in which cones were genetically ablated. Genetic manipulation of particular cell types in the retina may result in widespread effects. For example, removal of cones from the retina results in degeneration of the rods (Ying et al. 2000). Moreover, in a retinal degeneration model, RB cells form ectopic contacts with cones in the absence of rods (Peng et al. 2000). Taken together, this may hint that, in the coneless mice, wiring changes could take place, such that a larger number of cone bipolar cell types receive rod input than in the normal mouse, thus explaining the results observed in the coneless mice. However, no difference in the APB sensitivity of OFF responses between the normal and coneless retina was observed, making an indirect effect due to cone ablation unlikely.
It remains to be elucidated in which other species the alternative pathways are present and whether they play an important role. In the developing retina of the ferret, APB-resistant OFF responses have been recorded under dark-adapted conditions from the majority of OFF and a small percentage of ON-OFF GCs, although it is not known whether this pathway is also functional in the mature retina (Wang et al. 2001). In the cat retina, both ON and OFF responses recorded under scotopic conditions from brisk sustained (X) and brisk transient (Y) cells were completely blocked by APB, and OFF responses were blocked by strychnine (Müller et al. 1988). These two GC classes comprise only about 50% of all GCs of the cat retina, and responses from other GC types have not been characterized so far. However, expression of a GluR1 glutamate receptor subunit has been observed in dendrites of OFF CB cells contacting rod spherules of the cat retina, suggesting the possibility that this pathway may also be present in the cat retina (Kolodziej et al. 2003). In the dark-adapted monkey all light-mediated behavior is abolished on APB application, whereas in mesopic and photopic conditions, only the ON responses have been shown to be blocked by APB (Dolan and Schiller 1989). In addition, no contacts between rods and OFF cone bipolar cells have been reported in the primate retina (Boycott and Dowling 1969). However, it is possible that in the primate retina, where midget and parasol GCs make up 90% of the GCs, an alternative rod pathway is only present in some of the remaining 10% of the GCs.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. A. Protti, Discipline of Physiology, School of Medical Sciences and Inst. for Biomedical Research (F13), The Univ. of Sydney, NSW 2006, Australia (E-mail: dariop{at}physiol.usyd.edu.au)
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