ipRGCs express receptors for excitatory and inhibitory neurotransmitters
In all vertebrate retinas, conventional RGCs receive synaptic inputs from both bipolar and amacrine cells. To learn whether ipRGCs may be subject to such input, we first used voltage-clamp recordings to determine whether they possess receptors for the bipolar-cell transmitter glutamate, and the amacrine-cell transmitters GABA, glycine, and acetylcholine. We puffed receptor agonists onto ipRGCs and used baseline-subtracted voltage ramps to characterize the induced currents. To limit agonist effects to direct actions on the recorded cell, we blocked calcium-mediated synaptic release by using a cobalt-based Ringer (see Methods). The caesium-based internal solution, which we used to improve space clamp, would have precluded our detecting any metabotropically mediated potassium conductances evoked by these agonists. All ipRGCs responded to applied l-glutamate with a current that was inward at negative holding potentials and reversed, on average, at −5.6 ± 0.3 mV (mean ±s.e.m.; n= 15; Fig. 2), near the cationic Erev. Likewise, both GABA and glycine evoked currents in all ipRGCs tested and, as expected for fast ionotropic inhibitory currents, these reversed close to ECl (−57.3 ± 1.9 mV for GABA and −55.6 ± 0.9 mV for glycine; n= 15; Fig. 2). There were few if any nicotinic receptors on these cells, as neither acetylcholine (1 mm) nor the selective nicotinic agonist epibatidine (500 nm) evoked detectable currents (n= 9; not shown), although it should be noted that nicotinic receptors can desensitize rapidly and thus could have escaped detection. We conclude that ipRGCs possess the receptors for both excitatory bipolar-cell and inhibitory amacrine-cell transmitters.
Spontaneous synaptic inputs in darkness
To determine whether these receptors can be activated by synaptic input, we looked for spontaneous synaptic currents in cells bathed in a calcium-containing Ringer (Ames' medium) that permits synaptic transmission. In the absence of light stimulation, small spontaneous synaptic events could be observed under voltage clamp in all ipRGCs tested. These events reversed near −60 mV (n= 17; Fig. 3A). This is close to ECl, suggesting that, at rest, ipRGCs are bombarded by synaptic inputs that trigger inhibitory chloride conductances. To determine the transmitters mediating these inputs, we clamped ipRGCs close to the cationic Erev to minimize glutamate-induced currents and to study the inhibitory ones in relative isolation. Subsequent application of antagonists of ionotropic GABA and glycine receptors induced a net inward current and a substantial reduction in synaptic noise (n= 8; Fig. 3B). We interpret this as a blockade of a large outward chloride conductance attributable to the continuous barrage of GABAergic and/or glycinergic synaptic inputs.
Glutamatergic synapses were also spontaneously active in darkness as indicated by the spontaneous synaptic events that occurred during continuous application of the amacrine cell cocktail, which antagonized GABA, glycine and cholinergic receptors. These synaptic events reversed near 0 mV (n= 9; Fig. 3C), suggesting that they are synaptic cationic conductances, presumably triggered by glutamatergic inputs from bipolar cells. Because the cationic synaptic currents could not be observed clearly unless the GABAergic and/or glycinergic transmission was blocked (compare Fig. 3A and C), inhibitory synaptic inputs appear to dominate in darkness under these recording conditions, at least as viewed from the soma.
All ipRGCs exhibit synaptically mediated light responses
Having confirmed that ipRGCs receive functional chemical synapses, we next asked whether these inputs were capable of generating light responses in these cells. We also wanted to understand how any such ‘extrinsic’ responses differed from melanopsin-based (‘intrinsic’) light responses and from the light-evoked responses of conventional RGCs. To address these questions, we were obliged to alter our method for targeting and recording ipRGCs, because the intense epi-illumination we used to visualize fluorescent retrograde labelling severely adapted the rods and cones and may even have irreversibly bleached them. Thus, we turned to the multielectrode array (MEA) recording technique in which ipRGCs can be identified from among the large number of simultaneously recorded ganglion cells on the basis of functional criteria alone, with no need for fluorescence imaging (Tu et al. 2005). An additional advantage of the MEA is that, as an extracellular recording method, it avoids the intracellular dialysis that occurs with the whole-cell method, which could affect synaptic responses.
A typical MEA recording of an ipRGC appears in Fig. 4. The cell's identity as an ipRGC was evident from the persistence of a robust, sustained light response when rod/cone signalling was blocked, from the long onset latency of this response, and from the prolonged poststimulus discharge (Fig. 4, right). When synaptic blockers were omitted from the bathing solution (Fig. 4, left), the most dramatic difference was in the emergence of light responses to dim stimuli. Spikes were evoked by stimuli as much as six orders of magnitude dimmer than required to elicit firing through intrinsic phototransduction. This synaptically mediated response was generally very tonic, although near threshold it was relatively transient (Fig. 4 left, −6 and −5 log I). In addition, the synaptically mediated light response had distinctive kinetics. Whereas the intrinsically mediated response was very sluggish at the intensities tested (e.g. latency of 5.4 s for the +0.65 log I response; Fig. 4 right), the synaptically mediated response was brisk at all stimulus intensities (e.g. latency of 0.06 s for the +0.65 log I response; Fig. 4 left). Moreover, at −6 to −2 log I, the extrinsic response lacked the prominent poststimulus discharge of the intrinsic response. (The after-discharge at −1 log I in the control condition is not apparent in the presence of the rod/cone signalling blockers, suggesting a slight reduction in sensitivity for the intrinsic photoresponse in the drugs. This phenomenon was observed in most ipRGCs tested and was probably due to light adaptation, general rundown, and/or synaptic modulation of phototransduction gain; see the Discussion for more details.) Spontaneous activity also tended to be slightly higher when synaptic circuits were functional than when they were blocked. There is some variation in spike amplitude in the responses to the brighter light stimuli; such variation is also seen routinely in whole-cell recordings (Fig. 6A; see also Wong et al. 2005b). In both types of recordings, attenuation in spike amplitude is associated with strong activation and is thus presumably caused by inactivation of voltage-gated sodium channels, that is, depolarization block.
Figure 4. Multi-electrode array (MEA) recordings of synaptically mediated light responses in ipRGCs Extracellular recordings comparing spike responses of an ipRGC to light of various intensities with rod/cone-driven synaptic inputs left intact (left) or blocked (right). Log stimulus attenuation is indicated to the left. Note that all responses to weaker stimuli (−2 log attenuation and dimmer) and short-latency responses to brighter ones (−1 to +0.65 log I) were dependent on synaptic transmission, presumably because they reflect rod and/or cone influence on the recorded cell. Note also that intensities sufficient to recruit the intrinsic response (−0 and +0.65 log I; right) evoke responses with substantial poststimulus persistence, a well-established feature of melanopsin-dependent light responses.
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Figure 6. Whole-cell recordings of synaptically mediated light responses in ipRGCs A, in current-clamp recordings, the response of this ipRGC to prolonged light stimuli consisted of a transient, relatively weak depolarizing synaptic response (arrow) followed by a slower and larger depolarization that outlasted the stimulus (left). The transient response was synaptically mediated (extrinsic), because it was selectively abolished by superfusion with the rod/cone signalling blocker cocktail (right), whereas the larger, slower depolarization and poststimulus response were melanopsin based because they survived the synaptic blockade. Stimulus intensity =−3 log I. B, voltage-clamp recordings (Vhold=−70 mV) reveal the same two response components and confirm that the early, transient response (arrow) was selectively abolished by the rod/cone signalling blocker cocktail. Stimulus intensity =−1 log I.
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All ipRGCs recorded by this method exhibited the features of the extrinsic light responses evident in Fig. 4 (n= 82). In every case, the synaptically driven light response consisted of an increase in spike rate that was relatively transient at threshold but more sustained when evoked by brighter light (Fig. 5C, left). The extrinsic responses were also consistently 4–6 orders of magnitude more sensitive than the pharmacologically isolated intrinsic ones (mean thresholds: −5.56 ± 0.13 and −0.15 ± 0.09 log I, respectively). In addition, the latency of the extrinsic light response was always significantly shorter than that of the pharmacologically isolated melanopsin light response. Measured at +0.65 log I, the range of response latency of the extrinsic response was 0.05–0.06 s, while that of the intrinsic response was 0.4–14.9 s.
Figure 5. Differing kinetics of synaptically driven light responses in conventional and intrinsically photosensitive RGCs A, simultaneous extracellular recordings from two different cells from a single electrode of the MEA. The larger spikes come from a conventional RGC and the smaller spikes from an ipRGC. The conventional RGC's light-evoked responses were transient at all stimulus intensities in normal Ames' medium (left), and were completely abolished in the presence of the rod/cone signalling blocker cocktail (right; see Methods). The ipRGC's responses were sustained in control medium even at light intensities too dim to evoke the intrinsic, melanopsin-based response (compare −3 and −2 log I traces, left and right). B, a magnified version of the ‘−2’ response trace shown in A, left. The extrinsic light response of the ipRGC was more sustained than the light response of the conventional RGC, lasting throughout the duration of the light stimulus. C, a summary of the results from 12 ipRGCs (left) and 18 conventional RGCs (right). Action potentials from each trial were summed and plotted as a histogram with 0.5 s bins, and the largest-amplitude column from each cell was normalized to 1 (y axis in the ‘−0’ response traces). These histograms were averaged to generate the histograms shown. The error bars represent s.e.m. values.
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Thresholds for synaptically driven light responses were similar in conventional RGCs and ipRGCs, but such responses were consistently more sustained in ipRGCs. Figure 5A and B illustrates an example of this difference through simultaneous recording of a conventional RGC, which generated large-amplitude spikes, and an ipRGC, which emitted smaller action potentials. That only the latter unit was an ipRGC was evident from the persistence of its light response upon application of the rod/cone signalling blockers, whereas the large-amplitude unit was silenced (Fig. 5A, right). The ipRGC's light response in normal Ames' medium was sustained at every intensity except the weakest suprathreshold one (i.e. −4 log I), even when the stimulus was too dim to activate the intrinsic photoresponse (Fig. 5A left, ‘−3’ and ‘−2’, and Fig. 5B). By contrast, in normal Ames' medium (Fig. 5A, left), the conventional RGC generated transient ON responses lasting no more than 4 s at any stimulus intensity. Similar behaviour was evident in nearly all conventional ON and ON–OFF RGCs tested (n=∼40), with the responses returning to baseline firing rates within 6 s during prolonged light steps. Such a difference in response kinetics between ipRGCs and conventional RGCs is summarized in Fig. 5C.
Amacrine cells and ON and OFF bipolar cells trigger light responses in ipRGCs
Synaptically driven light responses could also be detected in whole-cell recordings. The advantage of this approach over the MEA is that subthreshold voltage responses and reversal potentials can be measured. We therefore used such recordings in combination with pharmacology to determine which types of presynaptic cells mediate extrinsic light responses of ipRGCs.
Of the 74 ipRGCs recorded in the whole-cell configuration, 66 (89%) cells' light responses to a full-field, subsaturating pulse of white light consisted of an extrinsic component in addition to the intrinsic, melanopsin-based photoresponse. The remaining 11% failed to show any extrinsic light responses. Because extrinsic responses were evident in every ipRGC recorded by the MEA method, we suspect that their absence in these whole-cell recordings is a technical artefact. As noted above, for the whole-cell approach, bright illumination must be used to detect the fluorescent retrolabelling so that presumptive ipRGCs may be targeted for recording. The resulting bleaching of rods and cones can be expected to weaken their influence on ipRGCs or, in extreme cases, to eliminate them altogether. Indeed, even when extrinsic light responses were observed in the whole-cell studies, they were far less sensitive than those recorded using the MEA. In fact, their thresholds were similar to that of the endogenous melanopsin-driven response (n > 20; not shown), so that stimuli intense enough to evoke extrinsic light responses usually also induced the endogenous light response. Another difference between the two recording methods was that the ipRGC extrinsic light responses obtained in the whole-cell mode were much more transient than seen in the MEA. Figure 6A compares the light responses of a single ipRGC recording in control Ames' medium (left) and in the presence of synaptic blockers (right). The most obvious synaptically mediated component of the light response was a small transient depolarization and brief burst of spikes immediately after stimulus onset (Fig. 6A, left; arrow). The slow depolarization at onset and the persistent poststimulus depolarization evident in the same trace persisted in synaptic blockade (Fig. 6A, right) and are thus attributable to intrinsic melanopsin-based phototransduction. Both extrinsic (arrow) and intrinsic response components were also apparent under voltage clamp (Fig. 6B). We speculate that such transient responses were driven primarily by cone photoreceptors, because (1) rod-driven responses would be much more sensitive than the intrinsic ipRGC response; (2) cone photoresponses are more transient than rod photoresponses; and (3) cone visual pigments are more resistant to bleaching than rhodopsin (Trevino et al. 2005). The more sustained and sensitive extrinsic light responses in the MEA recordings (see above) were probably generated mainly by rod photoreceptors.
To learn whether the extrinsic light responses of ipRGCs are mediated by amacrine cells, bipolar cells or both, we measured extrinsic light responses at various holding potentials under whole-cell voltage clamp. In normal Ames' medium, these responses occurred at light onset and consisted of conductance increases reversing at −57 ± 2 mV, close to the Erev for chloride (n= 6; Fig. 7). This implies that under these recording conditions, amacrine-cell inputs gating ionotropic inhibitory conductances dominate the synaptically mediated light responses of ipRGCs.
However, bipolar cells can also drive light responses in ipRGCs. During continuous incubation in the amacrine blocker cocktail (see Methods), nearly all ipRGCs still generated extrinsic responses at light ON (14 of 15 cells; 93%). These consisted of conductance increases reversing at −4.0 ± 3.3 mV (n= 5; Fig. 8A), suggesting a non-specific cationic basis. In addition, the amacrine-cell blockade revealed an OFF response in most cells (10 of 14; 71%), consisting of an inward current (reversing at −3.3 ± 3.7 mV) or depolarization when the light pulse was extinguished (Fig. 8A). Such OFF responses were almost never observed in control medium, and were invariably smaller than the ON responses (arrows in Fig. 8A, left and Fig. 8B, left; compare with Figs 6A and B, left and Fig. 7, left). A parsimonious interpretation of these results is that the ON and OFF responses reflect light-driven glutamatergic inputs from ON and OFF bipolar cells, respectively, and that these inputs become more prominent when presynaptic and/or postsynaptic inhibition are relieved by amacrine-cell blockade. Consistent with this hypothesis, further addition of a selective blocker of the ON-bipolar-cell light response (l-AP4, a group III metabotropic glutamate receptor agonist; Slaughter & Miller, 1981) selectively abolished the ON depolarization while enhancing the OFF depolarization (n= 8; Fig. 8B, right). It also revealed an ON hyperpolarization. This was presumably caused by light-induced reduction of glutamate release from OFF bipolar cells onto ipRGCs, because most fast inhibition was blocked. In conclusion, amacrine cells, ON bipolar cells and OFF bipolar cells synapse directly onto ipRGCs, and at least under these recording conditions, amacrine cells provide the dominant synaptic input both spontaneously (Fig. 3) and in response to light stimulation. Light can nonetheless evoke depolarization and spiking, presumably because at the resting potentials (∼−60 to ∼−75 mV), driving force is stronger for cationic than for chloride conductances.
We also examined the basis of synaptic drive to ipRGCs using the MEA method, since it appears less vulnerable to distortions of response sensitivity and kinetics than the whole-cell recording method. Because MEA recordings are obtained extracellularly, we could not measure reversal potentials and instead relied on pharmacological manipulations to determine the presynaptic inputs that evoke extrinsic light responses in ipRGCs. First, we sought to confirm that bipolar cells contribute to the ipRGC extrinsic light response by documenting their persistence in the presence of amacrine-cell blockers (with TTX omitted, to permit the generation of sodium spikes). A typical result is shown in Fig. 9. To limit the evoked responses to those mediated synaptically, and also to minimize bleaching of rods and cones, only relatively dim stimulus light intensities (up to −3 log I in this example) were tested. In normal Ames' medium (left), this cell gave a relatively weak and transient response at −5 log I, typical of near-threshold responses recorded in the MEA (see above). When the amacrine blocker cocktail was added to the bath (Fig. 9, right), two effects were noted. First, the ipRGC became spontaneously active in the dark. More importantly, the extrinsic light response not only survived the treatment but actually became stronger, resulting in a sustained response at −5 log I and pronounced depolarization block at higher light intensities (i.e. −4 and −3 log I). Similar results were obtained in all ipRGCs tested (n= 18). This result confirms that bipolar cells can drive sustained extrinsic light responses in ipRGCs. These effects of the cocktail also suggest that presynaptic and/or postsynaptic amacrine cell inputs inhibit both spontaneous and light-evoked excitatory bipolar cell inputs to ipRGCs, thus preventing ipRGCs from spiking spontaneously and their light responses from inducing depolarization block.
Figure 9. Bipolar cells can directly evoke sustained extrinsic light responses in ipRGCs Voltage responses of a single well-isolated ipRGC, recorded extracellularly with an MEA electrode, to a series of light intensities all of which were subthreshold for intrinsic phototransduction. In the presence of the amacrine blocker cocktail with TTX omitted (right), the extrinsic light response of this ipRGC was sustained, just as it was in control Ames' medium (left, ‘−4’ and ‘−3’). Under amacrine blockade, the extrinsic response became more robust and the responses to the −4 and −3 log I light flashes were so strong that depolarization block became evident. Amacrine blockade also increased the frequency of spontaneous spikes. Under both conditions, −6 log I light flashes did not evoke any response (not shown).
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In a second MEA experiment, we sought to confirm, under more physiological conditions, that the ON channel provides most of the input to generate the ipRGC extrinsic light response. Representative data are shown in Fig. 10A. Again, only relatively dim stimulus light intensities were tested to avoid activation of the intrinsic photoresponse and to minimize bleaching of rods and cones. Blocking the ON channel with l-AP4 abolished completely the increase in spiking during the light pulse for every cell tested (n= 10). In addition, for 7 of these 10 cells, ON-channel blockade induced a brief OFF response that was absent in control medium (Fig. 10A right, −2 log I, and Fig. 10B). This effect of l-AP4 has been reported for non-ipRGCs (e.g. Arkin & Miller, 1988) and is consistent with the enhancement by l-AP4 of the OFF depolarization in whole-cell current-clamp recordings (Fig. 8B). A likely explanation is that in the absence of ON-channel blockade, withdrawal of ON-bipolar-cell input at light OFF results in disfacilitation, which counterbalances and masks the excitation from the OFF channel; silencing the ON channel unmasks the OFF channel input. In conclusion, while the ipRGC extrinsic light response is driven predominantly by the ON channel, the OFF channel (presumably through direct inputs from OFF bipolar cells) also makes a small contribution.
Figure 10. The ipRGC extrinsic light response is generated primarily by the ON channel Effects of ON-channel blockade on extrinsic light responses of a single well-isolated ipRGC recorded extracellularly on the MEA. A, when 100 μm l-AP4 was applied to selectively block ON-bipolar-cell light responses and thus the rest of the ON channel, the extrinsic light response of this ipRGC was nearly completely abolished, and a few spikes were evoked at light offset (right). Notice that besides the larger spikes from the ipRGC, smaller spikes, presumably from other RGCs, were evoked at light offset in the presence of l-AP4 (seen most clearly in the −3 log I trace in the right column). B, a summary of the results from eight ipRGCs for the −2 log I responses, in control Ames' medium (top) and in 100 μm l-AP4 (bottom). Spikes from each trial were summed and plotted as a histogram with 0.5 s bins, and the largest-amplitude column from each cell was normalized to 1 (y axis in the ‘control’ response trace). These histograms were averaged to generate the histograms shown. The error bars indicate s.e.m. values.
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Mapping of the receptive field of the synaptically driven light response
To gain some insight into the anatomical distribution of the synaptic input to ipRGCs, we plotted receptive fields for the extrinsic light response (see Methods and Fig. 1A). A total of 15 ipRGCs were analysed using light spots that were subthreshold for the intrinsic response. The evoked responses, consisting of increases in firing during the presentation of the spot, appeared to be generated primarily in the ON channel, because virtually all of these responses were abolished in the presence of 100 μm l-AP4 (n= 4; not shown). All receptive fields had an oval shape similar to the example shown in Fig. 11A, with an average equivalent diameter of 957 ± 62 μm. For comparison, the receptive field of the melanopsin-based photoresponse was also mapped after eliminating extrinsic responses with the rod/cone signalling blockers (see Methods). The activation of intrinsic responses requires substantial spatial summation. We therefore replaced the mapping spot with an elongated bar stimulus. Also, because prolonged illumination is required to evoke an intrinsic response and because long interstimulus intervals are needed for recovery, we opted for a one-dimensional mapping protocol to limit the total time needed for mapping (see Methods and Fig. 1B). We analysed the melanopsin-based receptive fields of 18 ipRGCs, and the average width of these receptive fields was 820 ± 114 μm (Fig. 11B), which is not significantly different from the average diameter of the extrinsically generated receptive fields (Fig. 11A). Both measurements are substantially higher than the average dendritic field diameter of ipRGCs (∼500 μm; Berson et al. 2002). Because the resolution in both types of mapping experiments was 120 μm, the true values could have been overestimated by as much as 240 μm. In addition, some light scatter could have led to additional overestimation. The main point, however, is that the spatial extent of the excitatory synaptic input onto ipRGCs is comparable to that of the intrinsic light response, thus suggesting that ON bipolar cells make synaptic contacts onto the entire dendritic field of the ipRGCs.
Influence of synaptic inputs on temporal bandpass
The preceding experiments showed that a major effect of synaptic inputs on ipRGCs is an increase in the sensitivity of these cells to light. Because the extrinsic response of an ipRGC to a single light pulse is faster than its melanopsin response, we suspected that synaptic input would also improve the ability of ipRGCs to encode modulations of light intensity at relatively high temporal frequencies. To test this idea, we examined the effects of synaptic blockade on the whole-cell response to a flickering stimulus. In control medium, the flicker (1 s square-wave pulses at 0.33 Hz) evoked two response components: small and fast depolarizing events that were time-locked to the individual light pulses; and an underlying, slowly developing depolarization that outlasted the train of light pulses (Fig. 12A and B, left). The modulation at the stimulus frequency was synaptically mediated because it was effectively abolished by the application of synaptic blockers, leaving only a sluggish unmodulated depolarization generated by the endogenous melanopsin mechanism (Fig. 12A and B, right). A similar result was observed in all cells tested (n= 5).