Functional Stability of Retinal Ganglion Cells after Degeneration-Induced Changes in Synaptic Input

Rhythmic resting spike activity in RD RGCs

In normal (WT) RGCs, the resting spike activity in the absence of light is irregular with little underlying temporal structure (Pang et al., 2003; Murphy and Rieke, 2006; Sagdullaev et al., 2006; Margolis and Detwiler, 2007). In contrast, in RD mice, ON, OFF-T, and OFF-S RGCs displayed striking and consistent patterns of rhythmic resting spike activity (Fig. 2). All three cell types showed continuous rhythmic bursts of spikes with an interburst beating frequency of ∼10 Hz (Fig. 2A). Examination of subthreshold membrane potential revealed oscillatory depolarizations at the same frequency, each crested by 0–5 spikes (Fig. 2B, left). Such activity patterns were not observed in any recordings from WT cells of the same type (Fig. 2B, right). Histograms of interspike interval showed groups corresponding to the fundamental beating frequencies (∼12 Hz/0.08 s interval in these example cells), as well as the higher-frequency spike bursts (Fig. 2C). Spectral analysis revealed clear peaks in power corresponding to the beating frequencies (Fig. 2C, insets). The mean beating frequency in all recorded cells (P36–P50) was 9.0 ± 0.4 Hz (n = 35 total; 13 ON, 15 OFF-T, 7 OFF-S; range, 3–14 Hz). There were no differences in beating frequencies between ON and OFF cell types (p > 0.2) (Table 1). The beating frequency of any given cell was the same as measured in either current or voltage clamp (see Fig. 4); therefore, data from both types of recording was pooled for calculation of group means. The overall resting mean spike rate (including spikes that occur in bursts during rhythmic firing) was 24.9 ± 3.5 Hz (n = 25 total; 11 ON, 10 OFF-T, 4 OFF-S; range, 5–80 Hz). This is, on average, about twice the mean spike rate of WT cells (Margolis and Detwiler, 2007; Stasheff, 2008). It is also ∼2.5-fold higher than the beating frequency, which reflects the fact that rhythmic synaptic input typically generated more than a single spike (see Fig. 4). The high (>1) coefficients of variation (CV = SD/mean) are another consequence of the rhythmic nature of firing. These measures are summarized in Table 1. Persistent rhythmic spike activity was present in every recording from identified ON, OFF-T, and OFF-S RGCs in the P36–P50 age group as well as in cells from animals as old as P210, albeit at a slower rate. In older animals both the fundamental beating frequency and mean spike rate were reduced twofold to threefold (supplemental Fig. 3, available at www.jneurosci.org as supplemental material). Maintained rhythmic spike activity is a characteristic feature of RGCs in RD retina that is not seen in WT RGCs. It was present in whole-cell and cell-attached (loose patch) extracellular recordings regardless of the time of day or length of the experiment. These results indicate that the retina continues to generate output signals in both the early and later stages of degeneration-induced loss of photoreceptors.

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Figure 2.

Rhythmic resting spike activity in identified RD RGCs. A, Whole-cell current-clamp recordings from ON, OFF-T, and OFF-S RGCs in RD retina. B, Activity on an expanded time scale. Note the underlying oscillatory membrane depolarizations present in RD (left, black) but not WT (right, gray) cells. C, Interspike interval histograms from cells in B. Solid line, RD; dotted line, WT. Insets show the power spectra for RD RGCs (same cells as in B). Note the clear presence of peak frequencies at 12–14 Hz in these cells.

Oscillatory synaptic input underlies altered resting activity

The aberrant activity that gives rise to rhythmic spiking in RD RGCs could be generated either intrinsically, by voltage-dependent conductances that give rise to pacemaker currents, or extrinsically, by reverberant synaptic input. To test the possibility that synaptic mechanisms are the source of rhythmic activity, RGC membrane potential was held at −70 mV in whole-cell voltage clamp and resting input currents were measured. Under these conditions, the recorded current showed ongoing clear-cut amplitude fluctuations in ON, OFF-T, and OFF-S cells (Fig. 3) at about the same frequency as rhythmic spiking activity (Fig. 2). Baseline currents of this nature were only seen in RGCs from RD retina: membrane current recorded under the same conditions from WT cells was dominated by low-amplitude baseline synaptic noise with few resolvable discreet events (Fig. 3B). Power spectra of RD current recordings revealed peaks at ∼10 Hz that were not present in WT recordings (Fig. 3C) and generally matched the frequency of spike activity in current-clamp recordings from RD RGCs (Fig. 2). The correspondence between rhythmic variation in current and voltage (spiking) was examined explicitly by comparing sequential voltage- and current-clamp recordings from the same cells. The traces in Figure 4A illustrate the temporal coincidence between peak inward currents and peak depolarizations that evoked spike bursts in an ON and OFF RD RGC. Spectral analysis of these records reinforces this point by showing that power peaks at the same frequency in voltage and current recordings in both cell types (Fig. 4B). The strong correlation (r = 0.95; p < 0.001) between the peak frequency of spiking and the peak frequency of currents from all individual cells where both were recorded (n = 18 total; 7 ON, 8 OFF-T, 3 OFF-S) is apparent in the plot of Figure 4C.

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Figure 3.

Rhythmic resting synaptic currents in identified RD RGCs. A, Whole-cell voltage-clamp recordings of resting synaptic currents in ON, OFF-T, and OFF-S RGCs from WT (left, gray) and RD (right, black) retinas (V_hold = −70 mV). B, Currents on an expanded time scale. Note the strong periodicity in RD currents from all three cell types and different dynamics of currents in ON and OFF cells. C, Power spectra of RD (black) and WT (gray) currents (the same cells as in B). The multiple peaks in RD spectra reflect harmonics of the fundamental beating frequency of ∼10 Hz.

The fluctuating current recorded from voltage-clamped RGCs in RD retina were reduced by >90% by exposure to a mixture of synaptic antagonists (in μm) 50 l-APB, 20 CNQX, 50 APV, 1 strychnine, and 50 picrotoxin (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). This is additional evidence that the rhythmic currents are generated extrinsically by spontaneous variation in synaptic inputs. The baseline fluctuations in current were still present, however, in the presence of l-APB alone, an mGluR6 agonist, indicating that the source of the persistent synaptic activity that gives rise to the oscillatory activity is downstream of the photoreceptor-to-ON bipolar synapse (supplemental Fig. 1, available at www.jneurosci.org as supplemental material).

Balance of excitation and inhibition in RD RGCs

Thus far, the experiments have shown that rhythmic synaptic input drives firing in both ON and OFF RGCs in the RD retina. Differences in the amplitude and dynamics of synaptic inputs to ON and OFF cells are also apparent: at rest (V_hold = −70 mV), there are monophasic inward currents in ON cells and biphasic currents in OFF-T and OFF-S cells. This could reflect a different balance of excitatory (inward) and inhibitory (outward) synaptic currents in these cell types, consistent with the different allotment of excitatory and inhibitory synaptic input to ON and OFF cells in WT RGCs (Pang et al., 2003; Murphy and Rieke, 2006) (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). To examine more closely the effect of photoreceptor degeneration on the balance of synaptic inputs to RD RGCs, membrane currents were recorded in voltage clamp from RGCs held at membrane potentials that were close to the reversal potential for excitatory (0 mV) and inhibitory (−70 mV) synaptic currents (Taylor et al., 2000; Pang et al., 2003; Murphy and Rieke, 2006). Recordings of resting currents in ON and OFF RD RGCs at −70 and 0 mV are shown in Figure 5A. In ON cells, large-amplitude rhythmic excitatory currents (V_hold = −70 mV) were reduced by changing the holding potential to 0 mV. In OFF cells, the small-amplitude rhythmic inward currents recorded at −70 mV were replaced by large-amplitude outward currents at V_hold = 0 mV (Fig. 5A). The difference in the recorded currents at these two holding potentials in these three cell types was quantitated using spectral analysis. It is apparent in the power spectra plotted in Figure 5B that the peak power at the cells beat frequency was largest in currents recorded at −70 mV in the ON cell and at 0 mV in the OFF cells. Data pooled from a collection of 12 RGCs in RD retina (4 ON, 5 OFF-T, 3 OFF-S) showed that there is a strong correlation (r = 0.96; p < 0.001) between the frequency of peak power of the currents recorded at −70 and 0 mV (Fig. 5C, top). These results indicate that excitatory and inhibitory inputs are active at the same fundamental frequency in ON and OFF type RD RGCs. The ratio of inhibitory-to-excitatory power in the ON and OFF cell types is very different, however, with OFF cells receiving >10-fold stronger inhibition than ON cells (Fig. 5C, bottom). This is consistent with previous measurements of the relative weights of inhibitory and excitatory conductances in ON and OFF RGCs in the WT retina (Pang et al., 2003; Murphy and Rieke, 2006) and supports the conclusion that the balance of excitatory and inhibitory inputs to ON and OFF RGCs is mostly conserved in RD retina.

Similarity of intrinsic firing properties in WT and RD RGCs

Because the continuous generation of rhythmic synaptic input and spike activity might be expected to provoke compensatory changes in cellular physiology (Gorter et al., 2002; Frick and Johnston, 2005), spike generation and firing properties of WT (Margolis and Detwiler, 2007) and RD RGCs were compared. In the presence of synaptic blockers the intrinsic characteristics of electrically evoked spike activity in ON, OFF-T, and OFF-S RGCs were indistinguishable in RD and WT retinas (Fig. 6). Both ON and OFF cells in RD retina showed similar changes in firing frequency with increasing amplitude current injection, as well as similar spike-frequency adaptation and capacity for sustained firing (Fig. 6A). The maximum stimulated firing frequencies were ∼300–400 Hz and not different in RD compared with WT in any cell type (Fig. 6C) (ON: WT, 317 ± 45 Hz, n = 6; RD, 341 ± 46 Hz, n = 3; p = 0.7; OFF-T: WT, 315 ± 52 Hz, n = 5; RD, 362 ± 39 Hz, n = 4; p = 0.7). Action potential durations were submillisecond in all recordings (WT, n = 12; RD, n = 14).

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Figure 6.

Similarity of intrinsic electrical properties in WT and RD RGCs. A, Voltage (V_m) responses to injected current (Iinj) for ON (top) and OFF-T (bottom) cells recorded in whole-cell current-clamp mode in the presence of synaptic blockers (in μm: 50 l-APB, 20 CNQX, 50 APV, 1 strychnine, and 50 picrotoxin). Arrows indicate presence of rebound firing in both WT and RD OFF-T cells. B, Four superimposed responses to negative current injection. The strength of the responses is proportional to the level of hyperpolarization. C, Average maximum firing frequency and ADP strength for ON (left; n = 6 and 3) and OFF-T cells (right; n = 5 and 4, respectively).

Like in WT retina, RD ON RGCs lacked the ability for rebound firing (Fig. 6B), even when driven to very negative voltages (<−100 mV). Consistent with this, afterdepolarization (ADP) amplitude was approximately twofold to fourfold smaller in ON (<5 mV) than OFF cells (>10 mV). In three RD ON cells, ADP amplitude was smaller than in WT ON cells (WT, 3.7 ± 0.3 mV, n = 6; RD, 1.9 ± 0.4 mV, n = 3; p < 0.05).

OFF cells in RD retina showed characteristic WT-like responses to negative current injection. Importantly, they continued to exhibit rebound excitation after a hyperpolarizing potential change (Fig. 6A, arrows, B). This is a distinguishing difference between ON and OFF RGCs in WT retina (Margolis and Detwiler, 2007) (D. J. Margolis, A. J. Gartland, T. Euler, W. Denk, and P. B. Detwiler, unpublished observation). ADP amplitudes in RD OFF-T cells were large and not different from WT (WT, 13.3 ± 1.9 mV, n = 5; RD, 12.3 ± 1.0 mV, n = 4; p = 0.7) (Fig. 6C). Rebound duration and the relationship to the strength of negative current injection were also essentially the same in RD and WT RGCs (Fig. 6B). Three of three RD OFF-S cells also showed WT-like intrinsic properties, including rebound firing (data not shown).

Together, these results indicate that the distinct differences in intrinsic electrophysiological properties of ON and OFF RGCs are still present in RD retina. RGC intrinsic firing properties are stable despite altered ongoing synaptic input.

Similarity of dendritic Ca²⁺ signals in WT and RD RGCs

To test further for changes in the intrinsic properties of RGCs we compared the functional expression of voltage-gated Ca²⁺ channels in the dendrites of RGCs from WT and RD retina. Changes in Ca²⁺-dependent fluorescence were measured from line scans across RGC proximal dendrites using two-photon fluorescence microscopy. In both ON and OFF cells, depolarizing current injection generated spikes and increased intracellular Ca²⁺ (Fig. 7A,B), resulting from activation of high-voltage-activated Ca²⁺ channels. Figure 7 also shows that OFF, but not ON, cells have the additional capacity for rebound spike excitation at the termination of a hyperpolarizing voltage step. As shown previously (Margolis and Detwiler, 2007), rebound excitation in OFF cells results from deinactivation of low-voltage-activated (LVA) Ca²⁺ channels, making them available for activation when the voltage is stepped in the depolarized direction back to the resting potential. The experiments illustrated in Figure 7, A and B, were repeated on a number of ON and OFF cells and the results quantified by measuring the relative fluorescence change per spike (ΔF/F per spike) (see Materials and Methods). The bar graphs in Figure 7C show that there were no significant differences in the fluorescence change per spike in WT versus RD ON and OFF RGCs. In ON cells from WT and RD retina, ΔF/Fs per spike were 3.5 ± 0.8 and 3.2 ± 0.7%, respectively (n = 4 and 3, respectively; p = 0.8) and in OFF cells it was 2.9 ± 0.8 and 2.5 ± 0.8%, respectively (n = 4 and 3, respectively; p = 0.7). For spikes evoked by rebound firing, ΔF/F per spike was 25.3 ± 7.6 and 27.5 ± 13.8% (n = 5 and 3; p = 0.9) in OFF cells from WT and RD retina, respectively (Fig. 7C). The results of these experiments support the conclusion that photoreceptor degeneration and the associated subsequent changes in retinal physiology do not affect the expression and basic properties of voltage-evoked dendritic Ca²⁺ signals in either ON or OFF RGCs.

Altered firing resulting from aberrant synaptic input

To evaluate the effect of the absence of photoreceptors on the physiology of selected ganglion cells, the properties ON, OFF-T, and OFF-S RGCs in the WT and RD mouse retinas were compared. The distinctive rhythmic voltage fluctuations and spike activity present in the RD retina is not present in these cell types in WT retina (Pang et al., 2003; Murphy and Rieke, 2006; Sagdullaev et al., 2006; Margolis and Detwiler, 2007). Nor has this activity been reported under physiological conditions in any other type of adult mammalian RGC. Thus, the periodic spike activity at a characteristic frequency of ∼10 Hz represents a specific form of aberrant activity present during the early stages of retinal degeneration. Two recent reports showing increased glutamate signaling (Marc et al., 2007) and overall spiking activity (Stasheff, 2008) in RD retina are consistent with this view. We found rhythmic activity in older mice (up to P210), but at a reduced rate. Thus, rhythmic retinal output may be a general feature of retinal degeneration throughout progression of the disease.

The results showed that the rhythmic spike activity was caused by oscillatory synaptic input in both ON, OFF-T, and OFF-S RGCs. This indicates that the loss of photoreceptor input to the retina and/or the synaptic alterations that occur during retinal degeneration result in recurrent episodic release of neurotransmitter across parallel ON and OFF retinal pathways. Available evidence argues against the possibility that the activity occurs in phase across the full mosaic of RGCs. Electroretinogram recordings from RD mice (Strettoi et al., 2002) and humans (Francis et al., 2005) with retinal dystrophy show no signs of oscillatory potentials, which would be expected to be seen if the entire retina were “beating” synchronously. There is also no sign of synchronous activity in multielectrode recordings from RD mouse retina (Stasheff, 2008), consistent with the rhythmic retinal activity being asynchronous at least on a spatial scale of >200 μm, the spacing of the multielectrode array.

It is unclear what effect ongoing RGC spike activity may have on postsynaptic visual targets in the brain, but it is known that the RD retina retains functional synaptic connections with higher visual centers (Bi et al., 2006; Chen et al., 2006). Thus, it is possible that the persistent retinal activity we have observed in the blind retina is the source of photopsia in patients with retinitis pigmentosa who describe the sensation of seeing random flashes or shimmering lights in the absence of visual stimulation (Lepore, 1990; Amos, 1999; Murtha and Stasheff, 2003).

Maintenance of dendritic structure, firing properties and Ca²⁺ signaling

Despite the cellular changes that occur during retinal degeneration (Marc et al., 2003; Jones and Marc, 2005), the dendrites of three identified RGC types retain their characteristic branching and stratification patterns. That morphological changes are known to occur in horizontal and bipolar cells of RD retina (Strettoi and Pignatelli, 2000) and generally in response to altered synaptic input in other cell types (Deitch and Rubel, 1984; Kossut and Juliano, 1999), suggests that RGCs have a remarkable degree of morphological stability. These cells, the output cells of the retina, are also functionally stable. This is supported by the fact that the characteristic differences in the intrinsic electrical properties and dendritic Ca²⁺ signals between ON and OFF RGCs are the same in WT and RD retinas. Although there is little evidence of plasticity in the adult retina (Xia et al., 2007), it is nevertheless surprising that the irreversible and long term disruption of normal retinal architecture and function has no effect on the fundamental properties of ON and OFF output cells. The inherent stability of RGCs may be designed to ensure the maintenance of normal retina circuitry during prolonged changes in illumination that occur diurnally. Light, for example, by suppressing transmitter release from photoreceptors deprives the retina of photoreceptor output, which is also a consequence of photoreceptor loss in retinal degeneration.

The function of rhythmic activity: a link between cell survival and Ca²⁺ homeostasis?

What is the function of RGC activity after photoreceptor loss? One possibility is that RGCs must be active to survive. Neuronal survival depends on maintaining appropriate intracellular Ca²⁺ concentration. Because reducing spike activity by direct hyperpolarization changes Ca²⁺ dynamics in both ON and OFF cells (Fig. 8), it is likely that persistent spike activity plays a role keeping Ca²⁺ at a level that is compatible with cell survival. It has been proposed that a self-excitatory process plays a role in cell survival during retinal degeneration (Jones and Marc, 2005). The presence of persistent synaptic input at a fixed “resonant” frequency across both ON and OFF retinal pathways, and the majority of randomly sampled RGCs (Stasheff, 2008), is consistent with this proposal. Oscillatory activity may therefore represent an intrinsic retinal mechanism necessary for cell survival, and may be a general strategy for keeping neurons alive in the absence of normal synaptic transmission. This hypothesis could be tested by comparing the survival of RGCs in RD retina in the presence and absence of long-term synaptic blockade using intraocular injection of slow release antagonists.