Nonlinear dendritic integration of electrical and chemical synaptic inputs drives fine-scale correlations

Journal name:
Nature Neuroscience
Volume:
17,
Pages:
1759–1766
Year published:
DOI:
doi:10.1038/nn.3851
Received
Accepted
Published online

Abstract

Throughout the CNS, gap junction–mediated electrical signals synchronize neural activity on millisecond timescales via cooperative interactions with chemical synapses. However, gap junction–mediated synchrony has rarely been studied in the context of varying spatiotemporal patterns of electrical and chemical synaptic activity. Thus, the mechanism underlying fine-scale synchrony and its relationship to neural coding remain unclear. We examined spike synchrony in pairs of genetically identified, electrically coupled ganglion cells in mouse retina. We found that coincident electrical and chemical synaptic inputs, but not electrical inputs alone, elicited synchronized dendritic spikes in subregions of coupled dendritic trees. The resulting nonlinear integration produced fine-scale synchrony in the cells' spike output, specifically for light stimuli driving input to the regions of dendritic overlap. In addition, the strength of synchrony varied inversely with spike rate. Together, these features may allow synchronized activity to encode information about the spatial distribution of light that is ambiguous on the basis of spike rate alone.

At a glance

Figures

  1. Gap junctions between ganglion cells mediate fine-scale correlated activity.
    Figure 1: Gap junctions between ganglion cells mediate fine-scale correlated activity.

    (a) A cartoon cross-section of the inner retina, depicting glutamatergic bipolar cells terminals (blue triangles) synapsing onto electrically coupled RGCs, labeled in the Hb9::eGFP retina (green cells; for simplicity only the ON dendrites are drawn). The flash used to stimulate the photoreceptors (not shown) is illustrated by the yellow bar. The vertical and horizontal arrows indicate chemical and reciprocal electrical synapses, respectively. (b) Spike trains evoked by a high-contrast spot (300-μm diameter, 10 trials), measured simultaneously from a pair of neighboring cRGCs (C1 and C2), are synchronized on a broad timescale by the stimulus. (c) A high temporal resolution view of a portion of the ON response shown in b illustrates fine-scale correlated spikes (highlighted by gray boxes, 2 ms) that are independent of the stimulus. (d) A cross-correlogram that indicates the distribution of spike times measured in C2 relative to each spike in C1 (0.5-ms bins). Inset shows the fine-scale correlated peaks in the cross-correlogram at higher temporal resolution. (e) The bimodal peaks (within ±2 ms) in the cross-correlogram were abolished in the presence of the gap junction blocker 18βGA (25 μM). Correlations were also weaker between Hb9+ cells in Cx36−/− retina (Supplementary Fig. 5). (f) Polar plots (top left) indicating the peak spike rate (normalized to the preferred direction) evoked by stimuli moving in eight directions, measured simultaneously in a pair of uncoupled directionally selective ganglion cells (C1 and C2) coding different directions. Gaussian approximations of their receptive fields, indicating the high degree of receptive field overlap between C1 and C2 (bottom left, Online Methods). Right, cross-correlogram for light-evoked spike activity for this pair of uncoupled RGCs. D, dorsal; N, nasal; V, ventral; T, temporal.

  2. Simulated coupled spikelets do not act at the soma to drive correlated spiking.
    Figure 2: Simulated coupled spikelets do not act at the soma to drive correlated spiking.

    (a) Spiking responses in a pair of cRGCs (C1 red, C2 blue) in which both cells were injected with constant depolarizing current (~200 pA) through the patch electrode. (b) The area shaded in gray in a is shown at higher temporal resolution. The arrow points to a coupled spikelet (driven by C1) revealed during a spike failure in C2. (c) A cross-correlogram computed for spike trains measured in the pair of cRGCs shown in a. (d) Representative responses of an ON-OFF directionally selective RGC to a 1-s light flash while a Poisson-distributed train of simulated spikelets (indicated on top) of varying amplitudes (indicated on the left) were injected into the soma. The insets show the voltage responses to the spikelet injections at higher magnification (scale bars represent 5 mV, 100 ms). (e) Plots of the distribution of peak membrane depolarizations evoked by different-sized current injections. p is the fraction of spikelets in each bin. (f) CI between injected spikelets and light-evoked action potentials plotted as a function of delay (n = 6 cells).

  3. Gap junction inputs on their own do not trigger dendritic spikes.
    Figure 3: Gap junction inputs on their own do not trigger dendritic spikes.

    (a) Spike trains evoked in C1 (black, 200-pA depolarizing current step injected through the patch electrode) drove coupled spikelets in C2 (red), which were exclusively mediated by gap junctions (spikelets are shown on a magnified scale). (b) Local application of TTX blocked somatic spikes (top, black), revealing dendritic spikes (middle, blue) that were abolished when TTX was applied over the entire cell (bottom). (c) A plot of the interspike interval (dendritic spikes (blue) and somatic action potentials (gray), n = 91 action potentials and 139 dendritic spikes from 4 cells) illustrates a ~5-ms refractory period for somatic action potentials, but not dendritic spikes. (d) Overlays of somatic action potentials (black), dendritic spikes (blue) and coupled spikelets (red), shown on the same scale (n = 10 events; left). The time derivative of these events is shown on the right. (e) Normalized versions of the traces shown in d, emphasizing the different kinetics of the somatically measured events. (f) The maximum rate of change in voltage plotted against the peak voltage for the three types of events (35 events are plotted for each type). For this plot, light-evoked somatic action potentials and dendritic spikes were from the same cell that was hyperpolarized to increase the failure of somatic action potentials (Supplementary Fig. 6), whereas coupled spikelets were measured using the protocol shown in a.

  4. Dendritic spikes appear to mediate gap junction-dependent fine-scale synchronization.
    Figure 4: Dendritic spikes appear to mediate gap junction–dependent fine-scale synchronization.

    (a) Top, an infrared image depicting the recording configuration (a fluorescent image is overlaid to show Hb9+ GFP cells in blue). The panels below show fluorescent images of the Alexa 488–filled somata of neighboring cRGCs (green channel shown in blue), the local TTX puff imaged in a separate channel (Alexa 594 was included in the puff pipette, red channel shown in yellow) and an overlay of these images. (b) Cross-correlograms computed for light-evoked spike trains from a pair of cRGCs in control conditions (left) and when TTX was locally applied over the region of dendritic overlap (right). (c) Responses to drifting gratings (top, four trials) are shown for a paired recording in which TTX was locally puffed over the soma of C2. Somatic action potentials (APs) were measured extracellularly from C1 (red traces) and dendritic spikes were measured intracellularly from C2 (blue traces; see Supplementary Fig. 9 for experimental details). Individual extracellular spikes (C1, red) and dendritic spikes (C2, blue) are shown below at a higher resolution (ten events are shown for each). (d) A cross-correlogram (0.5-ms bins) plotting the distribution of dendritic spike times (measured in C2) relative to action potentials in C1 (set at time 0). The inset shows a cross-correlogram of the same events in d, but sampled at a higher time resolution (0.1-ms bins), illustrating that the dendritic spikes in C2 showed an increased probability of occurring shortly after a spike in C1.

  5. Gap junction-mediated correlations are spatially restricted to overlapping dendritic regions.
    Figure 5: Gap junction–mediated correlations are spatially restricted to overlapping dendritic regions.

    (a) An illustration of the experimental protocol depicting small spot stimuli positioned to stimulate common or non-overlapping regions of the receptive fields of neighboring cRGCs. (b) The location of the three spots are overlaid over the experimentally measured receptive fields, estimated with one-dimensional Gaussian functions (Online Methods). (c) The light-evoked spiking response of the pair of neurons shown in b following stimulation of either common (spot 1) or uncommon regions (spot 2 + 3). (d) Representative cross-correlograms constructed from spike trains during stimulation of common (left) or uncommon regions (right). (e) The average CI values for activity driven by common and uncommon input as shown in d. Error bars, s.e.m.

  6. Correlation strength varies inversely with spike rate.
    Figure 6: Correlation strength varies inversely with spike rate.

    (a) Representative spiking responses to moving gratings of high, mid and low contrast recorded from a pair of cRGCs (top, four trials are shown) from which cross-correlograms (bottom) were computed. (bd) Spike rate was modulated by changing contrast, direction or using pharmacology. The average peak spike rate versus CI for individual pairs is plotted for the different conditions (as labeled). Examples in which the average peak spike rate of the two cells decreased (red = control versus 20 μM NBQX; pink = control versus 50 μM AP5; purple = control versus 50 μM tubocurare) or increased (blue = control versus 50 μM picrotoxin; green = picrotoxin high contrast versus picrotoxin low contrast) are shown in d.

  7. Correlated action potentials carry information that is independent of the spike rate.
    Figure 7: Correlated action potentials carry information that is independent of the spike rate.

    (a) A cross-correlogram computed from spike trains evoked by a high-contrast bar (100 × 400 μm, left), the same bar presented at lower contrast (middle) or when a gap in the stimulus was used to decrease stimulation of the overlapping regions between the pair of cRGCs (right), as indicated in the cartoon (top). Note that varying the contrast of the bar produced a similar change in the spike rate of cRGCs as changing the pattern of the stimulus. (b,c) Plots of the average peak spike rate (black) and CI (red) for responses to bars of high or low contrast and for bars without or with a gap, respectively. Error bars, s.e.m.

  8. Broad stimulus-driven and fine-noise correlations in cRGCs.
    Supplementary Fig. 1: Broad stimulus-driven and fine-noise correlations in cRGCs.

    a, A cross-correlogram from the same pair of cells shown in Fig. 1d, but shown on a broader timescale (± 100ms) to reveal slower stimulus driven correlations. b, The same raw cross correlogram as shown in Fig. 1d (left), as well as the shuffled trials shift predictor (middle) and the shift-predictor subtracted (right, Corrected) correlations.

  9. Fine-scale gap junction-mediated correlations are present for both ON and OFF responses of cRGCs.
    Supplementary Fig. 2: Fine-scale gap junction–mediated correlations are present for both ON and OFF responses of cRGCs.

    a, A raster plot of the light-evoked spiking response (5 repeats are shown) of a pair of cRGCs (C1-red and C2-blue) to a high contrast flash of light (indicated on bottom). b, Cross-correlograms for both ON (left) and OFF (middle) responses exhibit fine-scale correlations. For data presented in the paper, we pooled data from ON and OFF responses (right).

  10. Pharmacologically blocking gap junctions inhibits fine-scale correlations, but does not block broad timescale stimulus-dependent correlations.
    Supplementary Fig. 3: Pharmacologically blocking gap junctions inhibits fine-scale correlations, but does not block broad timescale stimulus-dependent correlations.

    a, Control (left) and 18β-glycyrrhetinic acid (18βGA, 25 µM, right) treated responses from a pair of cRGCs. The top traces represent 4 trails of drifting gratings moving above a pair of neighbouring cRGCs (C1, red = cell 1; C2, blue = cell 2). Gaussian fits of the spike rate are plotted below. b, Cross-correlograms for the relevant control and 18βGA treated responses. The zoom in (bottom left) shows that the peak in the correlogram is a fine-scale correlation at < ± 2 ms, which is eliminated with application of 18βGA, while broad timescale stimulus driven correlations persist.

  11. Uncoupled RGCs do not exhibit fine-scale correlations.
    Supplementary Fig. 4: Uncoupled RGCs do not exhibit fine-scale correlations.

    a, A cross-correlogram (± 200 ms), showing the broad, stimulus driven correlation between a pair of uncoupled directionally selective ganglion cells. Note the absence of an additional fine-scale peak in the graph at ± ~2 ms.

  12. Coupling between cRGCs and fine-scale correlations are reduced in Cx36 knockout retina.
    Supplementary Fig. 5: Coupling between cRGCs and fine-scale correlations are reduced in Cx36 knockout retina.

    a, Example paired current clamp recordings from wt (top) and Cx36 knockout retina (bottom). The cartoon (top left) shows the experimental paradigm, where hyperpolarizing and depolarizing currents are injected into cell 1 (black). The change in membrane potential upon current injection into C1 is plotted for C1 (left, black) and C2 (right, red). Note that the amount of voltage change driven by gap junctions in C2 is reduced in Cx36 knockout. b, Plots of cross-correlograms for light evoked spike activity in cRGCs in wildtype retina (top) and Cx36 knockout retina (bottom), revealing that correlated spiking is greatly reduced in the absence of Cx36.

  13. Depolarization induced correlations persist in a cocktail of synaptic receptor blockers.
    Supplementary Fig. 6: Depolarization induced correlations persist in a cocktail of synaptic receptor blockers.

    a, The light response of a cRGC in control conditions (top) and in the presence of a cocktail of synaptic receptors blockers (bottom). The cocktail included: NBQX (20 µM, to block AMPA/Kainate receptors), AP5 (50 µM, to block NMDA receptors), tubocurare (50 µM, to block acetylcholine receptors), L-AP4 (50 µM, to block mGluR6 receptors), picrotoxin (50 µM, to block GABAA receptors), TPMPA (50 µM, to block GABAC receptors) and strychnine (5 µM, to block glycine receptors). The yellow bars indicate the duration of the light stimulus. b, Shows the correlated spiking responses of a pair of cRGCs when simultaneously depolarized by ~10 mV through the patch electrode (top). The bottom trace is a higher magnification view of the area show in grey. c, A cross-correlogram computed for the spike trains shown in (b). Note, as with the light response, the size of the bimodal peaks varied for different pairs, likely due to a combination factors including differences in the level of depolarization between two cells, the precise properties of the dendro-dendritic connections (which could potentially give rise to impedance mismatches) and intrinsic excitability.

  14. Dendritic spikes are revealed by hyperpolarizing the soma through the patch electrode.
    Supplementary Fig. 7: Dendritic spikes are revealed by hyperpolarizing the soma through the patch electrode.

    a, The light response of a cRGC is shown in control (top) and when hyperpolarized with a -150 pA current injection (bottom). b, The area highlighted in grey in (a) is shown at higher resolution (top). Plotting dV/dt allows the clear differentiation between single dendritic spikes (left), multiple dendritic spikes that sum (note that dendritic spikes do not to have a refractory period; middle, grey arrow) and somatic action potentials (right).

  15. Local dendritic application of TTX does not affect the shape of somatic action potentials.
    Supplementary Fig. 8: Local dendritic application of TTX does not affect the shape of somatic action potentials.

    a, A diagram outlining the experimental protocol, in which TTX is locally puffed over the overlapping dendritic regions between two neighbouring cRGCs. b, The average spike shapes for a pair of cRGCs before (blue) and during (red) dendritic TTX application.

  16. Detecting dendritic spikes by inhibiting somatic Na+ channels.
    Supplementary Fig. 9: Detecting dendritic spikes by inhibiting somatic Na+ channels.

    a, Intracellular recording of responses of a cRGC to drifting gratings while TTX was puffed locally over the soma reveals dendritic spikes (asterisks) and larger spike like events that represent incompletely blocked somatic spikes (see Oesch et al., 2005 for similar results in the rabbit retina). b, The time derivative of the voltage trace shown in (a). Events with peak amplitudes falling between the blue dotted lines were unequivocally identified as dendritic spikes and chosen for cross-correlation analysis (Fig. 4d in main text). Larger events that potentially could have arisen from the soma (due to the incomplete block of somatic Na+ channels by TTX) were discarded in this analysis. c, A histogram of the peak amplitude of spike-like events during somatic TTX application (>500 events).

  17. Dendritic spikes in post-junctional cells are synchronized with somatic action potentials in pre-junctional cells.
    Supplementary Fig. 10: Dendritic spikes in post-junctional cells are synchronized with somatic action potentials in pre-junctional cells.

    a, An average cross-correlogram, from 4 pairs, of the relationships between somatic spikes in one cell and dendritic spikes in a coupled neighbor (the red line indicates the 95% confidence interval). Somatic action potentials in cell 1 are set at time 0, meaning that peaks at positive time values represent dendritic spikes in cell 2 that consistently occurred after somatic action potentials in cell 1.

  18. Both common and uncommon inputs drive broad stimulus-driven correlations, but not fine-scale correlations.
    Supplementary Fig. 11: Both common and uncommon inputs drive broad stimulus-driven correlations, but not fine-scale correlations.

    a, A diagram of the experimental paradigm, where cells are either driven with a spot that stimulates common input (spot 1) or with two spots that stimulate uncommon inputs (spots 2+3). b, A cross-correlogram for a pair of cRGCs receiving common input. c, A cross-correlograms the same pair of cRGCs receiving uncommon input. This pair is the same pair shown in Fig. 5d.

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Author information

  1. Present address: Department of Neurobiology, Friedrich Miescher Institute, Basel, Switzerland.

    • Stuart Trenholm
  2. These authors contributed equally to this work.

    • Stuart Trenholm &
    • Amanda J McLaughlin

Affiliations

  1. Department of Biology, University of Victoria, Victoria, British Columbia, Canada.

    • Stuart Trenholm,
    • Amanda J McLaughlin &
    • Gautam B Awatramani
  2. Department of Physics, Princeton University, Princeton, New Jersey, USA.

    • David J Schwab
  3. Department of Physiology and Biophysics, University of Washington, Seattle, Washington, USA.

    • Maxwell H Turner &
    • Fred Rieke
  4. Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Robert G Smith

Contributions

This study was designed by S.T., D.J.S., A.J.M., F.R., M.H.T. and G.B.A. All of the experiments were performed by S.T. and A.J.M. (except for those presented in Fig. 2d–f, which were performed by M.H.T.). The results were analyzed by S.T., A.J.M., M.H.T. and D.J.S. The paper was written by S.T., D.J.S., M.H.T., F.R., R.G.S. and G.B.A.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Broad stimulus-driven and fine-noise correlations in cRGCs. (78 KB)

    a, A cross-correlogram from the same pair of cells shown in Fig. 1d, but shown on a broader timescale (± 100ms) to reveal slower stimulus driven correlations. b, The same raw cross correlogram as shown in Fig. 1d (left), as well as the shuffled trials shift predictor (middle) and the shift-predictor subtracted (right, Corrected) correlations.

  2. Supplementary Figure 2: Fine-scale gap junction–mediated correlations are present for both ON and OFF responses of cRGCs. (89 KB)

    a, A raster plot of the light-evoked spiking response (5 repeats are shown) of a pair of cRGCs (C1-red and C2-blue) to a high contrast flash of light (indicated on bottom). b, Cross-correlograms for both ON (left) and OFF (middle) responses exhibit fine-scale correlations. For data presented in the paper, we pooled data from ON and OFF responses (right).

  3. Supplementary Figure 3: Pharmacologically blocking gap junctions inhibits fine-scale correlations, but does not block broad timescale stimulus-dependent correlations. (289 KB)

    a, Control (left) and 18β-glycyrrhetinic acid (18βGA, 25 µM, right) treated responses from a pair of cRGCs. The top traces represent 4 trails of drifting gratings moving above a pair of neighbouring cRGCs (C1, red = cell 1; C2, blue = cell 2). Gaussian fits of the spike rate are plotted below. b, Cross-correlograms for the relevant control and 18βGA treated responses. The zoom in (bottom left) shows that the peak in the correlogram is a fine-scale correlation at < ± 2 ms, which is eliminated with application of 18βGA, while broad timescale stimulus driven correlations persist.

  4. Supplementary Figure 4: Uncoupled RGCs do not exhibit fine-scale correlations. (54 KB)

    a, A cross-correlogram (± 200 ms), showing the broad, stimulus driven correlation between a pair of uncoupled directionally selective ganglion cells. Note the absence of an additional fine-scale peak in the graph at ± ~2 ms.

  5. Supplementary Figure 5: Coupling between cRGCs and fine-scale correlations are reduced in Cx36 knockout retina. (118 KB)

    a, Example paired current clamp recordings from wt (top) and Cx36 knockout retina (bottom). The cartoon (top left) shows the experimental paradigm, where hyperpolarizing and depolarizing currents are injected into cell 1 (black). The change in membrane potential upon current injection into C1 is plotted for C1 (left, black) and C2 (right, red). Note that the amount of voltage change driven by gap junctions in C2 is reduced in Cx36 knockout. b, Plots of cross-correlograms for light evoked spike activity in cRGCs in wildtype retina (top) and Cx36 knockout retina (bottom), revealing that correlated spiking is greatly reduced in the absence of Cx36.

  6. Supplementary Figure 6: Depolarization induced correlations persist in a cocktail of synaptic receptor blockers. (131 KB)

    a, The light response of a cRGC in control conditions (top) and in the presence of a cocktail of synaptic receptors blockers (bottom). The cocktail included: NBQX (20 µM, to block AMPA/Kainate receptors), AP5 (50 µM, to block NMDA receptors), tubocurare (50 µM, to block acetylcholine receptors), L-AP4 (50 µM, to block mGluR6 receptors), picrotoxin (50 µM, to block GABAA receptors), TPMPA (50 µM, to block GABAC receptors) and strychnine (5 µM, to block glycine receptors). The yellow bars indicate the duration of the light stimulus. b, Shows the correlated spiking responses of a pair of cRGCs when simultaneously depolarized by ~10 mV through the patch electrode (top). The bottom trace is a higher magnification view of the area show in grey. c, A cross-correlogram computed for the spike trains shown in (b). Note, as with the light response, the size of the bimodal peaks varied for different pairs, likely due to a combination factors including differences in the level of depolarization between two cells, the precise properties of the dendro-dendritic connections (which could potentially give rise to impedance mismatches) and intrinsic excitability.

  7. Supplementary Figure 7: Dendritic spikes are revealed by hyperpolarizing the soma through the patch electrode. (55 KB)

    a, The light response of a cRGC is shown in control (top) and when hyperpolarized with a -150 pA current injection (bottom). b, The area highlighted in grey in (a) is shown at higher resolution (top). Plotting dV/dt allows the clear differentiation between single dendritic spikes (left), multiple dendritic spikes that sum (note that dendritic spikes do not to have a refractory period; middle, grey arrow) and somatic action potentials (right).

  8. Supplementary Figure 8: Local dendritic application of TTX does not affect the shape of somatic action potentials. (53 KB)

    a, A diagram outlining the experimental protocol, in which TTX is locally puffed over the overlapping dendritic regions between two neighbouring cRGCs. b, The average spike shapes for a pair of cRGCs before (blue) and during (red) dendritic TTX application.

  9. Supplementary Figure 9: Detecting dendritic spikes by inhibiting somatic Na+ channels. (159 KB)

    a, Intracellular recording of responses of a cRGC to drifting gratings while TTX was puffed locally over the soma reveals dendritic spikes (asterisks) and larger spike like events that represent incompletely blocked somatic spikes (see Oesch et al., 2005 for similar results in the rabbit retina). b, The time derivative of the voltage trace shown in (a). Events with peak amplitudes falling between the blue dotted lines were unequivocally identified as dendritic spikes and chosen for cross-correlation analysis (Fig. 4d in main text). Larger events that potentially could have arisen from the soma (due to the incomplete block of somatic Na+ channels by TTX) were discarded in this analysis. c, A histogram of the peak amplitude of spike-like events during somatic TTX application (>500 events).

  10. Supplementary Figure 10: Dendritic spikes in post-junctional cells are synchronized with somatic action potentials in pre-junctional cells. (126 KB)

    a, An average cross-correlogram, from 4 pairs, of the relationships between somatic spikes in one cell and dendritic spikes in a coupled neighbor (the red line indicates the 95% confidence interval). Somatic action potentials in cell 1 are set at time 0, meaning that peaks at positive time values represent dendritic spikes in cell 2 that consistently occurred after somatic action potentials in cell 1.

  11. Supplementary Figure 11: Both common and uncommon inputs drive broad stimulus-driven correlations, but not fine-scale correlations. (87 KB)

    a, A diagram of the experimental paradigm, where cells are either driven with a spot that stimulates common input (spot 1) or with two spots that stimulate uncommon inputs (spots 2+3). b, A cross-correlogram for a pair of cRGCs receiving common input. c, A cross-correlograms the same pair of cRGCs receiving uncommon input. This pair is the same pair shown in Fig. 5d.

PDF files

  1. Supplementary Text and Figures (1.66 MB)

    Supplementary Figures 1–11

  2. Supplementary Methods Checklist (395 KB)

Additional data