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Regulation of interneuron excitability by gap junction coupling with principal cells

Nature Neuroscience volume 16, pages 1764–1772 (2013)Cite this article

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Abstract

Electrical coupling of inhibitory interneurons can synchronize activity across multiple neurons, thereby enhancing the reliability of inhibition onto principal cell targets. It is unclear whether downstream activity in principal cells controls the excitability of such inhibitory networks. Using paired patch-clamp recordings, we show that excitatory projection neurons (fusiform cells) and inhibitory stellate interneurons of the dorsal cochlear nucleus form an electrically coupled network through gap junctions containing connexin36 (Cxc36, also called Gjd2). Remarkably, stellate cells were more strongly coupled to fusiform cells than to other stellate cells. This heterologous coupling was functionally asymmetric, biasing electrical transmission from the principal cell to the interneuron. Optogenetically activated populations of fusiform cells reliably enhanced interneuron excitability and generated GABAergic inhibition onto the postsynaptic targets of stellate cells, whereas deep afterhyperpolarizations following fusiform cell spike trains potently inhibited stellate cells over several hundred milliseconds. Thus, the excitability of an interneuron network is bidirectionally controlled by distinct epochs of activity in principal cells.

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Figure 1: Asymmetric electrical coupling between DCN fusiform and stellate cells.
Figure 2: Fusiform cells generate spikelets in electrically coupled stellate cells.
Figure 3: Fusiform cell action potentials propagate into the distal apical dendrites.
Figure 4: Control of the stellate cell membrane potential by fusiform cells and auditory nerve activity.
Figure 5: Optogenetic activation of fusiform cells depolarizes stellate cells.
Figure 6: Fusiform cells control the spike rate of stellate cells.
Figure 7: The timing of fusiform cell activity bidirectionally controls stellate cell spike output.
Figure 8: Fusiform cells generate inhibition in the DCN.

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Acknowledgements

We thank M. Roberts and S. Kuo for preliminary observations that led us to search for electrical coupling in the DCN; M. Bateschell and R. Larisch for help with mouse colony management and genotyping; C. Borges-Merjane (Vollum Institute/Oregon Hearing Research Center) for initially genotyping the Thy1-ChR2 mice and providing the cerebellum slices used in Supplementary Figure 2; H.-W. Lu for writing the macros used to analyze the calcium imaging data; S. Foster for performing the auditory brainstem response measurements; and K. Bender, W. Giardino and N. Sawtell for critical comments on the manuscript. Funding was provided by US National Institutes of Health grants R01DC004450 to L.O.T., F31DC012222 to P.F.A. and P30 DC005983 to the Oregon Hearing Research Center.

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Authors and Affiliations

  1. Neuroscience Graduate Program, Oregon Health and Science University, Portland, Oregon, USA

    Pierre F Apostolides

  2. Vollum Institute and Oregon Hearing Research Center, Oregon Health and Science University, Portland, Oregon, USA

    Pierre F Apostolides & Laurence O Trussell

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P.F.A. collected the data. P.F.A. and L.O.T. designed the study, analyzed and interpreted the results and wrote the manuscript.

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Correspondence to Laurence O Trussell.

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Integrated supplementary information

Supplementary Figure 1 The gap junction blocker MFA abolishes electrical coupling.

a) Average traces from a coupled fusiform-stellate cell pair (upper and lower traces, respectively). Black traces are the baseline period and red traces are after 15-30 min of incubation in the gap junction blocker MFA (100-200 μM). The increased amplitude in the stellate cell prejunctional waveform after MFA likely reflects a contribution of gap junction channels to the basal leak conductance. b) Summary of the coupling coefficients from six pairs before and after application of MFA. Open circles represent individual experiments, red points are mean±1SEM. Left and right panels represent fusiform-to-stellate and stellate-to-fusiform directions, respectively. MFA significantly reduced the coupling coefficient in the fusiform-to-stellate cell direction from 0.10±10.02 to 0.008±1 0.002 (t(5)=4.74, p=0.005), whereas the stellate-to-fusiform coupling dropped from 0.018±10.003 to 0.002±10.001 (t(5)=4.63, 0.006, paired t-tests).

Supplementary Figure 2 DCN stellate cells are weakly electrically-coupled.

a) Top: Example average traces from an electrically-coupled pair of DCN stellate cells. Lower panel: Summary data plotting the coupling coefficients for 9 coupled pairs. The apparent directional asymmetry in some pairs is probably due to intrinsic variability in basal input resistance, as the group average (red point) falls on the unity line. The homologous coupling coefficient between DCN stellate cells (0.024±0.005, average of both directions) was significantly weaker than fusiform-to-stellate cell coupling (F(2,123)=23.82, p=0.0006, One way ANOVA + Dunnett's multiple comparisons tests) but not significantly different from stellate-to-fusiform coupling (p=0.998). The calculated junctional conductance for homologous coupling between DCN stellate cells was 0.14±0.04 nS. b) Top: Example average traces from a pair of cerebellar molecular layer interneurons. 9/18 pairs showed coupling, and the coupling coefficient (0.15±0.02) was significantly greater than in DCN stellate cells (t(17)=6.83, p<0.0001, unpaired t-test). These values are in line with previous reports13. The junctional conductance for coupling between cerebellar stellate cells was 0.52± 0.07 nS.

Supplementary Figure 3 Fusiform cells are weakly electrically coupled to one another.

a) Example average traces from a pair of fusiform cells. Current injection in one cell results in a small hyperpolarization of the other cell. Black and red traces are before and after addition of the gap junction blocker MFA, confirming that this effect is due to gap junction coupling. b) Summary data showing the coupling coefficient of 20 similar pairs. Red symbol is mean±1SEM. Dotted gray line is unity. Homologous coupling between fusiform cells (0.014±10.001, average of both directions) was significantly weaker than fusiform-to-stellate cell coupling (F(3,143)=22.41, p<0.0001, one way ANOVA and Dunnett's multiple comparison test), but not significantly different than stellate-to-fusiform or stellate-to-stellate coupling (p=0.74 and p=0.95, respectively). The junctional conductance for this data set was 0.50±10.07 nS. c) Summary data showing that 100-200 μM MFA significantly reduced electrical coupling between fusiform cells (n=3 pairs; t(2)=9.5, p=0.01, paired t-test.) Open symbols are individual experiments, red is mean±1SEM. Asterisks denote statistical significance. d) Average traces from a fusiform cell pair in a Cx36-/- mouse. Electrical coupling was absent in fusiform cell pairs recorded in Cx36-/- mice (0/20 connected. c2(1)=22.64,p<0.0001 compared to wild-type mice.)

Supplementary Figure 4 Stellate cells do not express ChR2

a) Voltage-clamped fusiform (left) or stellate cell (right) at different membrane potentials. The -67 and +53 mV traces are highlighted for comparison in black and mauve, respectively. Peak and steady-state photocurrents in fusiform cells are outward at +53 mV, while stellate cell responses remain inward at all potentials. Traces from a Thy1-ChR2 mouse; similar results were observed in VGluT2-ChR2 mice. b) Average IV relationship for photocurrent responses in fusiform and stellate cells from ChR2 mice (n=10 cells each, solid and open symbols, respectively). Stellate cell responses were always inward, whereas fusiform cell photocurrents showed reversal. The positive reversal potential in fusiform cells may be due in part to electrically-coupled fusiform cells that are also depolarized during the light stimulus (Supplemental Figure 3). Moreover, the dendritic arbor of fusiform cells (see example cell in Figure 3, main text) may result in space-clamp limitations. c) Differential block of fusiform and stellate cell light responses by the gap junction blocker MFA. Average photocurrent responses in a simultaneously recorded (uncoupled) fusiform/stellate cell pair from a Thy1-ChR2 mouse during baseline (black) and after 200 μM MFA (red). The peak photocurrent in the fusiform cell was unaffected, whereas the stellate cell response was entirely abolished. MFA caused a small reduction in the steady-state fusiform cell response, and blocked the small outward current upon light offset. This may result from electrical coupling of adjacent fusiform cells activated during the light stimulus. d) Summary data showing the effects of 100-200 μM MFA on photocurrents in fusiform and stellate cells. The leftmost points show the fraction remaining in MFA (normalized to a pre-drug baseline) for the peak and steady-state amplitudes of the fusiform cell photocurrents. Black lines connect individual experiments, red points are mean±1SEM. MFA had no effect on peak photocurrents (98±1 4% remaining, n=7. t(6)=0.34, p=0.7, paired t-test). MFA slightly reduced the steady state photocurrent in 4/7 experiments, although this value was not statistically significant in grouped data (82±1 8% remaining. t(6)=2.09, p=0.08, paired t-test). The rightmost data points represent the fraction remaining of the steady-state postjunctional photocurrent in stellate cells. MFA completely abolished photocurrents in stellate cells (3±1 1% remaining, n=9. t(8)=3.93, p=0.004, paired t-test).

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Apostolides, P., Trussell, L. Regulation of interneuron excitability by gap junction coupling with principal cells. Nat Neurosci 16, 1764–1772 (2013). https://doi.org/10.1038/nn.3569

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