A supercritical density of Na(+) channels ensures fast signaling in GABAergic interneuron axons

Abstract

Fast-spiking, parvalbumin-expressing GABAergic interneurons, a large proportion of which are basket cells (BCs), have a key role in feedforward and feedback inhibition, gamma oscillations and complex information processing. For these functions, fast propagation of action potentials (APs) from the soma to the presynaptic terminals is important. However, the functional properties of interneuron axons remain elusive. We examined interneuron axons by confocally targeted subcellular patch-clamp recording in rat hippocampal slices. APs were initiated in the proximal axon ~20 μm from the soma and propagated to the distal axon with high reliability and speed. Subcellular mapping revealed a stepwise increase of Na(+) conductance density from the soma to the proximal axon, followed by a further gradual increase in the distal axon. Active cable modeling and experiments with partial channel block revealed that low axonal Na(+) conductance density was sufficient for reliability, but high Na(+) density was necessary for both speed of propagation and fast-spiking AP phenotype. Our results suggest that a supercritical density of Na(+) channels compensates for the morphological properties of interneuron axons (small segmental diameter, extensive branching and high bouton density), ensuring fast AP propagation and high-frequency repetitive firing.

Figure 1

Confocally targeted subcellular patch-clamp recording from axons of hippocampal fast-spiking, parvalbumin-expressing GABAergic interneurons. (a) Confocal image of a BC in the dentate gyrus filled with Alexa Fluor 488 during the experiment. Confocal stack maximum projection. (b) Same cell filled with biocytin during recording and labeled with 3,3′-diaminobenzidine as chromogen. White arrows indicate the axon trajectory. In a, b, recording pipettes are illustrated schematically. (c, d) Train of APs evoked by a 1-s depolarizing current pulse applied at the soma (c) and the axon (at 177 μm; d). Black traces, somatic voltage and corresponding current. Blue traces, axonal voltage and corresponding current. Data in a–d are from the same cell. (e) Double immunolabeling for parvalbumin (a selective marker of fast-spiking GABAergic interneurons) and myelin basic protein (MBP, a specific marker of myelination). Left, parvalbumin; center, MBP; right, overlay. Note the absence of colocalization between the two markers in the granule cell layer, suggesting that BC axons are largely unmyelinated. Experiments were repeated on 5 animals, giving consistent results. White arrows indicate the outer border of the granule cell layer. Lower micrographs are expanded versions of upper images.

Figure 2

Proximal initiation and fast, reliable propagation of APs in interneuron axons. (a) Simultaneous recording from the soma and the axon of a fast-spiking, parvalbumin-expressing BC during a long somatic current pulse. Black traces, somatic voltage and corresponding current; blue traces, axonal voltage and corresponding current. The axonal recording site was located 24 μm from the soma. Bottom traces show expanded views of the first and the 20^th AP in the high-frequency train. (b) Another simultaneous recording from the soma and the axon, in which the axonal recording site was located 315 μm from the soma. (c) Plot of latency between somatic and axonal AP against distance of the axonal recording site from the soma for the first AP (upper graph) and the 20^th AP (lower graph). Data from 62 axon–soma recordings. Data were fit with a bilinear function (continuous red line), giving a minimal latency at 20 and 22 μm and a propagation velocity of 0.52 m s⁻¹ and 0.49 m s⁻¹ for the first and the 20^th AP, respectively. Filled circles, recordings from axon varicosities; open circles, recordings from axon shafts. (d) Simultaneous recording from the soma and the axon of a BC during a high-frequency train of short somatic current pulses (120 stimuli at 100 Hz; 3 ms, 1 nA). Black, somatic voltage; blue, axonal voltage. The axonal recording site was located 222 μm from the soma. (e) Plot of instantaneous somatic and axonal AP frequency during the stimulation train (same cell as shown in d). (f) Plot of ratio of number of APs in the axon over number of APs in the soma, plotted against distance of the axonal recording site. Data from 28 simultaneous axon–soma recordings.

Figure 3

A high Na⁺ channel density in interneuron axons. (a) Na⁺ current in an outside-out patch in the soma, proximal axon (50 μm), and distal axon (315 μm from the soma). Na⁺ currents were evoked by a sequence of a prepulse to −120 mV followed by a test pulse to 0 mV (bottom). Black trace, somatic patch; blue traces, axonal patches. (b) Summary plot of Na⁺ conductance density ($\overline{g_{Na}}$) against distance from the soma. Data from 48 axonal recordings and 24 somatic recordings. (c) Summary bar graph showing $\overline{g_{Na}}$ in the soma (24 patches), proximal axon (< 100 μm, 37 patches) and distal axon (≥ 100 μm, 11 patches). Bars indicate mean ± SEM, circles represent data from individual experiments. * indicates 0.01 ≤ P < 0.05; ** indicates P < 0.01. (d) Top, overlay of 300 axonal Na⁺ current traces, superimposed with the average (red). Center, superimposition of the difference of individual current traces from the mean. Bottom, voltage-clamp protocol for evoking Na⁺ current. (e) Plot of variance versus mean from the experiment shown in (d). Red curve represents a parabolic function fit to the data points. (f) Summary bar graph of single-channel conductance and maximal open probability of Na⁺ channels (both at 0 mV). Bars indicate mean ± SEM, circles represent data from individual experiments. (g) Summary plot of Na⁺ channel density against distance from the soma. Symbol code in b, c, and g: Black symbols, somatic patches; blue symbols, axonal patches; filled circles, recordings from axon varicosities; open circles, recordings from axon shafts.

Figure 4

Dependence of reliability and velocity of AP propagation on axonal Na⁺ conductance density in an active BC model. (a, b) Plot of reliability of AP propagation, shown as color coding of the surface of the reconstructed neuron for $\overline{g_{Na}}$ = 200 pS μm⁻² (a) and 500 pS μm⁻² (b); proximal and distal $\overline{g_{Na}}$ were changed in parallel. Color scale bar indicates the reliability of propagation in the axon (inset, right); dendrites are depicted in black to indicate the lack of active AP propagation. Gray sphere represents the proximal region of the axon. (c, d) AP propagation reliability–distance plots (c) and contour plot of the average slope of the reliability–distance relation as a function of proximal and distal $\overline{g_{Na}}$ (d). Red circles and numbers in d indicate the correspondence with the graphs shown in c. Numbers right-adjacent to contour lines in d indicate distance dependence of AP propagation reliability (in mm⁻¹; values in the range ± 0.02 were represented as 0; see gray scale bar on the right). In a–d, 100-Hz trains of 10 brief stimuli were applied to the soma and the number of successfully propagated APs in the axon was plotted against distance. (e, f) Latency–distance plots (e) and contour plot of AP propagation velocity as a function of proximal and distal $\overline{g_{Na}}$. Dashed green lines in e indicate the results of linear regression for distances > 100 μm. Numbers right-adjacent to contour lines in f indicate AP propagation velocity (in m s⁻¹; see gray scale bar on the right). Red circles and numbers in f indicate the correspondence with the graphs shown in e. Inset in f shows one-dimensional representation for simultaneous changes in proximal and distal $\overline{g_{Na}}$. In e, f, single brief stimuli were used to evoke APs. All simulations were performed on cell 2 of Nörenberg et al..

Figure 5

A supercritical Na⁺ channel density in the axon ensures fast AP propagation. (a) Simultaneous axon–soma recordings in control conditions, in the presence of 2 nM TTX, and after washout. Upper traces are displayed at absolute voltage scale, lower traces are shown normalized to the same peak amplitude to facilitate comparison of the rising phase. (b) Confocal stack maximum projection of a recorded BC. Recording pipettes are illustrated schematically. (c) Plot of latency between axonal and somatic AP against experimental time during application of 2 nM TTX (horizontal bar). (d) Summary graph of the effects of 2 nM TTX on latency between somatic and axonal AP. Data from 6 simultaneous axon–soma recordings at distances of 91 to 222 μm. Data from the same experiment were connected by lines. Open circles, data from the cell shown in Supplementary Fig. 10. (e) Effects of 2 nM TTX on reliability of AP propagation during a high-frequency train (5 stimuli at 100 Hz; 5 ms, 0.7 nA). Data in (a–c, e) were obtained from the same experiment (axonal recording site 100 μm from the soma). (f) Na⁺ current recorded from an outside-out patch isolated from the axon 66 μm from the soma before and after bath application of 2 nM TTX. Na⁺ currents were evoked by a sequence of a prepulse to −120 mV followed by a test pulse to 0 mV. (g) Plot of Na⁺ current peak amplitude against experimental time during application of 2 nM TTX (horizontal bar) from the same experiment as shown in (f). Each data point represents the average of 6 consecutive peak current values. (h) Summary graph of the effects of 2 nM TTX on Na⁺ peak current. Data from 5 axonal outside-out patches. Data from the same experiment were connected by lines. In d and h, * indicates 0.01 ≤ P < 0.05 and ** indicates P < 0.01.

Figure 6

A supercritical Na⁺ channel density contributes to the fast-spiking AP phenotype. (a, b) Effects of Na⁺ conductance density on fast-spiking AP phenotype in a BC model. Example traces (a) and contour plot (b) of maximal AP frequency as a function of proximal and distal $\overline{g_{Na}}$. Red circles and numbers in b indicate the correspondence with the graphs shown in a. White numbers right-adjacent to contour lines in b indicate maximal AP frequency (in Hz; see gray scale bar on the right). APs were evoked by 500-ms current pulses (0.5 – 5 nA, 0.5 nA steps; 2.5 nA in a). (c–f) Effects of 2 nM TTX on fast-spiking AP phenotype in experimental conditions. (c) Train of APs evoked by long current pulses under control conditions (top, black trace) and in the presence of 2 nM TTX (bottom, red trace). (d) Plot of AP frequency in spike trains evoked by a 1-s depolarizing current pulse against experimental time during application of 2 nM TTX (horizontal bar). 500-pA current pulses were applied throughout. Data in c–e are from the same cell. (e) Frequency–current relation in control conditions (black filled circles), in the presence of 2 nM TTX (red circles), and after washout (black open circles). Note that 2 nM TTX slightly, but reversibly, reduced the maximal AP frequency. (f) Summary graph of the effects of 2 nM TTX on maximal AP frequency. Data from 10 BCs. Data from the same experiment were connected by lines. ** indicates P < 0.01.