Differential effects of axon initial segment and somatodendritic GABAA receptors on excitability measures in rat dentate granule neurons

Abstract

GABA(A) receptors are found on the somatodendritic compartment and on the axon initial segment of many principal neurons. The function of axonal receptors remains obscure, although it is widely assumed that axonal receptors must have a strong effect on excitability. We found that activation of GABA(A) receptors on the dentate granule neuron axon initial segment altered excitability by depolarizing the voltage threshold for action potential initiation under conditions that minimally affected overall cell input resistance. In contrast, activation of somatic GABA(A) receptors strongly depressed the input resistance of granule neurons without affecting the voltage threshold of action potential initiation. Although these effects were observed over a range of intracellular chloride concentrations, average voltage threshold was unaffected when E(Cl) rendered GABA(A) axon initial segment responses explicitly excitatory. A compartment model of a granule neuron confirmed these experimental observations. Low ambient agonist concentrations designed to activate granule neuron tonic currents did not stimulate axonal receptors sufficiently to raise voltage threshold. Using excitatory postsynaptic current (EPSC)-like depolarizations, we show physiological consequences of axonal versus somatic GABA(A) receptor activation. With axonal inhibition, individual excitatory postsynaptic potentials (EPSPs) largely retained their amplitude and time course, but EPSPs that were suprathreshold under basal conditions failed to reach threshold with GABA(A) activation. By contrast, somatic inhibition depressed individual EPSPs because of strong shunting. Our results suggest that axonal GABA(A) receptors have a privileged effect on voltage threshold and that two major measures of neuronal excitability, voltage threshold and rheobase, are differentially affected by axonal and somatic GABA(A) receptor activation.

Fig. 1.

Strong axonal GABA_A channel activation increases voltage threshold. A: reverse fluorescence image of an Alexa Fluor–filled dentate granule neuron, with pipettes positioned for axon initial segment (AIS) and soma application of muscimol. Soma and AIS puffer pipettes are barely visible to the right of the cell and indicated by arrows. The whole cell recording pipette is indicated with an asterisk in the lumen, to the left of the soma. All 3 pipettes are outlined by dashed lines for clarity. Scale bar is 10 μm. B1: action potentials in response to current injections in the presence of muscimol (30 μM, red traces) interleaved with trials in the absence of muscimol (black traces). Muscimol applications started 50 ms before depolarizing current injection. Action potentials were elicited by 20 ms current injection from a resting potential of −70 mV. B2: from the same cell as in A1, responses to current injections in the presence (red traces) and absence (black traces) of interleaved somatic muscimol applications. B3: phase plot of control (black) and muscimol conditioned (red) action potentials shown in A1. V_m is membrane potential. Inset: voltage thresholds (V_t) as a function of sweep number. C: summary of voltage thresholds for control and muscimol conditions. Every symbol pair represents a single cell's average values for interleaved control and muscimol sweeps (n = 16). In this and subsequent figures, the open squares in the paired scatter plot represent the grand average of all the cells in the baseline and treatment conditions. Here and subsequent figures: *significantly different at the P < 0.05 level. D: representative traces used to obtain input resistance values. The arrow indicates the onset of a muscimol puff to either the AIS (red trace) or soma (blue trace), and the shift in baseline in the red trace before the voltage pulse results from the onset of GABA_A receptor activation. The lower waveform gives the voltage protocol (mV values are given to the left). E: average input resistance for control, axonal, and somatic muscimol application (n = 4), shown in D.

Fig. 2.

Effects of moderate axonal and somatic activation of GABA_A channels. A1: AIS applications of 5 μM muscimol (red traces) depolarized the voltage threshold compared with control (black traces). Inset: magnification of the action potential early upswing, with dotted horizontal lines indicating the average threshold voltage. A2: phase plots of control (black) and muscimol-conditioned (red) action potentials shown in A1. Inset: magnification of the rising part of phase plot, calibration bars are 10 mV/ms and 2 mV. B1: somatic muscimol application produced a delay in action potentials without affecting voltage threshold (inset, same calibration as in A1). B2: phase plots of traces shown in B1. Notice the change in the peak dV/dt, without an effect on voltage threshold (inset, same calibration as A2). C and D: summary of AIS (C) and somatic (D) muscimol applications, with every dot pair representing a single cell (n = 13). Closed symbols represent 5 μM muscimol; open symbols represent 10 μM muscimol. E: change in voltage threshold in response to axonal and somatic muscimol, from C and D. F: input resistance under control (no application), axonal, or somatic muscimol application (n = 5), n.s., not significantly different.

Fig. 3.

Evidence for compartamentalized axonal Cl⁻. A: averaged current traces elicited by voltage (250 mV/s) ramps in presence (gray) or absence (black) of 10 μM axonal AIS muscimol application. Inset: digitally subtracted muscimol-dependent component. The reversal potential was obtained from linear fit of current near the voltage axis crossing (black line). B: response to somatic muscimol application in the same cell as in A. Muscimol-evoked currents were larger than axonal-evoked currents and exhibited a more hyperpolarized reversal potential. C: summary of reversal potentials obtained in paired AIS and somatic applications of 10 (●) and 30 μM (○) muscimol.

Fig. 4.

Altered AIS voltage threshold effects with varied intracellular [Cl⁻]. A1: local muscimol applications with pipette [Cl⁻]_i elevated from 5 (Figs. 1–3) to 17.7 mM still depolarized voltage threshold. Red traces are muscimol; black traces are interleaved control sweeps. Inset: magnification of action potential upswing shows the depolarized threshold. A2: phase plots from traces in A1 in presence (red) and absence (black) of muscimol applications to the AIS. Inset: change in voltage threshold, calibration bars are 10 mV/ms and 2 mV. B1: in the same cell as A1, somatic muscimol application (red traces) depolarized membrane voltage before current injection but did not alter threshold (inset). B2: phase plots from traces in B1 in presence (red) or absence (black) of somatic application. Notice the decrease in peak dV/dt with muscimol, without changes in threshold (inset). C: summary of action potential threshold changes for control and muscimol application during axonal and somatic applications with a pipette Cl⁻ concentration of 17.7 mM (Med Cl⁻; n = 8). The bar graph also shows the lack of significant overall change in threshold for 18 cells filled with 33.4 (High Cl⁻; n = 18 for axons and 6 for somas), so that GABA_A receptor activation was explicitly excitatory.

Fig. 5.

Simulations of the effects of somatic and AIS GABA_A conductances. A: schematic of the cell created in the NEURON simulation environment to test the effect of AIS and somatic Cl⁻ conductance on action potential threshold. Labels indicate dendrite (D), soma (S), and axon (A). The 1st 2 axon compartments, representing the 40 μm long AIS, are shown, although the entire simulated axon extended 240 μm, tapering from 1.5 μm in diameter in the 1st 2 compartments to 0.75 μm diam. The gray shading indicates the region of high sodium channel density (5-fold higher than somatodendritic compartment). The diagonal hatches represent the compartment of the AIS (20–40 μm distal) in which the GABA conductance was introduced. B: action potentials obtained from simulated soma recordings of the model cell. The black trace is a baseline trace (no Cl⁻ conductance). The red trace shows the simulated action potential elicited in the presence of a sustained conductance introduced into the proximal AIS (E_Cl = −80 mV), 20–40 μm from the soma (0.003 S/cm²). The green trace represents the conductance introduced into the soma compartment. For the soma conductance, we decreased the conductance density to 0.001 S/cm² to keep the rheobase for the conditions within a similar range (90 pA for soma vs. 70 pA for AIS conductance). C: initial portions of phase plots obtained from the simulation with a Cl⁻ conductance introduced at the soma and with E_Cl at −80 and −30 mV for the green and pink traces, respectively. The dashed black trace is the baseline trace. The black and green phase plots were derived from the action potentials in B. The dotted horizontal line indicates the threshold of 10 mV/ms. D: phase plot rises show the effect of AIS Cl⁻ conductance on action potential threshold at 3 different E_Cl values, as indicated. The vertical dashed lines give the membrane potential at threshold. The light blue line (E_Cl = −30 mV) and the black dashed baseline line are superimposed and nearly indistinguishable. Both E_Cl = −57 mV (dark blue trace) and E_Cl = −80 mV (red trace) conditions yielded depolarization of action potential threshold. The red trace is derived from the red action potential in B.

Fig. 6.

Simulated action potential waveforms in the soma and AIS. We used the simulations depicted in Fig. 5 to probe additional details of the likely mechanism of the depolarized threshold measured in somatic recordings. We used simulations with E_Cl set at −80 mV and with the GABA conductance introduced in the AIS. A: the action potential waveforms from the distal initial segment (AIS; gray crosshatched compartment in Fig. 5A) and from the soma compartment. B and C: the rises of the action potential waveforms are highlighted. Under baseline conditions, the AIS waveform rises 1st (B), consistent with the high density of sodium channels. The simulations show that the situation partially reverses with the presence of the AIS GABA conductance (C). Now the somatic waveform (gray trace) rises 1st at the very earliest stages of initiation. The AIS remains shunted by the GABA conductance (pink), but soon catches up and eventually crosses the somatic waveform as the dense AIS sodium channels are recruited. The resulting dynamics yield the surprising result that the AIS voltage threshold in the simulation actually hyperpolarizes in the presence of the GABA conductance. D: magnification of the rising part of phase plots constructed from the 4 simulations shown in A illustrating the voltage thresholds (dashed horizontal line).

Fig. 7.

AIS GABA_A receptor activation is inhibitory in unperturbed granule neurons. A: slow sweep showing spontaneous spiking from a cell-attached recording of a granule neuron. The cell had been electroporated 15 min beforehand with Alexa Fluor dye for axon visualization. Downward deflections represent spontaneous action currents arising from spontaneous action potentials, shown in greater detail in the inset below. At the times indicated by red arrows, a 50 ms pulse application of muscimol was made to the initial segment 20–30 μm from the soma. B: summary of the firing frequency changes from the 3 epochs shown in A. For quantification, an 800 ms window before and after muscimol application was evaluated. Results are representative of 4 cells in which spiking was detected in the cell attached configuration.

Fig. 8.

Tonic activation of GABA_A receptors does not change voltage threshold. Action potentials were evoked every 10 s for 2 min, then 1 μM muscimol was applied for 2 min, and finally washed out for 1 min. Pipette chloride was standard low chloride concentration to facilitate detection of effects on threshold. A1: representative action potentials in the absence (black), presence (red), and washout (blue) of 1 μM muscimol. Inset: rising part of the phase plot, with voltage threshold indicated by the horizontal line at 10 mV/ms. A2: time course of voltage threshold change with time under the various experimental conditions. Same color codes as in A1. A3: time course of input resistance (R_m). The example shows 1 of the larger changes observed. B and C: average values for thresholds (B) and input resistance (C) for each condition (n = 10 cells).

Fig. 9.

Somatic and AIS muscimol differentially affect temporal summation of excitatory postsynaptic potential (EPSP)-like events (aPSPs) and input–output relationships. A1: muscimol application to the AIS failed to evoke an action potential during the 4th aPSP, which routinely reached threshold in interleaved control sweeps. Notice that aPSP size and shape are similar in the absence (red traces) and presence (black traces) of muscimol. Bottom: current waveform used to stimulate the cell. The peak amplitude was 175 pA. All insets are magnification of the boxed area, with calibration bars 20 mV and 2 ms. A2: somatic muscimol failed to evoke an action potential at the 4th aPSP. Notice the difference in aPSP peak and time course in the absence (black) and presence (red) of muscimol. B1: with larger current amplitude (250 pA peak amplitude), muscimol application to AIS just before the 2nd aPSP depolarized the voltage threshold and delayed firing. B2: somatic muscimol under the same current-injection conditions as B1 caused an action potential failure at the 2nd aPSP. All traces are from the same cell, which is representative of the 4 cells tested. C: input/output curves for a representative granule cell subjected to 200 ms current pulses of varied amplitudes, with muscimol applied for 50 ms to either the AIS or to the soma immediately before current onset. The y-axis gives the number of action potentials (APs) elicited by the given current amplitude. Muscimol concentration was 5 μM, applied either to the AIS (red circles) or to the soma (red squares). Baseline input/output curve is given as black circles. D: raw traces (top) and voltage thresholds (bottom) in the absence and presence of AIS muscimol application for the cell represented in C. Traces were matched for number of action potentials (8), which corresponded to a current amplitude of 200 pA for control (black trace) and 250 pA for muscimol (red trace). Bottom: voltage thresholds calculated from phase-plot analysis for each action potential in the train. The effect of muscimol on voltage threshold may be reduced compared with the effect in other figures as a result of the larger amplitude of current injection used. Increased current amplitudes tended to hyperpolarize action potential threshold. E: summary of effect represented by the example in C (n = 5). The number of action potentials at a fixed current amplitude is plotted. The current amplitude was that needed to elicit 50% of the maximum firing rate under baseline conditions (175 pA for the example in C). F: summary of effect represented by the example in D (n = 5). Average action potential threshold for the final 4 action potentials in each condition is plotted.