Propagation of GABA-induced hyperpolarization at the axon regulates AP generation.

<p>A, DAB staining of recorded neurons. Simultaneous recording from the soma and axon bleb were performed in a pyramidal neuron (left), and GABA was applied to the axon trunk (right). The axon length was 239 µm in this case. The distance between the iontophoresis site and the soma was 117 µm. Scale bar: 100 µm (left); 50 µm (right). B, The sign of the effect of GABA (hyperpolarization or depolarization) depended on the <i>V</i>_m. Top, traces were taken from the bleb. Bottom, traces were the corresponding responses at the soma. The <i>V_m</i> was clamped through somatic DC current injection. Asterisk indicates application of GABA to the main trunk. C, Left, application of GABA to the axon increased the amplitude but decreased the half-width of propagating APs. GABA iontophoresis hyperpolarized the <i>V</i>_m by 2.3±0.4 mV (n = 7). Right, similar results were obtained when <i>V</i>_m was hyperpolarized by 2.8±0.3 mV (n = 5) through DC current injection. *, P<0.05; **, P<0.01, paired t-test. D, Example traces showing activation of axonal GABA_A receptors reduced firing probability and frequency. The distances between the iontophoresis site and the soma were 100 µm (distal axon) and 18 µm (AIS). E, Left, repetitive firing recorded at an axon bleb induced by 400 pA DC current injection at the soma before (black) and after (red) GABA application to the axon trunk. The arrow indicates GABA iontophoresis. Middle, instantaneous firing frequency of APs decreased after GABA application (same data as shown in the left). Right, group data showing a decrease in the mean frequency of APs after GABA iontophoresis at the axon trunk. **, P<0.01, paired t-test. Low-Cl⁻ ICS was used in these experiments.</p

Activation of axonal GABA_A receptors shapes the AP waveform.

<p>A, Example traces showing the change in AP waveform after GABA iontophoresis to the axon bleb. <i>V</i>_m change was –3.9 mV in this bleb. The amplitude and the half-width of APs decreased to 95.6% and 86.8% of the control, respectively. B, Group data showing that activation of axonal GABA_A receptors shaped AP waveforms by regulating the amplitude and the half-width. Note that the recordings were performed under current-clamp and that the <i>V</i>_m could be manipulated by DC current injection. Black, GABA responses were hyperpolarizing; gray, depolarizing. The blebs were recorded with low-Cl⁻ ICS (7 mM [Cl⁻]_i). C, Increasing [Cl⁻]_i depolarized the <i>V</i>_m but still showed a shunting effect on AP waveforms. Amplitude and half-width were significantly reduced. Modified ICS (20 mM [Cl⁻]_i) was used for these recordings. ***, P<0.001, paired t-test. D, Bath application of PTX could block the GABA-induced <i>V</i>_m depolarization and its shunting effect on AP waveform. Modified ICS was used. E, Example traces showing that GABA application caused a shunting effect on APs evoked by electric shocks (asterisks), although GABA itself could evoke an AP (arrow). High-Cl⁻ ICS (75 mM [Cl⁻]_i) was used here.</p

The presence of GABA_A (but likely not GABA_B) receptors in the axon.

<p>A, Reversal potential of GABA responses (I_GABA) in the axon bleb. Left, representative currents induced by GABA application at different holding potentials (from –100 to –40 mV). At –60 mV (near reversal potential), GABA application induced no obvious change in baseline current (gray). Right, I-V curve of the GABA-induced responses shown on the left. B, I_GABA could be blocked by GABA_A receptor blocker PTX. Left, example traces before (black), during (gray) and after (Wash, dashed line) the bath application of PTX (25 µM). V_hold = –50 mV, GABA was applied via iontophoresis. Middle, time course of the effect of PTX. Right, group data showing the change of I_GABA during (n = 6) and after (n = 3) PTX application. The dashed line indicates 100% of control. C, Left, currents evoked by puffing baclofen (200 µM), a GABA_B receptor agonist, to the soma (16 psi, 15 ms). Right, no response was observed when baclofen was applied to the axon trunk (16 psi, 20 ms). D, Group data showing that GABA-induced currents at the axon blebs could not be blocked by the GABA_B receptor antagonist CGP 35348 (100 µM); however, PTX could diminish these responses. Different symbols indicate different cells.</p

GABA receptors are located at axon bleb and trunk.

<p>A, Left, schematic diagram of bleb recording and GABA iontophoresis in a pyramidal neuron. Positive (but not negative) pulses could induce current responses. V_hold = –50 mV; iontophoresis pulses: 200 nA, 5 ms; retention current: –10 nA. Right, whole-cell recording from an axon bleb (top, fluorescence image; bottom, DIC image). Scale bar: 20 µm. The sharp electrode was used for GABA iontophoresis. Alexa Fluor 488 was added to the patch pipette solution so that the recording pipette was visible. B, Plot of the normalized GABA response as a function of the distance between the bleb and the tip of the iontophoresis electrode. Different symbols indicate different cells. The measurement of distance <i>L</i> is shown in the schematic diagram in panel A (indicated by arrows). C, GABA-induced responses could be observed when GABA was applied to the bleb (site <i>a</i>) or the main axon trunk (site <i>c</i>). The distance between sites <i>a</i> and <i>c</i> was approximately 50 µm, whereas that between <i>a</i> and <i>b</i> was approximately 25 µm. V_hold = –80 mV; iontophoresis pulses: 200 nA, 5 ms.</p

Reversal potential of GABA responses (E_GABA) is more negative than the local RMP.

<p>A, Gramicidin perforated patch recording from an axon bleb. Arrow indicates the recorded bleb. Top, DIC image of the recording; middle, fluorescence image (unlabeled bleb); bottom, fluorescence image (labeled bleb, indicating rupture of patch membrane). Scale bar: 50 µm. B, Example traces showing GABA responses at different holding potentials (from –90 to –50 mV) before (black) and after the break-in (membrane rupture, gray). C, Comparison of E_GABA and RMP. Note that E_GABA at both the soma and the distal axon bleb were more hyperpolarized than their local RMP. *, P<0.05, paired t-test.</p

Action Potential Initiation in Neocortical Inhibitory Interneurons

<div><p>Action potential (AP) generation in inhibitory interneurons is critical for cortical excitation-inhibition balance and information processing. However, it remains unclear what determines AP initiation in different interneurons. We focused on two predominant interneuron types in neocortex: parvalbumin (PV)- and somatostatin (SST)-expressing neurons. Patch-clamp recording from mouse prefrontal cortical slices showed that axonal but not somatic Na⁺ channels exhibit different voltage-dependent properties. The minimal activation voltage of axonal channels in SST was substantially higher (∼7 mV) than in PV cells, consistent with differences in AP thresholds. A more mixed distribution of high- and low-threshold channel subtypes at the axon initial segment (AIS) of SST cells may lead to these differences. Surprisingly, Na_V1.2 was found accumulated at AIS of SST but not PV cells; reducing Na_V1.2-mediated currents in interneurons promoted recurrent network activity. Together, our results reveal the molecular identity of axonal Na⁺ channels in interneurons and their contribution to AP generation and regulation of network activity.</p></div

Reducing Na_V1.2 currents promotes the generation of recurrent network activity.

<p>(A) Bath application of PaurTx3 (PTx3) increased the occurrence frequency of spontaneous network activity in a prefrontal cortical slice maintained in Mg²⁺-free ACSF (with GABA-mediated inhibition preserved). (B) Group data of Mg²⁺-free experiments (<i>n</i> = 6). (C) PTx3 showed no effect on spontaneous network activity in the presence of GABA receptor blockers (50 µM PTX and 100 µM CGP35348). (D) Group data of experiments using GABA receptor blockers (<i>n</i> = 7). (E) A network-activity event evoked by an electrical stimulation to the tissue showing the measurement of duration. (F) Group data showing that PTx3 had no effect on the duration of the network activity evoked in either conditions. For (B), (D), and (F), paired <i>t</i> test, ** <i>p</i><0.01. Error bars represent s.e.m.</p

Spontaneous firing in SST neurons were suppressed by PTx3.

<p>(A) Example recordings from SST-PC and PV-PC pairs. PC and PV neurons showed no spontaneous activity during the refractory period between network-activity events; however, the SST neuron was constantly active. Spontaneous APs in the SST neuron could be substantially suppressed by bath application of 30 nM PTx3. (B) Group data showing that 30 nM PTx3 significantly decreased the frequency of spontaneous APs in SST neurons. (C) Puffing PTx3 (300 nM) at the soma had no effect on discharge probability in SST neurons (left), whereas puff at the AIS substantially decreased the firing probability (right). For (B) and (C), paired <i>t</i> test, ** <i>p</i><0.01. Error bars represent s.e.m.</p

Polarized distribution of Na_V1.1 and Na_V1.6 at the AIS of PV neurons.

<p>(A) Triple staining using antibodies for PV (blue), AnkG (red), and Na_V1.2 (green) revealed the absence of Na_V1.2 at the AIS of PV neuron (arrowheads). Note that neighboring PV-negative AIS (presumably from PCs, asterisks) show strong immunosignals for Na_V1.2. (B) Triple staining for PV, AnkG (green), and Na_V1.6 (red). Note that distal regions of AIS were heavily stained for Na_V1.6 (arrowheads). Neighboring axons (asterisks) also showed strong immunosignals. (C) Triple staining for PV, Na_V1.6, and Na_V1.1 shows polarized distribution of these subtypes at the AIS. (D) Plots of the averaged fluorescence intensity (± s.e.m., see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001944#s4" target="_blank">Materials and Methods</a>) as a function of distance from soma at the AIS. Data were obtained from triple-staining experiments similar to (C). Images are projections of confocal <i>z</i> stacks. Scale bars represent 10 µm. Error bars represent s.e.m.</p

Voltage dependence of somatic Na⁺ channels.

<p>(A) Schematic diagram of recording from somatic nucleated patch (i.e., giant outside-out patch of somatic membrane). (B) Example current traces evoked by activation voltage commands (top) in PV and SST nucleated patches. (C) Current traces evoked by the test pulse (0 mV) in the voltage protocol for channel inactivation. (D) Comparison of averaged peak Na⁺ currents and conductance density in nucleated patches. Error bars represent s.e.m. (E and F) Activation and availability curves of somatic Na⁺ currents in PV (red) and SST neurons (blue). (Insets) Comparison of the activation and inactivation <i>V</i>_1/2, showing no difference between the two cell types. Error bars represent s.e.m.</p