Estimation of AP initiation site at the AIS of PV and SST neurons.

<p>(A) Schematic diagram of simultaneous recording from the soma and the axon bleb. (B) Example recording from a PV neuron with an axon bleb formed 112 µm away from the soma. APs evoked by current injections at the soma (top) or axonal bleb (bottom). When the soma was stimulated, somatic AP generated earlier than axonal AP; in contrast, when the axon bleb was stimulated, axonal AP occurred earlier. (C) Similar to (B) except that the recording distance was 55 µm. Note that axonal APs preceded somatic APs in both conditions. (D) The estimated initiation sites (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001944#s4" target="_blank">Materials and Methods</a>) in PV were more proximal than that in SST neurons.</p

Difference in voltage dependence of axonal Na⁺ channels.

<p>(A) Projection of two-photon <i>z</i>-stack images of a GFP-positive PV neuron (left, black/white inverted). Note the axon bleb. (Right) Schematic diagram of the outside-out recording from patches excised from axon blebs. (B and C) Example current traces evoked by activation and inactivation voltage commands in PV and SST axonal patches. (D) Group data showing no significant difference in peak amplitude of axonal Na⁺ currents. However, in both types of neurons, the peak amplitudes of Na⁺ currents in outside-out patches of the axon were much greater than those in the soma. Error bars represent s.e.m. (E) Activation and availability curves for axonal Na⁺ currents. (F) Comparison of <i>V</i>_1/2 of activation and inactivation in the two cell types. * <i>p</i><0.05. Error bars represent s.e.m. (G) The <i>V</i>_1/2 of activation was plotted as a function of recording distances from the soma. The average <i>V</i>_1/2 (±s.e.m.) of somatic and axonal Na⁺ currents is shown for comparison.</p

Difference in voltage thresholds of APs in PV and SST neurons.

<p>(A) Projection of two-photon images showing recordings from PV-positive (top, B13 mouse) and SST-positive (bottom, GIN mouse) neurons in prefrontal cortical slices. Cells were loaded with Alexa Fluor 594 (red) through patch pipettes. (B) Firing patterns of the two recorded cells shown in (A). Traces are color-coded (PV, red; SST, blue). (C) Overlaid APs from PV and SST neurons. Note the difference in AP waveforms. (D) Phase plots of PV and SST APs evoked by brief current injections. Note the AIS and SD potential components. (E) Difference in peak amplitudes of d<i>V</i>/dt. (F) Dependence of AP thresholds on <i>V</i>_m levels. For (E) and (F), *** <i>p</i><0.001. Error bars represent s.e.m.</p

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

Polarized distribution of channel subtypes at the AIS of SST neurons.

<p>(A) Triple staining using antibodies for SST (blue), Pan-Na_V (red), and Na_V1.1 (green) show modest intensity of Na_V1.1 immunosignals at the AIS (arrowheads) and adjacent axon regions of SST neuron. Asterisks indicate a neighboring SST-negative axon (presumably PV axon) that was heavily stained. Nearby PC axons were not stained. (B) Triple staining for SST, Na_V1.6 (red), and Na_V1.1 (green) indicates co-localization of the two subtypes at the AIS. (C) Triple staining shows polarized distribution of Na_V1.2 (proximal region) and Na_V1.6 (distal region) at the AIS. (D and E) Plots of the averaged fluorescence intensity (± s.e.m.) as a function of distance from the soma. Data were obtained from triple-staining experiments similar to (B) and (C). Images are projections of confocal <i>z</i> stacks. Scale bars represent 10 µm. 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

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

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

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

Figure 4

<p>(A) Changes in extracellular pH <u>(pH_e)</u> in the PC and in mOT induced by ipsilateral MCA occlusion and reperfusion. Occlusion induced a rapid metabolic acidification of the extracellular microenviroment in PC, interrupted by a transient and mild basification (arrow) associated to the hypoxic spreading depression (HD, asterisk). No changes in extracellular [H⁺] were recorded in the mOT, that is not served by the MCA. (B) Simultaneous changes in extracellular potassium concentration ([K⁺]_o)and extracellular pH in the PC after MCA occlusion and reperfusion. An initial enhancement in [K⁺] was followed by a fast and large increase in [K⁺]_o. associated to the HD. The schematic drawing on the right illustrates the position of the two-barrel recording electrodes. The field responses (FP) recorded with the conventional extracellular barrel are also shown. The period of MCA occlusion is marked by the shaded area.</p