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

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

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

Experimental protocols.

<p>Ischemia was induced for 30 minutes, 2 hours after the <i>in vitro</i> placement of the isolated brain. NC were perfused for 1 h immediately either after the reopening of the vessel or 1 our later (protocol 2). The perfusion was followed by a wash-out period with a solution without NCs. At 5 hours <i>in vitro</i> the brains were fixed for immunohistochemistry. The bottom of the panel shows an example of simultaneous DC recordings from 4 different sites in an isolated guinea pig brain. Hypoxic depressions (HD) were recorded in the electrodes located in the regions vascularized by the occluded MCA. Potentials evoked by LOT stimulation before and during the first part of ischemia (arrowhead) disappeared when HD occurred, and recovered during MCA reperfusion. Evoked potentials in the hemisphere contralateral to MCA occlusion were not altered.</p

Mean NCs density (cells/cm²) in the MAP-2 positive zone contralateral (left column) and ipsilateral (middle column) to the ischemic side and in the MAP-2 negative zone of the ischemic hemisphere (right column) in 6 different experiments.

<p>On the left, the number of slices on which counts were made for each experiments is indicated.</p

Confocal fluorescence microscope images of PECAM-1 stained sections (50 µm thick) cut from the piriform region of brains fixed at the end of the electrophysiological experiment.

<p>CFDA green fluorescent NCs and brain vessels are shown at different magnifications. Calibration bar = 100 µm. NCs were observed in close proximity to cerebral vessels.</p