Inhibition of the Akt/PKB Kinase Increases Nav1.6-Mediated Currents and Neuronal Excitability in CA1 Hippocampal Pyramidal Neurons

In neurons, changes in Akt activity have been detected in response to the stimulation of transmembrane receptors. However, the mechanisms that lead to changes in neuronal function upon Akt inhibition are still poorly understood. In the present study, we interrogate how Akt inhibition could affect the activity of the neuronal Nav channels with while impacting intrinsic excitability. To that end, we employed voltage-clamp electrophysiological recordings in heterologous cells expressing the Nav1.6 channel isoform and in hippocampal CA1 pyramidal neurons in the presence of triciribine, an inhibitor of Akt. We showed that in both systems, Akt inhibition resulted in a potentiation of peak transient Na+ current (INa) density. Akt inhibition correspondingly led to an increase in the action potential firing of the CA1 pyramidal neurons that was accompanied by a decrease in the action potential current threshold. Complementary confocal analysis in the CA1 pyramidal neurons showed that the inhibition of Akt is associated with the lengthening of Nav1.6 fluorescent intensity along the axonal initial segment (AIS), providing a mechanism for augmented neuronal excitability. Taken together, these findings provide evidence that Akt-mediated signal transduction might affect neuronal excitability in a Nav1.6-dependent manner.

Given their canonical role in CNS signaling, ion channels stand out as plausible candidates that could be acted upon by Akt to exert regulatory effects on intrinsic neuronal properties; however as potential downstream targets of Akt, ion channels have only been marginally investigated in the brain. On account of their central role in initiating the action potential, the regulation of voltage-gated Na+ (Na v ) channels by Akt would be of particular consequence, as small changes in the activity of these ion channels can corrupt circuits and can lead to the unbalanced function of neuronal activity and aberrant behavior outputs. Posttranslational modifications such as phosphorylation play an essential role in regulating the function of Na v channels and have profound effects on the kinetics, subcellular localization, and trafficking of the channel, altering intrinsic the excitability and activity-dependent plasticity of the neurons [46]. Apart from a demonstration of a link between Akt and Na v 1.7 and Na v 1.8 in dorsal root ganglion (DRG) neurons [47] and demonstration of Akt importance in the regulation of Na v 1.1, which are critical for interneurons function [48], information on the relationship between Akt and Na v channels in the primary CNS neurons that are known to abundantly express the Na v 1.2 and Na v 1.6 channels is scarce. Providing evidence for a linkage between Akt and the Na v channel, we have previously elucidated a complex kinase network that involves a diverse array of proteins related to Akt that regulates the macromolecular complex of the Na v channel [49][50][51][52][53]. Pertinent to the present investigation, we have previously shown that the genetic and pharmacological manipulation of glycogen synthase kinase 3 (GSK3), a kinase whose activity is decreased upon phosphorylation by Akt, confers changes in the activity of the Na v channels and neuronal excitability [52]. Related to this, we have shown that the pharmacological manipulation of Wee1, a kinase whose activity potentially increases Akt activity, also confers changes in Na v channel activity [49,54]. Despite these reported regulatory effects on Na v channel activity conferred by manipulating the kinases that control Akt activity, the effects of directly altering Akt activity on Na v channel activity have been less well described.
To elucidate the latter and while focusing on the Akt-mediated regulation of the Na v 1.6 channel on account of it serving as one of the primary channel isoforms for CNS principal neurons, we employed a pharmacological inhibitor of Akt called triciribine in the present investigation to study how Akt inhibition affected Na v 1.6 channel activity and neuronal excitability. In voltage-clamp studies, the application of triciribine was shown to potentiate the peak transient Na current (I Na ) in heterologous cells expressing the Na v 1.6 channel and in CA1 pyramidal neurons in hippocampal slices. Consistent with these voltageclamp recordings, we correspondingly demonstrate that the pharmacological inhibition of Akt increases the excitability of CA1 pyramidal neurons. Using confocal microscopy, we additionally show that the observed changes in CA1 pyramidal neuron activity are accompanied by changes in Na v 1.6 pattern distribution at the axonal initial segment (AIS). When considered collectively, these results, in tandem with the results of previous investigations, demonstrate that the direct and indirect manipulation of Akt activity confers changes in Na v channel and neural activity, which has important implications for unraveling the complex signaling cascades that fine-tune neuronal excitability.

Effects of Pharmacological
Inhibition of Akt on Na v 1.6-Encoded Currents in In Vitro HEK-Na v 1.6 Cells To evaluate the effect of the Akt pathway on Na v 1.6-encoded currents, HEK293 cells that were stably expressing the human Na v 1.6 channel (HEK-Na v 1.6) were treated with either vehicle (0.1% DMSO) or triciribine (30 µM) for 60 min prior to performing whole-cell patch clamp recording. After forming a stable whole cell patch-clamp configuration in a submerged recording chamber, cells were subjected to a series of depolarizing pulses to evoke inward Na + currents ( Figure 1A). confers changes in Nav channel and neural activity, which has important implications for unraveling the complex signaling cascades that fine-tune neuronal excitability.

Effects of Pharmacological Inhibition of Akt on Nav1.6-Encoded Currents in In Vitro HEK-Nav1.6 Cells
To evaluate the effect of the Akt pathway on Nav1.6-encoded currents, HEK293 cells that were stably expressing the human Nav1.6 channel (HEK-Nav1.6) were treated with either vehicle (0.1% DMSO) or triciribine (30 µM) for 60 min prior to performing wholecell patch clamp recording. After forming a stable whole cell patch-clamp configuration in a submerged recording chamber, cells were subjected to a series of depolarizing pulses to evoke inward Na + currents ( Figure 1A).  . Pharmacological inhibition of Akt by triciribine leads to an increased I Na current density in HEK-293 cells expressing Na v 1.6. (A) Representative traces of I Na currents recorded from HEK-Na v 1.6 cells treated for 60 min either with DMSO (0.1%) (black) or 30 µM triciribine (red). (B) Current-voltage relationship showing increased peak I Na current densities in triciribine-treated HEK-Na v 1.6 cells (red, n = 11) compared to DMSO control (black, n = 11). (C) Peak current density at voltage step of −10 mV in DMSO and triciribine-treated HEK-Na v 1.6 cells. A 60 min treatment with triciribine (30 µM) significantly increased the Na v 1.6-mediated peak I Na current density (n = 11 in both group; ** p = 0.0076 with Student's unpaired t-test). (D,E). Voltage dependence of I Na activation (D) and steady-state inactivation (E) in DMSO and triciribine-treated HEK-Na v 1.6 cells. Treatment with triciribine did not affect voltage dependence of I Na activation or steady-state inactivation. Inset shows the inactivation protocol (scale bars: 20 mv/100 ms). Box plot shows: Range (Perc 25, 75), Mean, Median, Min, and Max. Results are summarized in Table 1. Table 1. Effects of triciribine on Na v 1.6 properties in HEK-Na v 1.6 cells a .
In the cells that had been pretreated with triciribine, we observed an upregulation of Na v 1.6-encoded currents that was evident throughout the majority of the current-voltage relationship (from −20 to +40 mV) ( Figure 1B). On average, we found that the Na v 1.6mediated peak current density measured at -10 mV ( Figure 1C) was significantly higher (−99.4 ± 14.6 pA/pF, n = 11, p = 0.0076) in the triciribine (30 µM) -treated HEK-Na v 1.6 cells compared to in the DMSO control (−47.8 ± 9.4 pA/pF, n = 11) ( Figure 1C). The voltage-dependence of the activation and of steady-state inactivation of Na v channels are critical parameters in determining intrinsic firing that affects the channel availability and the threshold of action potential. Thus, further analyses were performed to determine any potential effect of triciribine on Na v 1.6 channel kinetics ( Figure 1D,E). As shown in Figure 1D, the Na v 1.6 currents in the presence of triciribine exhibited an activation profile that was indistinguishable from the control group (p = 0.96). The V 1/2 of activation upon treatment with triciribine was −23.5 ± 2.1 mV (n = 10) compared to −23.6 ± 1.2 mV in the DMSO control group (n = 9) ( Table 1).
To determine whether triciribine modified the steady-state inactivation properties of the Na v 1.6 channels, cells from the two experimental groups were subjected to a standard two-step protocol, including a variable pre-step conditioning pulse followed by a test pulse. We found that the cells that had been treated with triciribine exhibited a V 1/2 of steady-state inactivation of −59.5 ± 3.1 mV (n = 8), which was not significantly different (p = 0.358) from the DMSO value of −62.9 ± 1.9 mV (n = 11) ( Figure 1E and Table 1). Taken together, these data, which are summarized in Table 1., demonstrate that the inhibition of the Akt pathway leads to an effect on the peak current densities without modifying the basic kinetic properties of Na v 1.6-mediated currents.

Effects of Pharmacological Inhibition of Akt on Na v 1.6-Encoded Currents and Neuronal Excitability in Hippocampal Pyramidal Neurons
To recapitulate the findings observed in the heterologous cells in neurons, whole-cell voltage-clamp recordings of I Na were performed in intact hippocampal CA1 pyramidal neurons in the acute brain slice preparation using the voltage-clamp protocol shown in Figure 2A. As described by Milescu et al. [55], a depolarizing pre-pulse step was used to inactivate Na v channels distant from the recording electrode, and a second step was applied shortly afterward to record the Na v channels close to the recording pipette (Figure 2A), similar to our previous publication [56]. Importantly, hippocampal CA1 pyramidal neurons represent a promising cellular target to be affected by changes in Akt signaling, as these cells abundantly express Na v 1.6 channels where they contribute to somatic I Na and intrinsic excitability, Na v 1.6 channels are abundantly expressed in hippocampal CA1 pyramidal neurons, where they contribute to somatic I Na and intrinsic excitability [57][58][59][60].  Table 2.
As described by Milescu et al. [55], a depolarizing pre-pulse step was used to inactivate Nav channels distant from the recording electrode, and a second step was applied shortly afterward to record the Nav channels close to the recording pipette (Figure 2A), similar to our previous publication [56]. Importantly, hippocampal CA1 pyramidal neurons represent a promising cellular target to be affected by changes in Akt signaling, as these cells abundantly express Nav1.6 channels where they contribute to somatic INa and intrinsic excitability, Nav1.6 channels are abundantly expressed in hippocampal CA1 pyramidal neurons, where they contribute to somatic INa and intrinsic excitability [57][58][59][60].
CA1 pyramidal neurons from slices treated with vehicle (DMSO) displayed an average peak INa density for the second test pulse of −49.5 ± 4.5 pA/pF (n = 8), whereas 20 µM triciribine treatment (60 min) induced a significantly increased peak INa current density of −99.4 ± 9 pA/pF (n = 10; p < 0.001; Figure 2B,C). Finally, we also measured the voltagedependence of the activation and of steady-state inactivation of Nav channels in CA1 pyramidal neurons. Our findings are consistent with the effect of triciribine treatment that we observed in heterologous cells expressing Nav1.6, namely that we did not find significant differences in these parameters between the Akt inhibitor (triciribine) treated and  Table 2. CA1 pyramidal neurons from slices treated with vehicle (DMSO) displayed an average peak I Na density for the second test pulse of −49.5 ± 4.5 pA/pF (n = 8), whereas 20 µM triciribine treatment (60 min) induced a significantly increased peak I Na current density of −99.4 ± 9 pA/pF (n = 10; p < 0.001; Figure 2B,C). Finally, we also measured the voltagedependence of the activation and of steady-state inactivation of Na v channels in CA1 pyramidal neurons. Our findings are consistent with the effect of triciribine treatment that we observed in heterologous cells expressing Na v 1.6, namely that we did not find significant differences in these parameters between the Akt inhibitor (triciribine) treated and control groups ( Figure 2D,E and Table 2). Taken together, these results suggest that Akt inhibition might increase neuronal excitability in CA1 neurons through Na v 1.6 channel modulation.
To test whether the pharmacological inhibition of Akt affected the excitability of the CA1 pyramidal neurons, acute hippocampal slices were prepared and treated with either triciribine (20 µM) or its vehicle (DMSO) for 60 min, after which whole-cell patch-clamp recordings were performed. Whole-cell current-clamp recordings were obtained from visually identified CA1 pyramidal neurons and voltage responses, and action potentials were evoked by 800 ms long (−20 to +210 pA, ∆ = +10 pA) current injections. In these experiments, we found that the triciribine-treated group showed increased action potential discharge ( Figure 3A-C) compared to the DMSO control group.
To test whether the pharmacological inhibition of Akt affected the excitability of the CA1 pyramidal neurons, acute hippocampal slices were prepared and treated with either triciribine (20 µM) or its vehicle (DMSO) for 60 min, after which whole-cell patch-clamp recordings were performed. Whole-cell current-clamp recordings were obtained from visually identified CA1 pyramidal neurons and voltage responses, and action potentials were evoked by 800 ms long (−20 to +210 pA, Δ = +10 pA) current injections. In these experiments, we found that the triciribine-treated group showed increased action potential discharge ( Figure 3A-C) compared to the DMSO control group.   Table 3. We then assessed whether the action potential current and voltage thresholds were altered by triciribine treatment, which were evaluated using the first measurable action potential induced by the above-mentioned current injection protocol. We found that the current threshold (the mean injected current required to evoke an action potential) was significantly lower in the triciribine-treated group 71.8 ± 12.5 pA (n = 11) compared to the DMSO control group (124 ± 17.1 pA (n = 10); p = 0.0221 with Student's t-test) ( Figure 3C and Table 3).
The voltage threshold appeared to be lower in the triciribine-treated group (−45.8 ± 2.4 mV, n = 11) compared to control cells (−41.8 ± 2.1, n = 10) but this difference was not statistically significant (p = 0.1517) ( Figure 3D and Table 3). Triciribine had no effects on other passive intrinsic properties and input resistance of the recorded cells (Table 3). Collectively considered, the results of these voltage and current-clamp recordings, in combination with the results shown in Figure 1, suggest that the pharmacological inhibition of Akt increases the intrinsic excitability of CA1 pyramidal neurons by potentiating the activity of their constituent Na v channels.

Pharmacological Inhibition of Akt with Triciribine
Alters the Length of the Na v 1.6 Immunofluorescence Labelling at the AIS Changes in neuronal excitability can occur in response to the increased localization of Na v channels as well as the relative enrichment of particular Na v channel isoforms at the action potential initiation site, the AIS. Recently, it has also been shown that treatment with triciribine alters the fluorescent intensity of βIV spectrin, a critical constituent of the AIS [61], suggesting that the inhibition of Akt may alter pattern distribution and the expression of other components of the AIS. Thus, we hypothesized that treatment with triciribine could be accompanied by a change in the pattern expression and distribution of Na v 1.6 channels, which were then enriched at the AIS in the hippocampal CA1 pyramidal neurons. To test this hypothesis, ex vivo brain slices containing the hippocampus were exposed to triciribine (20 µM) or vehicle (0.02% DMSO) for 2 h and were processed to determine the immunofluorescence analysis of the CA1 region ( Figure 4).  Cell bodies were labeled with the nuclear marker DAPI ( Figure 4A,D), with a guinea pig antibody against the neuronal marker NeuN ( Figure 4B,E), with a mouse monoclonal antibody against AnkyrinG, used as an AIS marker ( Figure 4C,F,H,K), and a mouse monoclonal antibody against Nav1.6 ( Figure 4C,F,G,J). As previously shown in other studies, AnkyrinG is enriched at the AIS of neurons [62,63], while Nav1.6 is found in both the soma ( Figure 4C,F) and the AIS ( Figure 4G,I,J,L). Following treatment with triciribine or DMSO, the immunofluorescence of Nav1.6 in AnkG positive AIS was traced, revealing a significant increase in the length of the Nav1.6 staining after treatment with triciribine compared Cell bodies were labeled with the nuclear marker DAPI ( Figure 4A,D), with a guinea pig antibody against the neuronal marker NeuN ( Figure 4B,E), with a mouse monoclonal antibody against AnkyrinG, used as an AIS marker ( Figure 4C,F,H,K), and a mouse monoclonal antibody against Na v 1.6 ( Figure 4C,F,G,J). As previously shown in other studies, AnkyrinG is enriched at the AIS of neurons [62,63], while Na v 1.6 is found in both the soma ( Figure 4C,F) and the AIS ( Figure 4G,I,J,L). Following treatment with triciribine or DMSO, the immunofluorescence of Na v 1.6 in AnkG positive AIS was traced, revealing a significant increase in the length of the Na v 1.6 staining after treatment with triciribine compared to DMSO control (p = 0.0104 as analyzed by a two-tailed nested t-test; Figure 4M). Conversely, the treatment with triciribine did not alter the fluorescent intensity of Na v 1.6 at the AIS as measured by the analysis of the cross-section of the AIS ( Figure 4I,L,N). Overall, these results demonstrate that triciribine causes an increase in the length of Na v 1.6 staining in the AIS, which is in line with previous results showing that the elongation of the AIS is associated with an increase in neuronal excitability [64,65].

Discussion
Previous studies have characterized a complex kinase network that regulates the activity of the Na v 1.6 channel macromolecular and resultantly modulates neuronal activity [50,52,61]. In this network, the phosphorylation of either the Na v 1.6 channel or its auxiliary proteins by various kinases has been shown to exert powerful regulatory effects on macromolecular complex assembly of the Na v 1.6 channel and its activity. Pertinent to the present investigation, we have shown that GSK3, a kinase whose activity is decreased through phosphorylation by Akt, directly phosphorylates the Thr1936 of the Na v 1.6 channel [52]. Through this phosphorylation of the Na v 1.6 channel, GSK3 is able to regulate the channel's activity, which resultantly confers changes in the excitability of neurons in clinically relevant brain regions, such as the nucleus accumbens, part of the striatum. Given this linkage between the Na v 1.6 channel and GSK3, coupled with the latter's activity being regulated by Akt, we postulated a signaling axis involving Na v 1.6, GSK3, and Akt. To further interrogate this postulated signaling cascade, we sought to characterize the effects of the direct pharmacological inhibition of Akt on the Na v 1.6 channel activity and on neuronal excitability in the present study.
Therefore, we provided functional evidence that Akt activity modulates the peak I Na density elicited by heterologous cells expressing the Na v 1.6 channel. Specifically, the treatment of HEK-Na v 1.6 cells with the Akt inhibitor triciribine increased their peak I Na density relative to treatment with vehicle (DMSO) (Figure 1). Our results also showed evidence for the modulatory effect of Akt activity on the intrinsic firing properties of CA1 pyramidal neurons. The observed effect of triciribine was twofold: (1) it increased the maximum number of action potential firing; and (2) it decreased the current threshold ( Figure 3). These effects of triciribine on the firing properties of the CA1 pyramidal neurons in tandem with the lack of effects on the passive electrical properties (Table 3) and the results observed in heterologous cells collectively point toward the pharmacological inhibition of Akt increasing neuronal excitability by increasing the number of available Na v channels [52,66,67]. Lending further evidence to the Na v channel modulation underlying triciribine's potentiation of the excitability of CA1 pyramidal neurons, our voltage-clamp experiments performed in CA1 pyramidal neurons in the intact slice preparation (Figure 2 and Table 2) show that triciribine increases the transient I Na of these cells, similar to the results observed in heterologous cells.
Previously, it has been shown that the activity of different kinases in the AIS or inhibition of these kinases have significant effects on the subcellular distribution of AIS proteins, which determines the shape and ion channel composition of AIS [59]. For instance, βIV spectrin is sensitive to kinase perturbation (Akt inhibition) at the AIS and the dendrites of primary hippocampal neurons [61]. Based on these findings, we complemented our electrophysiology studies with confocal imaging (immunohistochemistry) experiments to assess any potential changes in Na v 1.6 pattern distribution related to the functional phenotypes we observed in the hippocampal slices. We found that the inhibition of Akt in pyramidal neurons of the CA1 region leads to a lengthening of the Na v 1.6 fluorescent labelling along the AIS, which supports the reported augmented excitability phenotype observed in our electrophysiologic results from this cell type.
The combined findings of these electrophysiological and imaging studies further demonstrate that intracellular phosphorylation pathways have a direct effect on voltagegated Na + currents and that the manipulation of these kinase networks causes modifications in the neuronal activity. Recently, it was found that the activation of Akt can result in a robust decrease in the Na v 1.1-mediated sodium currents accompanied by significant changes in the inactivation of the channel. The effect of Akt was attributed to its ability to directly phosphorylate the Na v 1.1 channel [48]. Thus, the mechanism by which the pharmacological inhibition of Akt alters neuronal activity in our study could arise by directly preventing the kinase mediated phosphorylation of the Na v 1.6 channel, an indirect effect on the phosphorylation of the Na v 1.6 channel conferred by increasing the activity of GSK3, or a combination of these direct and indirect effects; however, future in vitro phosphorylation studies will be necessary to unequivocally address how the pharmacological inhibition of Akt alters the phosphorylation of the Na v 1.6 channel.
Overall, by employing a combination of electrophysiology and immunohistochemistry, we have further elucidated the cellular signaling cascades that regulate Na v channel activity and neuronal excitability. In particular, our results point toward Akt functioning as a central node in an AKT-GSK3-Na v 1.6 network, and that this pathway plays an important role in fine-tuning neuronal excitability. Considering the important role of Akt-mediated signaling in a diverse array of CNS conditions, including schizophrenia [68][69][70], depression [71], Alzheimer's disease [72,73], and Parkinson's disease [74][75][76][77], our results have important implications for refining our understanding of the molecular determinants of complex neuropsychiatric and neurodegenerative disorders.

Chemicals
Triciribine (EMD Chemicals, San Diego, CA, USA) was dissolved in 100% DMSO (Sigma-Aldrich, St. Louis, MO, USA) to a working stock concentration of 20 mM, aliquoted, and stored at −20 • C. Triciribine was further diluted as needed for experimental purposes.

Cell Culture
All reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA, unless noted otherwise. HEK-293 cells stably expressing human Na v 1.6 (HEK-Na v 1.6 cells) were maintained in a medium composed of equal volumes of DMEM and F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 0.05% glucose, 0.5 mM pyruvate, 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 500 µg/mL G418 (Invitrogen, Carlsbad, CA, USA) to ensure stable Na v 1.6 expression. Cells were maintained at 37 • C with 5% CO 2 . Whole-cell voltage-clamp recordings in heterologous cell systems were performed as previously described [78,79]. HEK-Na v 1.6 cells were dissociated and re-plated at lowdensities onto glass coverslips. After allowing 2-3 to ensure adherence, coverslips were transferred to a recording chamber filled with the following extracellular solution: (in mM): 140 NaCl, 3 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES, 10 glucose, pH: 7.3. For the control recordings, 0.15% DMSO was added to the extracellular solution. For recordings to assess the effects of pharmacological inhibition of Akt, 30 µM of triciribine was added to the extracellular solution. The concentration of DMSO was maintained at 0.15% in both groups. Cover slips were incubated in the extracellular solution containing either vehicle (DMSO) or 30 µM triciribine at room temperature for 60 min prior to the start of recordings. The pipette (intracellular) solution contained (in mM): 130 CH 3 O 3 SCs, 1 EGTA, 10 NaCl, 10 HEPES, pH: 7.3.

Electrophysiology
Data were only collected from cells forming a 1 GΩ or tighter seal at a holding potential of −70 mV. Membrane capacitance and series resistance were estimated by the dial settings on the amplifier. After break-in, capacitive transients and series resistances were carefully compensated by 70-80% at the beginning of every protocol. Cells exhibiting a series resistance of 20 MΩ or higher were excluded from the analysis. The data were acquired with pClamp/Clampex 7/9 (Molecular Devices, Sunnyvale, CA, USA) and digitized with a Digidata 1322A A/D converter (Molecular Devices, Sunnyvale, CA, USA) at a sampling rate of 20 kHz and filtered at 5 kHz prior to digitization and storage. All of the experimental parameters were controlled by Clampex 7/9 software (Molecular Devices).
Cells were maintained at −70 mV holding potential, and voltage-dependent inward currents were evoked by depolarizations to test potentials between −100 mV and +60 mV (∆ +5 mV) from a holding potential of −70 mV followed by a voltage pre-step pulse of −120 mV (Na v 1.6) ( Figure 1A-inset). Steady-state inactivation of the Na v channels was measured with a two-pulse protocol. From the holding potential (−70 mV), cells were stepped to varying test potentials between −120 mV and 20 mV (pre-pulse) prior to a test pulse to −10 mV.
For the whole cell patch-clamp recordings (performed in current-clamp mode), the prepared brain slices were transferred and submerged in a recording chamber and perfused with standard recording aCSF (see above), which was continuously oxygenated with 95% O 2 and 5% CO 2 (pH 7.4). The flow rate in the recording chamber was kept at 1.5 mL/min, and the bath temperature was maintained at 30-32 • C by an inline solution heater and temperature controller (TC-344B, Warner Instruments, Hamden, CT, USA). Additionally, 20 µM bicuculline; 20 µM NBQX; and 100 µM AP5 (Tocris, Bristol, UK) were applied to the recording solution to block synaptic activity. The whole-cell current-clamp recordings from visually identified pyramidal neurons were performed by using an Axopatch 200B or Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA). Borosilicate glass pipettes with a resistance of 3-6 MΩ were made using a Narishige PP-83 vertical Micropipette Puller (Narishige International Inc., Amityville, NY, USA). Current-clamp recording were performed with pipettes filled with internal solution containing (in mM): 145 K-gluconate, 2 MgCl 2 , 0.1 EGTA, 2 Na 2 ATP, and 10 HEPES (pH 7.2 with KOH; and osmolarity = 290 mOsm). The pipette potential was adjusted to zero and the pipette capacitance was compensated before seal formation. Membrane potentials were not corrected for the liquid junction potential.
Data were only collected from cells forming a 1 GΩ or tighter seal. The series resistance was carefully compensated at the beginning of every protocol (in voltage-clamp mode). The maximal series resistance we accepted was 25 MΩ. Data were acquired at 50 kHz and filtered at 2 kHz before digitization and storage. All of the experimental parameters were controlled using Clampex 9.2 software (Molecular Devices, Union City, CA, USA) interfaced to the electrophysiological equipment using Digidata 1320A or 1322A analog-to-digital interfaces (Molecular Devices). Voltage responses and action potentials were evoked by hyper and depolarizing current pulses (with a range of current injections from −20 pA to 210 pA with 800 ms pulses and a change in injected current of 10 pA between sweeps) from a starting holding membrane potential of −70 mV. Input-output relationships were plotted as the number of spikes against the given current step (Figure 3). Only spikes with overshoots were taken for analysis (as described in Scala, Nenov et al., 2018. [52]). In the whole-cell current-clamp recordings, n = 7 and n = 6 mice were allocated into the DMSO and triciribine-treated groups, respectively (n = number of cells as listed in the corresponding Figure 3). Finally, to confirm that the results seen in current-clamp recordings were mediated by changes in Nav channel activity, whole-cell voltage-clamp recordings of INa were performed (n = 3 mice were used (half of the slices were treated with triciribine parallel with DMSO as control treatment). On account of space clamp issues, recording fast-gating I Na in intact neurons is not possible using conventional voltageclamp protocols, with the main barrier being the inability to accurately record Na v channel activity in processes distant from the recording electrode. To circumvent this issue, we employed a protocol described by Milescu and colleagues [55] that uses a depolarizing prepulse step to inactivate Na v channels distant from the recording electrode that is followed shortly afterward with a second step to record Na v channels close to the recording pipette. Using this protocol, we reliably resolved well-clamped I Na of intact pyramidal neurons in acute brain slice preparation. The same extracellular solution used for current-clamp recordings was also used to record I Na with the addition of 120 µM CdCl 2 (Sigma-Aldrich, St. Louis, MO, USA) to block Ca 2 + currents. The intracellular solution for this recording contained the following (in mM): 100 Cs-gluconate (Hello Bio Inc., Princeton, NJ, USA); 10 tetraethylammonium chloride; 5 4-aminopyridine; 10 EGTA; 1 CaCl 2 ; 10 HEPES; 4 Mg-ATP; 0.3 Na 3 -GTP; 4 Na 2 -phosphocreatine; and 4 NaCl (Sigma-Aldrich, St. Louis, MO, USA) (pH = 7.4 and osmolarity = 285 ± 5 mOsm/L; CsOH used to adjust pH and osmolarity). The same cocktail of synaptic blockers as the one used for the current-clamp recordings was used. Transient I Na were evoked by using the voltage-clamp protocol shown in Figure 2 and as described elsewhere [55,56,81,82].

Electrophysiological Data Analysis
In voltage-clamp experiments, the current densities were obtained by dividing the Na + current (I Na ) amplitude by membrane capacitance. Current-voltage relationships were generated by plotting current density as a function of the holding potential. Conductance (G Na ) was calculated by the following Equation (1): where I Na is the current amplitude at voltage V m , and E rev is the Na + reversal potential. Activation curves were derived by plotting normalized G Na as a function of test potential and fitted using the Boltzmann equation. Equation (2): where G Na,Max is the maximum conductance, V a is the membrane potential of half-maximal activation, E m is the membrane voltage, and k is the slope factor. For steady-state inactivation, normalized current amplitude (I Na /I Na,Max ) at the test potential was plotted as a function of pre-pulse potential (V m ) and fitted using the Boltzmann equation. Equation (3): where V h is the potential of the half-maximal inactivation, and k is the slope factor. In current-clamp recordings, the maximum number of APs was determined by quantifying the maximum number of APs a CA1 pyramidal cell fired at any current step during the evoked protocol (evoked APs were measured in response to a range of current injections from −20 to +210 pA, 800 ms). The current threshold (I thr ) was defined as the current step at which at least one AP with overshoot was evoked ( Figure 3C). The voltage threshold (V thr ) was defined as the voltage at which the first-order derivative of the rising phase of the AP exceeded 10 mV/ms (from a plot of dV/dt versus V) ( Figure 3D). Passive membrane properties, such as input resistance (R in ) and membrane time constant (τ), were measured with current-clamp recordings with a membrane potential of −70 mV. To determine R in , the steady-state values of the voltage responses to a series of current steps from −120 to +20 pA with 20 pA increments/step and a duration of 500 ms were plotted as a voltagecurrent relationship. R in was calculated as the slope of the data points fitted with linear regression. Membrane τ was calculated by fitting a single exponential function to the first 100-150 ms at a −40-pA hyperpolarizing, 500 ms current step from the same series. Cm was estimated as tau/R in × gain of the amplifier (usually 1000) [52]. These data are also summarized in Table 3. Data analysis was performed using pClamp 9 (Clampfit 9) (Molecular Devices, Union City, CA, USA) and Origin 8.6 or Prism version 9.1.0, and the results were plotted with Origin 8.6 (OriginLab Corp., Northampton, MA, USA) or Prism version 9.1.0 (GraphPad Software, San Diego, CA, USA).

Confocal Imaging and Image Analysis
Confocal images were acquired with a Zeiss LSM-880 confocal microscope with a 63× oil immersion objective (1.4 NA). Multi-track acquisition was done with excitation lines at 405 for DAPI, 488 nm for Alexa 488, 561 nm for Alexa 568, and 633 nm for Alexa 647. Z-stacks were collected at z-steps of 0.43 µm for the first dataset, with a frame size of 1024 × 1024 and a pixel dwell time of 0.77 µs. All acquisition parameters, including photomultiplier gain and offset, were kept constant throughout each set of experiments. Acquired Z-stacks were sum-projected, and pixel intensity values were analyzed using Fiji ImageJ (fiji.sc). A total of four brain slices (n = 3 animals (DMSO) and n = 2 (triciribine)) were used for determining the length of Na v 1.6 fluorescence intensity along the AIS in DMSO and triciribine treated conditions, and a single animal was used for the cross-section analysis of Na v 1.6 staining at the AIS. For each condition, four z-stacks were acquired from each brain slice. With the experimenter blinded to the condition of each image, a region of interest (ROI) was highlighted on an overlay image of the somatic marker (NeuN) and the AIS marker (AnkyrinG) staining during the AnkyrinG positive processes. The "starting point" for the ROI was the point of reduced NeuN and increased AnkyrinG staining intensity, which corresponded to the AIS, as previously described [61]. The length of the AnkyrinG staining was considered the length of the AIS and was visually inspected to ensure that it corresponded to the length of Na v 1.6 staining (n = 119 cells (DMSO) and 139 cells (triciribine)).
The cross-section analysis was performed as previously described [84]. Briefly, an ROI was made at the middle of the AIS to measure the fluorescent intensity perpendicular to the AIS, including 2.5 mm on either side of the peak AIS intensity using an ImageJ plugin LRoi (available at https://sites.imagej.net/CIPMM-MolPhys/, accessed on 3 January 2022) at the middle of the AIS. Fluorescent intensity was normalized to the background fluorescence (F O ), which was calculated using the average fluorescent intensity of the first and last micron of the ROI (n = 16 cells (DMSO) and 20 cells (triciribine)). Data were tabulated in Excel and analyzed with GraphPad Prism 9. The linear filters available in Adobe Photoshop were applied to max projections of six optical slices from each condition for illustration purposes only.

Statistical Analysis
Results were expressed as mean ± standard error (SEM) or in box plots as Range (Perc 25, 75), Mean, Median, Min, and Max. The statistical significance of the observed differences among groups was determined by Student's t-test. In case of the number of evoked action potentials, repeated-measure two-way ANOVA with uncorrected Fisher's LSD was used.
For the immunohistochemistry, the statistical analysis was performed using a nested t-test to compare triciribine treatment to the DMSO control, with cells from individual slices that were considered to be technical replicates, with each bar in the figure representing a single brain slice ( Figure 4M). For the cross-section analysis, a two-way ANOVA was used to compare DMSO to triciribine treatment ( Figure 4N). The level of significance is listed in the figure legends for each experiment. Statistical analysis was performed using Origin 2021b software (OriginLab, Northampton, MA, USA) and GraphPad Prism version 9.1.0 (GraphPad Software, San Diego, CA, USA).

Institutional Review Board Statement:
The study was conducted according to the guidelines of the United States Department of Agriculture Animal Welfare Act, the NIH Guide for the Care and Use of Laboratory Animals, the American Association for Laboratory Animal Science, and UTMB Institutional Animal Care and Use Committee approved protocols (protocol code: 0904029D; approved January 2019).

Informed Consent Statement: Not applicable.
Data Availability Statement: Data included in this study are available upon request from the corresponding author.