Age-dependent expression of Nav1.9 channels in medial prefrontal cortex pyramidal neurons in rats.

Research Article

Developmental Neurobiology DOI 10.1002/dneu.22537

Title: Age-dependent expression of Nav1.9 channels in medial prefrontal cortex (mPFC) pyramidal neurons in rats

Running title: Nav1.9 channels in pyramidal neurons Author names and affiliations: Maciej Gawlak1, Bartłomiej Szulczyk2, Adam Berlowski3, Katarzyna Grzelka1, Anna Stachurska4, Justyna Pelka3, Katarzyna Czarzasta5, Maciej Malecki4, Przemyslaw Kurowski1, Ewa Nurowska1, Pawel Szulczyk*1 1

Laboratory of Physiology and Pathophysiology, Centre for Preclinical Research and

Technology, The Medical University of Warsaw, Poland. 2

Department of Drug Technology and Pharmaceutical Biotechnology, The Medical University of

Warsaw, Poland. 3

Student of the Faculty of Pharmacy, The Medical University of Warsaw, Poland.

4

Department of Molecular Biology, The Medical University of Warsaw, Poland.

5

Laboratory of Experimental and Clinical Physiology, Centre for Preclinical Research, Medical

University of Warsaw, Warsaw, Poland

*Corresponding author: Email: [email protected] (PS).

Acknowledgments This

study

was

sponsored

by

the

National

Science

Centre

Poland

[grant

nos.

2015/17/N/NZ4/02889, 2014/15/N/NZ4/04760, FW5/PM2/17].

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/dneu.22537 © 2017 Wiley Periodicals, Inc. Received: Jul 31, 2017; Revised: Sep 08, 2017; Accepted: Sep 10, 2017 This article is protected by copyright. All rights reserved.

Developmental Neurobiology

ABSTRACT Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na+ current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20-day-old), late adolescent (60-dayold) and adult (6-7-month-old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX-resistant, low-threshold, noninactivating and voltage-dependent Na+ current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats. KEYWORDS prefrontal cortex; pyramidal neurons; SCN11a; Nav1.9 channels, Nav1.9-like currents

INTRODUCTION Developmental changes that occur in the prefrontal cortex alter behavior (Yuan et al., 2015), and these behavioral changes may reflect the altered activity of prefrontal cortex neurons (Picken Bahrey and Moody, 2003). The activity of these neurons depends in part on the expression of ion channels, including Na+ channels (Seamans and Yang, 2004). Nine members of the Na+ channel family have been identified: Nav1.1 to Nav1.9. Na+ channel currents have also been classified according to their sensitivity to tetrodotoxin (TTX), i.e., as TTX sensitive (Nav1.1 to Nav1.4, Nav1.6 and Nav1.7) or TTX resistant (Nav1.5, Nav1.8 and Nav1.9) (Goldin et al., 2000; Catterall et al., 2005).

John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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Medial prefrontal cortex (mPFC) pyramidal neurons in rats express fast-inactivating voltagedependent Na+ channel currents, which are responsible for the depolarizing phase of action potentials (Maurice et al., 2001; Witkowski and Szulczyk, 2006). These channels are encoded by the Nav1.1 and Nav1.2 Na+ channel α subunits (Maurice et al., 2001). Low-threshold, slowly inactivating TTX-sensitive Na+ currents that are responsible for prolonged depolarization are also found in mPFC pyramidal neurons (Maurice et al., 2001; Gorelova and Seamans, 2015). These currents amplify excitatory postsynaptic potentials (Stuart and Sakmann, 1995) and regulate the membrane potential and cell excitability (Durstewitz et al., 1999; Hu et al., 2009). The Nav1.6 channel α subunit has been proposed to be responsible for prolonged depolarization (Maurice et al., 2001; Hu et al., 2009).

Nav1.9 channel currents are TTX resistant, become activated at or below the resting membrane potential, and possess very slow activation and inactivation kinetics (for a review: (Dib-Hajj et al., 2015)). The expression of Nav1.9 channels in the cortex is controversial. Nav1.9 channels have been shown to be expressed in small nociceptive dorsal root ganglion (DRG) neurons (Dib-Hajj et al., 1998; Tate et al., 1998; Cummins et al., 1999; Fang et al., 2002; Maruyama et al., 2004) and only residually in cortical neurons (Dib-Hajj et al., 1998). However, other studies have shown that these channels are expressed throughout the central nervous system (Blum et al., 2002; Blum and Konnerth, 2005; Subramanian et al., 2012; Wetzel et al., 2013; Black et al., 2014). Recently, it was reported that Nav1.9 channels are expressed in layer V mPFC pyramidal neurons (Kurowski et al., 2015) and that Nav1.9 transcripts are expressed in layer V mPFC tissue (Radzicki et al., 2017) of young rats. The purpose of this study was to evaluate the presence of Nav1.9 channels in mPFC pyramidal neurons and to determine the developmental trajectory of their expression in layer II and V mPFC pyramidal neurons in young (20 days old), late adolescent (60 days old) and adult (6 months old) rats. METHODS The experimental procedures used in this study adhered to the EU (22 September 2010, Directive 2010/63/EU), Polish (26 February 2015, Official Journal of Laws of 2015, No. 266) and First Local Ethics Committee for Animal Experimentation in Warsaw guidelines for the ethical use of



animals. Tissues were obtained from young (18 to 23 days old), late adolescent (58 to 62 days old) and old (5-6 months old) male WAG Cmd (Spear, 2000, 2013) rats that were obtained from a local animal facility.

Electrophysiology The young rats were decapitated under ethyl chloride anesthesia. The brains were dissected and placed in cold (1-4 °C) oxygenated extracellular solution. The solution was bubbled with carbogen (95% O2 and 5% CO2) and contained (in mM) NaCl (130), KCl (2.5), NaH2PO4 (1.25), NaHCO3 (25), glucose (10), MgCl2 (7), and CaCl2 (0.5). The pH of the solution was 7.4 (adjusted with N-methyl-d-glucamine [NMDG]), and its osmolality was 330 mOsm/kg H2O (adjusted with sucrose). Coronal slices (300 µm thick) of prefrontal cortex tissue were prepared using a vibratome (Leica VT1200S, Germany). The slices were stored at room temperature (21–22 °C) for up to 8 h in a solution that contained (in mM) NaCl (130), KCl (2.5), NaH2PO4 (1.25), NaHCO3 (25), glucose (10), MgCl2 (2), and CaCl2 (2). This solution was also bubbled with carbogen (osmolality of 310 mOsm/kg H2O and pH of 7.4).

Whole-cell current recordings from dispersed pyramidal neurons Dispersed pyramidal neurons were prepared as described previously (Ksiazek et al., 2013). Portions of slices (0.6–0.9 mm from the midline, 3–5 mm below the upper cortical surface and 2.2–3.5 mm anterior to the bregma) (Van Eden and Uylings, 1985; Uylings et al., 2003; Hoover and Vertes, 2007; Paxinos and Watson, 2007) were cut and placed in a solution that was bubbled with oxygen and contained (in mM) NaCl (135), HEPES-Cl [N-(2-hydroxyethyl)piperazine-N'(2-ethanesulfonic acid)] (10), KCl (5), MgSO4 (1), CaCl2 (0.1), glucose (10) and protease type XIV (1 mg/ml, Sigma-Aldrich, Poland). The pH of the solution was adjusted to 7.4 with NaOH, and its osmolality was 300 mOsm/kg H2O. Enzymatic reactions lasted 15–30 min at 32 °C. The enzymatic activity was stopped by replacing the solution containing the slices 3 times with an identical solution without proteases. Portions of the slices were dispersed using Pasteur pipettes. Next, the dispersed neurons were placed in a recording chamber (type RC-24E, Warner Instr., Connecticut, Hamden, USA) coated with poly-L-lysine hydrobromide (50 mg/ml) that was placed on the stage of an inverted microscope (Olympus Corporation IX2, Japan). Neurons were


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identified using differential interference contrast (DIC). Pyramidal neurons selected for recording had a smooth, three-dimensional appearance, including a triangular shape, a short axon at the base of the soma, and residual basal and apical dendrites (as in Fig 1B in (Witkowski and Szulczyk, 2006)). Whole-cell current recordings were performed with a pipette solution that contained (in mM) CsF (110), CsCl (20), HEPES (10), NaCl (10), MgCl2 (2), and EGTA (2). The pH of the pipette solution was 7.2 (adjusted with CsOH), and its osmolality was 285 mOsm/kg H2O (adjusted with sucrose). The dispersed neurons were perfused by gravity at a speed of 2 ml/min. This solution contained the following compounds (in mM): NaCl (130), MgCl2 (2), CaCl2 (1.5), HEPES (10), CdCl2 (0.1), TEA-Cl (tetraethylammonium chloride, 30), 4-AP (4-aminopyridine, 1) and TTX (tetrodotoxin citrate, 0.0005 or 0.002). The pH of the solution was 7.4, and the osmolality was 330 mOsm/kg H2O. When required, 120 mM NaCl was replaced with 120 mM choline-Cl to decrease the concentration of Na+ in the extracellular solution. Recordings were performed at room temperature (21–22 °C). Currents from dispersed neurons were recorded using an Axopatch 1D amplifier and pClamp software (Molecular Devices, USA). Patch pipettes were produced from borosilicate glass capillaries (O.D. 1.5 mm, I.D. 0.86 mm) using a P-87 puller (Sutter Instrument Inc., Novato, CA, USA). The junction potential was zeroed with the pipette tip dipped in the bath. The electrode capacitance was compensated using the circuit of the amplifier. The membrane ruptured spontaneously or by suction. The access resistance ranged from 5 to 7 MΩ. Currents were digitized at 50 kHz and filtered with a pole Bessel filter (5 kHz). Series resistance compensation of 80% was applied. In dispersed neurons, currents were evoked by a voltage ramp protocol from -110 mV to +60 mV over 680 ms. The ramp was preceded by a -110 mV prepulse for 500 ms. The holding potential was -70 mV. Na+ currents were isolated by subtracting a straight line fit of the fraction of the trace that lacked the voltage-dependent component (usually the fraction recorded at potentials more negative than -80 mV) from the whole trace (linear leak subtraction).

To generate the activation and inactivation curves, currents were evoked by rectangular voltage steps of 100 ms or 200 ms preceded by 1000 or 2000 ms prepulses. For the activation curve,



rectangular voltage steps ranging from -110 mV to -20 mV were preceded by a -110 mV prepulse. For the inactivation curve, the rectangular voltage step was constant (-40 mV) and was preceded by prepulses ranging from -110 to -10 mV. The conductance (G) was calculated according to the equation G=I/(V-Vr), where I is the peak current at voltage V, and Vr is the reversal potential calculated from the Nernst equation. A Boltzmann function in the form of 1/(1+exp[(V-V1/2)/k]) obtained by a nonlinear least-squares fitting routine (GraphPad Prism 7.00) was used to fit the G/Gmax (activation) or I/Imax (inactivation) curves, where V1/2 is the voltage of half-activation or inactivation, and k is a slope factor that corresponds to the change in voltage that causes an e-fold increase/decrease in the plotted function.

Whole-cell current recordings from layer II and V pyramidal neurons in slices Slices for electrophysiological recordings were prepared as previously described (Kurowski et al., 2015). Prior to the current recordings, the slices were kept for 40 min in a warm (34 °C) extracellular solution containing the following (in mM): NaCl (130), KCl (2.5), NaH2PO4 (1.25), NaHCO3 (25), MgCl2 (2), CaCl2 (2) and glucose (10). The solution was bubbled with carbogen. The pH of the solution was 7.4, and the osmolality was 330 mOsm/kg H2O. Currents were recorded in the same solution that also included blockers of glutaminergic and GABAergic transmission (50 µM AP-5, 10 µM DNQX and 50 µM picrotoxin) and a blocker of TTX-sensitive Na+ currents (TTX; tetrodotoxin citrate, 0.5 µM or 2 µM). Ca++ ions were omitted from the extracellular solution. When required, NaCl (130 mM) was replaced with choline-Cl (130 mM). When the effect of Cd++ ions on the currents recorded from pyramidal neurons in the slices was tested, NaH2PO4 was removed from the extracellular solution. The pipette solution for whole-cell current recordings was the same as that for the dispersed neuron recordings. The slices were placed in a recording chamber (RC-24E, Warner Instruments, LLC, MA, USA) on the stage of an upright Nikon microscope (Eclipse E600FN; Nikon Instech Co., Ltd., Japan). The neurons were viewed using infrared DIC optics with a 40x water immersion objective, a camera (C7500-50) and a camera controller (C2741-62) from Hamamatsu Photonics K.K. (Japan). The junction potential was zeroed with the pipette tip immersed in the bath. After a gigaseal was formed, the electrode capacitance was compensated. The membrane ruptured spontaneously or by suction. The access resistance ranged from 5–7 MΩ. Recordings were performed at 35 °C.


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Voltage-clamp recordings were obtained from layer II or layer V pyramidal neurons of the infralimbic and prelimbic mPFC (see Fig 4A). Currents were recorded with a MultiClamp 700A amplifier and a DigiData 1332A converter. pClamp 9.0 software was used (Molecular Devices, CA, USA). The current recordings were digitized at 20 kHz and filtered at 2 kHz. In neurons in slices, currents were evoked by a voltage ramp protocol from -110 mV to +60 mV over 680 ms. The ramp was preceded by a -110 mV prepulse for 500 ms. The holding potential was -70 mV. Na+ currents were isolated by subtracting a straight line fit of the fraction of the trace that lacked the voltage-dependent component (usually the fraction recorded at potentials more negative than -80 mV) from the whole trace.

Real-time quantitative PCR (qPCR)-based studies of gene expression The rats were deeply anesthetized with an overdose of Morbital (sodium pentobarbitone 133.3 mg/ml and pentobarbitone 26.7 mg/ml, i.p.; Biovet-Pulawy, Poland) and decapitated. The mPFC on both sides, including the prelimbic and infralimbic areas, was dissected out, placed in RNAlater Stabilization Solution (Life Technologies) and stored at -80 °C until analysis. The samples were reverse transcribed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). The amount of RNA transcript was quantified using real-time PCR; reactions were performed using a 7500 Fast Real-Time PCR System (Life Technologies) and HGPRT as a reference gene. Gene expression was evaluated with SCN11a-specific (Life Technologies, Rn00570487_m1), SCN10a-specific (Life Technologies, Rn00568393_m1), SCN1a-specific (Life Technologies, Rn00578439_m1), and HPRT-specific (Life Technologies, Rn01527840_m1) TaqMan assays. qPCRs were run in triplicate as singleplex reactions in a volume of 20 µl using TaqMan Universal Master Mix II, no UNG (Life Technologies) and approximately 10-11 ng total RNA that had been reverse transcribed to cDNA using a HighCapacity cDNA Reverse Transcription kit with RNase Inhibitor (Life Technologies). All realtime qPCR-based studies of gene expression were performed using the method for relative quantification without a standard curve based on the 2-∆∆CT formula. For each sample, the expression of target genes was normalized to that of the reference gene. The real-time qPCR data were analyzed using software designed for the 7500 Fast Real-Time PCR System (Life Technologies). The SCN1a results are shown in Supplementary material 1.



Confocal microscopy The rats were administered an overdose of Morbital (sodium pentobarbitone 133.3 mg/ml and pentobarbitone 26.7 mg/ml, i.p.; Biovet-Pulawy, Poland) and perfused through the right atrium and ascending aorta with phosphate-buffered saline (PBS) at room temperature followed by 4% paraformaldehyde in PBS. Subsequently, the brains and the DRG were removed. The isolated tissues were postfixed in 4% paraformaldehyde in PBS (at 4 °C) for 24 h. Next, the tissues were incubated in an increasing gradient of sucrose solutions (in PBS) for cryoprotection. Serial coronal frozen sections (40 µm thick) were then cut through the mPFC, including the infralimbic and prelimbic regions, using a freezing microtome (Leica CM1850UV). The free-floating mPFC sections in cryoprotectant were stored at -20 °C until immunohistochemistry processing (Gorlewicz et al., 2009; Ładno et al., 2017). One mPFC section from each young rat, late adolescent rat and adult rat were treated together in a single staining/washing chamber to ensure that the slices from the different age groups were all treated under the same staining conditions. The free-floating sections were washed with PBS and then blocked with 5% goat serum in PBS with 0.01% Triton X-100 (PBST). The free-floating sections were then incubated with guinea pig polyclonal anti-Nav1.9 (1:200, Alomone Labs, catalog number AGP-030) and chicken polyclonal anti-Map2 (1:200, Abcam, catalog number ab5392) primary antibodies on a shaker overnight at 4 °C. Unbound antibodies were washed out with PBST. Alexa Fluor® 488-conjugated goat anti-chicken IgG (1:200, Life Technologies, catalog number: A-11039) and Alexa Fluor® 568-conjugated goat anti-guinea pig IgG (1:200, Life Technologies, catalog number: A-11075) secondary antibodies were applied sequentially at room temperature for 2 h. After the incubation period for each antibody, the sections were washed with PBST (4 times for 5 min each). After the sections were washed, they were mounted and coverslipped. Immunofluorescence measurements were performed with an Olympus X1000 confocal microscope. The same parameter settings were used for all measurements of Nav1.9 signals (e.g., laser power, the voltage applied to the photomultiplier tubes, and pinhole size). Confocal images within the mPFC area were acquired from 4-5 fields along layer II and from 4-5 fields along layer V. Each field (1024x1024 pixel resolution) was scanned at 0.5-µm intervals along the Z axis. In each field, 4-5 pyramidal neurons were identified (based on their triangular shape and the presence of a thick apical dendrite pointing towards the cortical surface).


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The DRG were cut (20 µm thick) and mounted on adhesive slides (Superfrost™ Ultra Plus, Thermo Scientific). The slides were then stored at -20 °C until they were processed for immunofluorescence using the same protocol used for Nav1.9 staining of the brain sections. The DRG sections were also stained with a fluorescent Nissl stain (NeuroTrace 535/455 blue fluorescent Nissl stain; Invitrogen). The DRG labeling results are shown in Supporting information (S2 Fig). The antibody specificity was assessed for both the brain and DRG (biological positive control) staining by making the following three modifications to the protocol: (1) the primary antibody was omitted from the serum solution during the overnight incubation, (2) normal guinea pig IgG (Santa Cruz Biotech sc-2711, 5 µg/ml) was used instead of the anti-Nav1.9 antibody, and (3) the primary antibody was preabsorbed for 24 h with a blocking peptide (according to the supplier manual, i.e., 1 µg of peptide per 1 µg of antibody; peptide delivered by Alomone Labs). The images were processed using the Fiji image processing package, and the data were analyzed using GraphPad InStat software v3.06 (GraphPad Software, Inc., La Jolla, CA, USA).

Statistical analysis Data representing the immunolabeling of Nav1.9 channels in pyramidal neurons were collected from 18 animals (6 young, 6 late adolescent and 6 adult, Fig 5C). Each data point (obtained from 1 animal) represents the average of 22-27 neurons. One-way ANOVA (for correlated samples) followed by Tukey’s multiple comparisons test was used to compare observations among the different age groups (young vs late adolescent vs adult animals) separately for each layer (II and V). Paired t-tests were used to compare differences between layers II and V in young, late adolescent and adult rats (t-tests were followed by the Benjamini–Hochberg procedure for multiple comparisons with a false discovery rate of 0.05, (McDonald, 2015)). To ensure the homogeneity of variance within the groups of young, late adolescent, and adult animals, three brain sections were used for each antibody staining procedure. One brain section from a young rat, one from a late adolescent rat and one from an adult rat were exposed to identical conditions during antibody staining by processing all three brain sections in the same container. For this reason, we used a version of ANOVA for correlated samples (data obtained during the same staining procedure were matched). Homogeneity of variance was checked using Bartlett's test (α=0.05).



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To analyze mRNA levels, ANOVA with Tukey’s post hoc procedure was applied. Paired t-tests were used to determine whether whole-cell currents obtained in different conditions were significantly different. When a paired t-test was applied (for fluorescence or electrophysiology data), the assumption that the between-group differences were normally distributed was confirmed using the modified Anderson-Darling A* test for normality (A*=A2(1+0.75/n+2.25/n2), where n is the sample size) with α=0.05 (Stephens, 1979). The data are presented as the mean±standard error of the mean. For t and F tests, the degrees of freedom are given in parenthesis.

Chemical compounds The chemical compounds used for this study were purchased from Sigma-Aldrich (4-AP, CdCl2, choline-Cl, CsF, CsCl, EGTA, paraformaldehyde, poly-L-lysine hydrobromide, and TEA-Cl), Tocris (DNQX, HEPES, and picrotoxin), Latoxan France (TTX) or Polskie Odczynniki Chemiczne.

RESULTS TTX-resistant Na± currents in mPFC pyramidal neurons TTX-resistant Na+ currents in dispersed neurons We sought to obtain electrophysiological evidence that TTX-resistant, low-threshold Nav1.9-like Na+ currents are present in mPFC pyramidal neurons, as suggested by our earlier study (Kurowski et al., 2015). Therefore, whole-cell membrane currents were recorded from dispersed mPFC pyramidal neurons from young rats. Better conditions for voltage-clamp recordings can be achieved using dispersed pyramidal neurons than neurons in slices because dispersed pyramidal neurons lack long processes (Witkowski and Szulczyk, 2006). The extracellular solution contained Na+ (TTX, 0.0005 or 0.002 mM) and Ca++ (CdCl2, 0.1 mM) channel blockers. The concentration of Cd++ ions was low to avoid significant inhibition of Nav1.9-type Na+ channels (Coste et al., 2007). Moreover, the extra- and intracellular solutions contained K+ channel blockers. Currents were evoked by a depolarizing ramp (described in Methods, Fig 1A). The recorded current consisted of leak and voltage-dependent components (Fig 1Ba). The threshold and amplitude of the voltage-dependent component were evaluated after the leak component was subtracted from the recorded ramp current (Fig 1Bb). When the Na+ ion concentration in the


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extracellular solution was decreased from 130 (Fig 1C, +Na+) to 10 mM (Fig 1C, -Na+), the voltage-dependent component was attenuated; its mean amplitude decreased from 90.2±10.3 pA to 8.7±1.3 pA (n=15 neurons, paired t(14)=8.351, P=0.0345, Fig 1D). Its threshold in the presence of Na+ ions in the extracellular solution was -74.6±1.6 mV (n=15). We also recorded Na+ currents evoked by rectangular voltage steps from -110 mV to -20 mV in 10 mV increments lasting 100 or 200 ms. The steps were preceded by a -110 mV prepulse lasting 1000 or 2000 ms to remove voltage-dependent inactivation of the recorded currents. Original current traces evoked by voltage steps to -80, -70, -50, and -40 mV (Fig 1E inset) are shown in Fig 1E. Nav1.9 and Nav1.8 neuronal TTX-resistant Na+ currents can potentially be recorded in our experimental conditions (compare (Coste et al., 2004, 2007) and see below). The Nav1.8 current fully inactivates in less than 10 ms (Browne et al., 2009; Osorio et al., 2014). Therefore, we measured the mean current amplitude between 30 and 50 ms after the onset of the voltage steps (time span at which Nav1.8 current was fully inactivated). The V1/2 of current activation was -54.53±4.2 mV (n=10), and the slope factor was 14.6±3.7 mV (n=10, Fig 1G, black circles). In the same experimental condition, currents were evoked by 100 or 200-ms voltage steps to -40 mV. The voltage threshold of the Nav1.8 current is above approximately -40 mV; therefore, a voltage step to -40 mV did not activate the Nav1.8 current (Coste et al., 2004). The voltage steps to -40 mV were preceded by prepulses lasting 1000 or 2000 ms and in 10 mV increments from 110 mV to -10 mV. Fig 1F demonstrates original currents evoked by steps to -40 mV preceded by -90-, -60-, -50-, and -40-mV prepulses (inset to Fig 1F). The recorded current exhibited steady-state inactivation. The V1/2 of steady-state inactivation was -62.4±1.3 mV (n=8), and the slope factor was 9.1±1.2 mV (n=8, Fig 1G, black triangles). Whole-cell currents evoked by a voltage step to -40 mV preceded by a 2000 ms prepulse (-100 mV) were fitted with two components, one noninactivating and one exponentially decaying with a time constant of 12.6±0.43 ms (n=10). The contribution of the noninactivating component was 18%. Currents evoked by the voltage steps were abolished when Na+ ions were replaced by choline. The voltage-dependent currents evoked by a voltage ramp or voltage steps most likely represented low-threshold, TTX-insensitive Na+ currents. TTX-resistant Na+ currents in neurons in slices



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When whole-cell voltage-clamp recordings are made from neurons with long processes, appropriate voltage control cannot be obtained beyond the soma due to the lack of “space clamp” (Williams and Mitchell, 2008). Despite this limitation, we recorded whole-cell membrane currents from layer II and V pyramidal neurons in slices obtained from young rats to determine whether TTX-resistant Na+ currents could be detected in these cells. Similar recordings were performed in other studies (Gorelova and Yang, 2000; Astman et al., 2006). Currents were evoked by the same depolarizing ramp that was applied to study Na+ currents in dispersed neurons (Fig 2Aa). The recording pipette contained Cs+ ions, and Ca++ ions were omitted from the extracellular solution, which also contained TTX and synaptic transmission blockers. When the extracellular solution contained a high concentration of Na+ ions, the voltage ramp evoked leak and voltage-dependent currents. The voltage-dependent component was present in layer II (not shown) and V (Fig 2Ab, +Na+, shown after leak subtraction) pyramidal neurons. When the concentration of Na+ ions in the extracellular solution was decreased from 156.25 mM to 26.25 mM, the current amplitude in layer II and V pyramidal neurons significantly decreased from 144.1±16.5 pA to 18.3±9.6 pA (n=7, paired t(6)=7.441, P=0.0003, Fig 2Ba) and from 258.3±50.5 pA to 38.6±10.4 pA (n=8, paired t(7)=5.271, P=0.0012, Fig 2Ab –Na+, Fig 2Bb), respectively. The thresholds of this current were -68.1±2.5 mV (n=7) and -68.4±1.8 mV (n=8) in layer II and V pyramidal neurons, respectively.

It was previously demonstrated that TTX-resistant Nav1.9 currents in DRG neurons are inhibited by Cd++ ions (Coste et al., 2007). We tested the effects of Cd++ ions (0.4 mM) on the amplitude of TTX-resistant Na+ currents in layer V mPFC pyramidal neurons in slices obtained from young rats. Currents were evoked by a depolarizing ramp from -110 mV to +60 mV over 680 ms. The extracellular solution contained TTX and 156 mM Na+. Ca++ ions were absent in the extracellular solution. The ramp was preceded by a -110-mV prepulse for 500 ms (Fig 2Ca). In the absence (Cd++, Fig 2Cb) and presence (+Cd++, Fig 2Cb) of Cd++ ions, the current amplitude was 262.5±57.0 pA and 131.8±36.7, respectively. The presence of Cd++ ions significantly decreased the amplitude of the TTX-resistant Na+ current (n=7, paired t(6)=3.934, P=0.0077, Fig 2Cc). Taken together, our results demonstrate that Nav1.9-like channel currents are expressed in layer II and layer V mPFC pyramidal neurons. Because the amplitude of the recorded currents was small and current recordings from neurons in slices from older animals are problematic, the


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expression levels of Nav1.9 gene products were tested along the developmental trajectory with real-time PCR and confocal microscopy.

Nav1.9 gene expression in mPFC tissue in young, late adolescent and adult rats Using real-time qPCR, we identified SCN11a mRNA (which encodes the Nav1.9 channel), SCN10a mRNA (which encodes the Nav1.8 channel) and SCN1a mRNA (which encodes the Nav1.1 channel) in mPFC tissue from young (n=7), late adolescent (n=7) and adult (n=7) rats. The normalized expression levels of SCN11a mRNA were 1.44±0.10 (x10-3, n=7), 0.64±0.03 (x10-3, n=7) and 0.63±0.08 (x10-3, n=7) in the young, late adolescent and adult rats, respectively. The expression in the young rats was 2.23-fold and 2.34-fold higher than the expression in the late adolescent and adult rats, respectively (Fig 3A; ANOVA, F(2,18)=37.7, P0.05). We also tested the expression levels of SCN1a mRNA in mPFC tissue. SCN1a encodes Nav1.1 channels, which are found in mPFC pyramidal neurons (Maurice et al., 2001). The expression levels of SCN1a mRNA were not significantly different among tissues isolated from young, adolescent and adult rats (Supporting information, S1 Fig). These results indicate that mPFC expression of the SCN11a and SCN10a genes, which encode Nav1.9 and Nav1.8 channels, respectively, was lower in late adolescent and adult animals than in young animals. However, expression of the SCN1a gene in the mPFC tissues of young, adolescent and adult rats was not related to age.

Expression of Nav1.9 channels in mPFC pyramidal neurons



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The specificity of Nav1.9 antibodies (which we applied to cortical slices) was validated using cells that are known to express Nav1.9 channels. Others have repeatedly reported that small DRG neurons exhibit prominent Nav1.9 expression, while large DRG neurons express little or no Nav1.9 channel protein (Dib-Hajj et al., 1998; Tate et al., 1998; Cummins et al., 1999; Fang et al., 2002; Maruyama et al., 2004). The pattern of DRG neuron staining with the Nav1.9 channel antibodies applied in our study (see Supporting information, S2 Fig) was exactly the same as previously demonstrated by other authors (Dib-Hajj et al., 1998; Tate et al., 1998; Fang et al., 2002). Therefore, the anti-Nav1.9 antibodies applied in our study likely correctly recognize Nav1.9 channels in cortical neurons. The Nav1.9-stained mPFC sections could be divided into five layers: I, II, III, V and VI (marked with Roman numerals, Fig 4A), consistent with previous studies (Cunningham et al., 2002; van Aerde and Feldmeyer, 2015). Neurons were labeled with Nav1.9 (Fig 4ADG) and a Map2 fluorescent stain (Fig 4CEH). Merged labeling is shown in Fig 4F and I. Control immunostaining with primary anti-Nav1.9 antibody omitted is demonstrated in Figure 4B. The same area as in Figure 4B stained for MAP-2 is shown in Figure 4C. Numerous pyramidal neurons with prominent apical dendrites were found in layer II (Fig 4DEF) and layer V (Fig 4GHI). All layer II and layer V pyramidal-like neurons (with prominent apical dendrites) showed Nav1.9 immunoreactivity.

Single Nav1.9-immunostained pyramidal neurons in layer II (Fig 5Aabc) and layer V (Fig 5Adef) in young (Fig 5Aad), late adolescent (Fig 5Abe) and adult (Fig 5Acf) rats were scanned along the Z axis in 0.5-µm intervals. The section in which the diameter of the nucleus was maximal was chosen for analysis. The mean gray value of the Nav1.9-immunostained area was manually delineated in layer II (for example, Fig 5Aa, Ba, single arrow) and layer V (for example, Fig 5Ad, Bb, double arrow), measured with Fiji software and corrected for background staining. The mean gray value of layer II pyramidal neurons in the late adolescent (720.2±141.9, n=6 ) and adult (731.1±109.2, n=6) rats was significantly lower than that in the young (1322.0±109.0, n=6) rats (repeated ANOVA, F(1.646, 8.228)=28.27, P=0.0003, Tukey’s multiple comparisons P0.05, Fig 5C, closed triangles). The mean gray value of layer II (open triangles) pyramidal neurons was significantly greater than that of layer V (closed triangles) pyramidal neurons in the young rats (paired t(5)=5.581, P=0.0025, after correction for multiple comparisons with a false discovery rate of 0.05, P0.05) and adult rats (paired t(5)=2.582, P=0.0493, after correction with a false discovery rate of 0.05, P>0.05, Fig 5C).

DISCUSSION The results of this study indicate that Nav1.9 channels are expressed in mPFC pyramidal neurons and that the expression of these channels is lower in late adolescence and adulthood than in preadolescence. The electrophysiological properties of Nav1.9 currents have been well defined through current recordings in small DRG neurons, which express these channels. The very low threshold, lack of significant inactivation with time, presence of a window current, and activation and inactivation kinetics of the current described in our study are similar to the current properties of Nav1.9 in rat (Coste et al., 2004, 2007) and mouse (Cummins et al., 1999; Maruyama et al., 2004) DRG neurons. The Nav1.9–like current amplitude was markedly lower than the total Na+ current amplitude in mPFC pyramidal neurons (Witkowski and Szulczyk, 2006; Szulczyk et al., 2012). Two types of voltage-dependent neuronal Na+ currents, Nav1.8 and Nav1.9 TTX-resistant currents, could be potentially evoked in our experimental conditions. Recently, the presence of



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Nav1.8-like currents has been reported in cortical neurons (Szulczyk and Nurowska, 2017). Moreover, in our study, we demonstrated that Nav1.8 channel mRNA is present in mPFC tissue (see our Fig 3B). Therefore, our measurements were assessed under conditions that would exclude the involvement of any Nav1.8 current. In particular, the experiments were performed in the presence of fluoride ions in the intracellular solution. In the presence of these ions, the activation and inactivation kinetics of the Nav1.9 current shift to more negative potentials, while the kinetics of the Nav1.8 current remains unchanged. This lack of change facilitates the separation of the Nav1.9 current from the Nav1.8 current in cells expressing both channels (Coste et al., 2004; Cummins et al., 1999; Maruyama et al., 2004). To evaluate the activation kinetics, the mean current amplitude was measured 30-50 ms after voltage step onset, i.e., when Nav1.8 channels are already time-dependently inactivated (Szulczyk and Nurowska, 2017). To construct inactivation curves, currents were evoked by a voltage step of -40 mV, which does not activate Nav1.8 but is well above the threshold for the Nav1.9 current (Cummins et al., 1999; Coste et al., 2004, 2007; Maruyama et al., 2004; Szulczyk and Nurowska, 2017). mPFC pyramidal neurons have frequently been shown to exhibit persistent, low-threshold and TTX-sensitive Na+ currents (Gorelova and Yang, 2000; Maurice et al., 2001; Astman et al., 2006). A TTX-resistant noninactivating Na+ current has also been detected in cortical pyramidal neurons, particularly when experiments were performed in the absence of Cd++ ions in the extracellular solution (compare (White et al., 1993) and (Zhang, 2003).

DRG neurons are the only cells in which the expression of Nav1.9 channels has been unequivocally confirmed. DRG neurons include small neurons, which express Nav1.9 channels, and large neurons, which do not or only residually express Nav1.9 channels. This expression pattern of Nav1.9 channels in DRG neurons has been confirmed in laboratories in which different Nav1.9 antibodies and different staining methods were applied (for example, Dib-Hajj et al., 1998; Tate et al., 1998; Fang et al., 2002). Therefore, the specificity of the Nav1.9 antibodies that we applied in our study was tested on DRG neurons. The pattern of DRG neuron staining in our study was exactly the same as in earlier studies by other authors. These findings indicated that the antibodies effectively detected Nav1.9 channels. mPFC pyramidal neurons in our study were immunolabeled with Nav1.9 antibodies, results consistent with those of earlier studies indicating the expression of these channels in the central nervous system (Dib-Hajj et al., 1998; Blum et al.,


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2002; Blum and Konnerth, 2005; Subramanian et al., 2012; Wetzel et al., 2013; Black et al., 2014; Kurowski et al., 2015; Radzicki et al., 2017).

mPFC neurons control fear memory (Peters et al., 2009), innate anxiety (Saitoh et al., 2014) and working memory (Goldman-Rakic, 1999) in rats. The working memory process has been proposed to be based on prolonged depolarizations (also termed up-states or self-maintained depolarized states) in layer II (Gonzalez-Islas and Hablitz, 2003) and layer V (O'Donnell, 2003) mPFC pyramidal neurons. Prolonged depolarizations have also been found to be ubiquitous in neurons during NREM sleep and anesthesia (Crunelli and Hughes, 2010; Beltramo et al., 2013) and in the primary sensory cortex (Petersen et al., 2003). Nav1.9 channels, the expression of which we demonstrated in the mPFC, are ideally suited to support self-maintained depolarized states in pyramidal neurons because their activation evokes depolarization, they do not inactivate over time, and their threshold is close to the resting membrane potential.

In late adolescence, the mPFC becomes morphologically and functionally mature (Andersen et al., 2000; Spear, 2000, 2013; Bhatt et al., 2009; Little and Carter, 2013). At this stage of development, mPFC pyramidal neurons exhibit decreased spine density (Petanjek et al., 2011; Gourley et al., 2012; Koss et al., 2014), reduced D1 and D2 receptor expression (Andersen et al., 2000), and decreased synaptic density (Huttenlocher and Dabholkar, 1997) compared with neurons in young animals. This phenomenon reflects synaptic and neuronal (structural) pruning of the prefrontal cortex. In our case, maturation of the mPFC manifested as a decrease in Nav1.9 channel expression in layer II and layer V mPFC pyramidal neurons in late adolescent and adult rats compared to that in young animals. In addition, the Nav1.9 mRNA expression level in mPFC tissue was found to be more than two-fold lower in the late adolescent and adult animals than in the young rats. The results of a previous study indicated that symptoms (increased pain sensitivity) of gain-in-function Nav1.9 channelopathies are present in young patients but vanish in adolescent and adult patients, suggesting that Nav1.9 channels are less effective in the DRG neurons of adolescent and adult patients than in those of young patients with this channelopathy (Zhang et al., 2013; Okuda et al., 2016). This result and the findings of our study are indicative of a possible concomitant decrease in the effectiveness of Nav1.9 channels in DRG and mPFC pyramidal neurons of adolescent and adult subjects compared to that of young subjects.



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Layer II and layer V pyramidal neurons differ in both their activity patterns (Boudewijns et al., 2013; van Aerde and Feldmeyer, 2015) and their morphology (Ferreira et al., 2015; van Aerde and Feldmeyer, 2015). Pyramidal neurons in these layers also possess different reciprocal connections with subcortical structures. Layer II neurons project to the amygdala and receive strong synaptic input from the hippocampus (Degenetais et al., 2003) and amygdala (Little and Carter, 2013), while layer V pyramidal neurons project to the periaqueductal gray (Ferreira et al., 2015) and receive input from the hippocampus (Degenetais et al., 2003). Moreover, layer V pyramidal neurons seem to be more strictly controlled by acetylcholine (Gulledge et al., 2009) and adenosine A1 receptors (van Aerde et al., 2015) than layer II pyramidal neurons. Our study showed that Nav1.9 channel expression levels were greater in layer II than in layer V pyramidal neurons in young animals. Conversely, Nav1.9-like currents were larger in layer V pyramidal neurons than in layer II pyramidal neurons. This difference may be due to the larger soma and dendritic surface area of layer V than that of layer II pyramidal neurons (van Aerde and Feldmeyer, 2015), which may express Nav1.9 channels. Presumably, the density of Nav1.9 channel expression is greater in layer II pyramidal neurons than in layer V pyramidal neurons; however, the total number of Nav1.9 channels is greater in layer V pyramidal neurons than in layer II pyramidal neurons. We conclude that Nav1.9 Na+ channels are expressed in layer II and layer V mPFC pyramidal neurons and that the expression of these channels is lower in late adolescence and adulthood than in preadolescence. FIGURE LEGENDS. Fig 1. Current recordings from dispersed mPFC pyramidal neurons. (A) Voltage ramp applied to evoke currents in pyramidal neurons. (B) Current-voltage relation in pyramidal neurons. (a) Current evoked by the voltage ramp shown in “A”. (b) Current evoked by the voltage ramp after subtraction of the leak current. The leak current is marked by a line fit to the portion of the current trace shown in “a” that was more negative than -80 mV. The arrow in “b” indicates the threshold of the voltage-dependent component of the inward current. (C) Leaksubtracted current recorded from dispersed pyramidal neurons before (a) and after (b) the 120 mM Na+ in the extracellular solution was replaced with 120 mM choline ions. (D) Mean


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amplitude of inward currents recorded from dispersed pyramidal neurons before and after 120 mM NaCl (130 Na+) in the extracellular solution was replaced with 120 mM choline-Cl (10 Na+). (E) Current traces evoked by rectangular voltage steps to -80, -70, -50, and -40 mV lasting 100 ms and preceded by a 2000-ms prepulse to -110 mV. The voltage protocol is shown in the inset. (F) Current traces evoked by a voltage step to -40 mV lasting 200 ms and preceded by prepulses to -90, -60, -50, and -40 mV lasting 2000 ms. (G) Plot of the normalized Na+ conductances for each voltage step (ordinate) vs the amplitude of the voltage step (abscissa, black circles). Normalized peak currents evoked by the test pulse (ordinate) are plotted as a function of the prepulse voltage step (abscissa, black triangles).

Fig 2. Current recordings from mPFC pyramidal neurons in slices. (Aa) Voltage ramp applied to evoke currents in pyramidal neurons. (b) Leak-subtracted current recorded from layer V pyramidal neurons in slices when the concentrations of Na+ ions in the extracellular solution were 156.25 mM (+Na+) and 26.25 mM Na+ (-Na+). (B) Mean amplitude of inward currents recorded from layer II (a) and layer V (b) pyramidal neurons before and after 130 mM NaCl (156 Na+) in the extracellular solution was replaced with 130 mM choline-Cl (26 Na+). (C) Effect of Cd++ ions on Na+ currents in layer V pyramidal neurons. (a) The voltage ramp applied to evoke currents. (b) Currents recorded in the absence (-Cd++) and presence (+Cd++, 0.4 mM) of Cd++ ions in the extracellular solution. (c) Mean amplitude of Na+ currents recorded in the absence (0 Cd++) and presence of Cd++ (0.4 Cd++) ions in the extracellular solution.

Fig 3. mRNA expression of the SCN11a (A) and SCN10a (B) genes in young, late adolescent and adult rats.

Fig 4. Immunofluorescence staining of prefrontal cortex neurons in young rats. (A) mPFC neurons stained for Nav1.9 protein. The cortical layers are marked with Roman numerals. (B) Control immunostaining with primary anti-Nav1.9 antibody omitted. (C) The same area as in B stained for MAP-2. (DEF) Stained layer II mPFC neurons. (GHI) Stained layer V mPFC neurons. (DG) Staining for Nav1.9 channels. (EH) Staining for Map2. (FI) Merged staining for Nav1.9 and Map2. Nav1.9-positive neurons are shown in red (ADG). Map2-positive neurons are shown in green (CEH). Double-positive neurons are shown in yellow/brown (FI).



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Scale bars, 250 µm (A), 100 µm (BC) and 50 µm (D-I). M – medial, L – lateral, D – dorsal and V – ventral.

Fig 5. Age-related changes in Nav1.9 signal intensity in layer II and layer V pyramidal neurons. (A) Images of Nav1.9-stained layer II (abc) and layer V (def) pyramidal neurons in young (ad), late adolescent (be) and adult (cf) rats. Scale bar – 50 µm. Nav1.9-positive neurons are shown in red (Aabcdef). The single (a) and double (d)-arrows indicate examples of layer II and layer V pyramidal neurons, respectively. The same neurons are shown in Bab. (B) Examples of delineated immunostained areas of layer II (a, single arrow) and layer V (b, double arrow) pyramidal neurons. (C) Mean gray value of the Nav1.9 signal in stained layer II (open triangles) and layer V (closed triangles) pyramidal neurons in young, late adolescent and adult rats. Vertical axis – mean gray value. Scale bars, 50 µm (A) and 10 µm (B).


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REFERENCES Andersen SL, Thompson AT, Rutstein M, Hostetter JC, Teicher MH. 2000. Dopamine receptor pruning in prefrontal cortex during the periadolescent period in rats. Synapse 37:167-169. Astman N, Gutnick MJ, Fleidervish IA. 2006. Persistent sodium current in layer 5 neocortical neurons is primarily generated in the proximal axon. J Neurosci 26:3465-3473. Beltramo R, D'Urso G, Dal Maschio M, Farisello P, Bovetti S, Clovis Y, Lassi G, Tucci V, De Pietri Tonelli D, Fellin T. 2013. Layer-specific excitatory circuits differentially control recurrent network dynamics in the neocortex. Nat Neurosci 16:227-234. Bhatt DH, Zhang S, Gan WB. 2009. Dendritic spine dynamics. Annu Rev Physiol 71:261-282. Black JA, Vasylyev D, Dib-Hajj SD, Waxman SG. 2014. Nav1.9 expression in magnocellular neurosecretory cells of supraoptic nucleus. Exp Neurol 253:174-179. Blum R, Kafitz KW, Konnerth A. 2002. Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9. Nature 419:687-693. Blum R, Konnerth A. 2005. Neurotrophin-mediated rapid signaling in the central nervous system: mechanisms and functions. Physiol 20:70-78. Boudewijns ZS, Groen MR, Lodder B, McMaster MT, Kalogreades L, de Haan R, Narayanan RT, Meredith RM, Mansvelder HD, de Kock CP. 2013. Layer-specific high-frequency action potential spiking in the prefrontal cortex of awake rats. Front Cell Neurosci 7:99. Browne LE, Clare JJ, Wray D. 2009. Functional and pharmacological properties of human and rat NaV1.8 channels. Neuropharmacology 56:905-914. Catterall WA, Goldin AL, Waxman SG. 2005. International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev 57:397-409.



Coste B, Crest M, Delmas P. 2007. Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons. J Gen Physiol 129:57-77. Coste B, Osorio N, Padilla F, Crest M, Delmas P. 2004. Gating and modulation of presumptive NaV1.9 channels in enteric and spinal sensory neurons. Mol Cell Neurosci 26:123-134. Crunelli V, Hughes SW. 2010. The slow (

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