Muscarinic receptor control of pyramidal neuron membrane potential in the medial prefrontal cortex (mPFC) in rats

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MUSCARINIC RECEPTOR CONTROL OF PYRAMIDAL NEURON MEMBRANE POTENTIAL IN THE MEDIAL PREFRONTAL CORTEX (mPFC) IN RATS

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P. KUROWSKI, M. GAWLAK AND P. SZULCZYK *

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Laboratory of Physiology and Pathophysiology, Centre for Preclinical Research and Technology, The Medical University of Warsaw, Banacha 1B, Warsaw 02-097, Poland

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Abstract—Damage to the cholinergic input to the prefrontal cortex has been implicated in neuropsychiatric disorders. Cholinergic endings release acetylcholine, which activates nicotinic and/or G-protein-coupled muscarinic receptors. Muscarinic receptors activate transduction systems, which control cellular effectors that regulate the membrane potential in medial prefrontal cortex (mPFC) neurons. The mechanisms responsible for the cholinergic-dependent depolarization of mPFC layer V pyramidal neurons in slices obtained from young rats were elucidated in this study. Glutamatergic and GABAergic transmission as well as tetrodotoxin (TTX)-sensitive Na+ and voltage-dependent Ca++ currents were eliminated. Cholinergic receptor stimulation by carbamoylcholine chloride (CCh; 100 lM) evoked depolarization (10.0 ± 1.3 mV), which was blocked by M1/M4 (pirenzepine dihydrochloride, 2 lM) and M1 (VU 0255035, 5 lM) muscarinic receptor antagonists and was not affected by a nicotinic receptor antagonist (mecamylamine hydrochloride, 10 lM). CCh-dependent depolarization was attenuated by extra- (20 lM) or intracellular (50 lM) application of an inhibitor of the bc-subunit-dependent transduction system (gallein). It was also inhibited by intracellular application of a bc-subunit-binding peptide (GRK2i, 10 lM). mPFC pyramidal neurons express Nav1.9 channels. CCh-dependent depolarization was abolished in the presence of antibodies against Nav1.9 channels in the intracellular solution and augmented by the presence of ProTx-I toxin (100 nM) in the extracellular solution. CCh-induced depolarization was not affected by the following reagents: intracellular transduction system blockers, including U-73122 (10 lM), chelerythrine chloride (5 lM), SQ 22536 (100 lM) and H-89 (2 lM); channel blockers, including Ba++ ions (200 lM), apamin (100 nM), flufenamic acid (200 lM), 2-APB (200 lM), SKF 96365 (50 lM), and ZD 7288 (50 lM); and a Na+/Ca++ exchanger blocker, benzamil (20 lM). We conclude that muscarinic M1 receptor-dependent depolarization in mPFC pyramidal neurons is evoked by the activation of Nav1.9 channels and that the signal transduction pathway

involves G-protein bc subunits. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.

Key words: prefrontal cortex, pyramidal neurons, muscarinic receptors, bc subunits, Nav1.9 channels, rats. 10

*Corresponding author. Tel: +48-22-1166161. E-mail address: [email protected] (P. Szulczyk). Abbreviations: CCh, carbamoylcholine chloride; DMSO, dimethyl sulfoxide; DRG, dorsal root ganglia; EGTA, ethylene glycol-bis-(2-ami noethylether)-N,N,N0 ,N0 -tetraacetic acid; HEPES-Cl, N-(hydroxyethyl) piperazine-N-(2-ethanesulfonic acid)-Cl; mPFC, medial prefrontal cortex; NMDG, N-methyl-D-glucamine; PBS, phosphate-buffered saline; TRP, transient receptor potential; TTX, tetrodotoxin. http://dx.doi.org/10.1016/j.neuroscience.2015.07.023 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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Damage to the cholinergic inputs to the prefrontal cortex has been implicated in several neuropsychiatric disorders, including schizophrenia, Alzheimer’s disease and other forms of senile dementia. These pathologies are characterized by impaired cognitive functions, including learning, attention and working memory (Scarr et al., 2013). Consistent with experimental findings, acetylcholinesterase inhibitors have established efficacy in treating cognitive impairment (Hasselmo and Sarter, 2011). In rats, muscarinic cholinergic input to medial prefrontal cortex (mPFC) neurons controls learning (Barker and Warburton, 2008), attentional processes (Kozak et al., 2006) and working memory (Chudasama et al., 2004). Acetylcholine signaling activates ionotropic nicotinic and metabotropic muscarinic G-protein-coupled receptors. Five subtypes of muscarinic receptors have been identified, M1–M5. The muscarinic M1, M3 and M5 receptors control cellular effectors by activating Gq proteins and phospholipase C. The M2 and M4 receptors are coupled to the Gi/Go family of G proteins and inhibit adenylyl cyclase. The transduction pathways of both systems involve cytoplasmic second messengers (Felder, 1995). Evidence also indicates that muscarinic receptors can control membrane effectors in a membrane-delimited fashion by releasing G-protein bc subunits without the participation of cytoplasmic second messengers (Herlitze et al., 1996; Nemec et al., 1999; Olianas and Onali, 2000). Tonic (prolonged) activation of muscarinic receptors evokes depolarization of mPFC pyramidal neurons (Andrade, 1991; Haj-Dahmane and Andrade, 1996, 1998; Carr and Surmeier, 2007) and of numerous types of neurons outside the prefrontal cortex (for example, Fisahn et al., 2002; Zhang et al., 2012). Phasic (shortlived) application of muscarinic agonists to mPFC pyramidal neurons evokes primarily hyperpolarization, followed by depolarization (Gulledge and Stuart, 2005).

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Different conductances have been proposed to be responsible for muscarinic-dependent depolarization in mPFC pyramidal neurons: a decrease in Kir currents (Carr and Surmeier, 2007); an increase in Na+ ion permeability due to the activation of non-selective and voltage-dependent cationic currents (Haj-Dahmane and Andrade, 1996); and the activation of hyperpolarizationactivated currents (Ih) (Thuault et al., 2013). A preferentially Ca++-dependent, non-selective cationic current is responsible for muscarinic-dependent depolarization in neurons outside the prefrontal cortex (Fisahn et al., 2002; Zhang et al., 2012). Gulledge and Stuart (2005) indicated that phasic and short-lived stimulation of M1 muscarinic receptors activates SK potassium channels, which leads to the hyperpolarization of mPFC pyramidal neurons. The data presented above indicate that the mechanism responsible for cholinergic receptor control of the membrane potential of mPFC pyramidal neurons remains controversial. The purpose of our study was to define the mechanism responsible for modulation of the membrane potential of mPFC pyramidal neurons in young rats during prolonged application of a cholinergic agonist.

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EXPERIMENTAL PROCEDURES

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The experimental procedures used in this study adhered to institutional and international guidelines regarding the ethical use of animals and were performed with the approval of the Second Local Ethics Committee for Animal Experimentation in Warsaw (Decision 1/2009). The experiments were performed on the neurons of young (18- to 22-day-old) male rats (WAG Cmd; Spear 2000) obtained from a local animal house.

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Membrane potential recordings

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Brain slices were prepared as previously described (Witkowski and Szulczyk, 2006; Witkowski et al., 2008; _ Ksiazek et al., 2013; Szulczyk, 2015). After the induction of anesthesia using ethyl chlorate, the animals were decapitated, and the brains were removed and placed in cold (0–4 °C) oxygenated extracellular solution containing the following compounds: sucrose (234 mM); KCl (2.5 mM); NaH2PO4 (1 mM); glucose (11 mM); MgSO4 (mM); N-(hydroxyethyl)piperazine-N-(2-ethanesulfonic acid)-Cl (HEPES-Cl; 15 mM); and ascorbic acid (1 mM). The pH was adjusted to 7.4 with NaOH, and the osmolality was 300 mOsm/kg H2O. Coronal slices (300-lm thick) were prepared from cerebral prefrontal tissue using a vibratome (Vibratome Line, Leica VT1200S, Nussloch, Germany). The slices were incubated for 15 min in warm (34 °C) extracellular solution that was infused with 95% O2 and 5% CO2 and contained the following compounds: NaCl (130 mM); KCl (2.5 mM); glucose (10 mM); NaHCO3 (25 mM); NaH2PO4 (1.25 mM); MgCl2 (1 mM); and CaCl2 (2 mM). The solution had a pH of 7.4 and an osmolality of 320 mOsm/kg H2O. Next, the slices were stabilized at room temperature in the same solution for

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at least 60 min before they were transferred to the recording chamber. During the recordings, the slices were perfused with the same extracellular solution supplemented with GABAergic and glutamatergic transmission blockers (50 lM picrotoxin, 10 lM DNQX and 50 lM AP-4). To block tetrodotoxin (TTX)-sensitive and voltage-dependent Na+ currents, the extracellular solution was supplemented with TTX (1 lM). To eliminate Ca++ currents, the extracellular solution was supplemented with ethylene glycol-bis-(2-aminoethyle ther)-N,N,N0 ,N0 -tetraacetic acid (EGTA, 2 mM), and the Ca++ concentration in the extracellular solution was decreased from 2 to 0.1 mM. To test the effect of lithium ions on the activity of the Na+/Ca++ exchanger and on Na+-dependent K+ currents, NaCl (130 mM) was replaced with LiCl (130 mM) in the extracellular solution. The slices were placed in a bath chamber (RC-24E, Warner Instruments, LLC, Hamden, MA, USA) on the stage of an upright Olympus microscope (BX51WI, Olympus Corporation, Tokyo, Japan) and were maintained at 34.0 °C. Images of neurons were captured using infrared differential interference contrast microscopy with a 40 water-immersion objective, a camera (C7500-51) and a camera controller (C2741-62) from Hamamatsu Photonics K. K. (Hamamatsu City, Japan). Recordings were obtained from mPFC pyramidal neurons 550–700 lM below the cortical surface. In young rats, this area of the mPFC corresponds to layer V pyramidal neurons (Kawaguchi and Kubota, 1997; Gonza´lez-Burgos and Barrionuevo, 2001). The neurons selected for recordings had a triangular shape and a prominent apical dendrite. The experiments were performed using a Multiclamp 700B, a Digidata 1440 and pClamp 10.4 software (Molecular Devices, Sunnyvale, CA, USA). In the majority of the experiments, membrane potentials were recorded using the gramicidin perforated-patch recording method under current-clamp conditions (Akaike and Harata, 1994). In these experiments, the pipette solution contained the following compounds: potassium gluconate (105 mM); KCl (20 mM); HEPES-Na+ (10 mM); EGTA (0.1 mM); and gramicidin (10–20 lg/ml). The solution had a pH of 7.25 and an osmolality of 280 mOsm/kg H2O. A stock solution of gramicidin was prepared in dimethyl sulfoxide (DMSO). The concentration of DMSO in the pipette solution did not exceed 0.01%. The open-tip resistance was 5–7 MX. The junction potential was zeroed when the pipette tip was immersed in the bath. The progress of membrane perforation by gramicidin was monitored by observing a gradual decrease in access resistance. Recordings began 15–20 min after the formation of the giga seal. The access resistance was periodically inspected, and the recording was discarded if the resistance rapidly decreased. The ‘‘classical’’ whole-cell current-clamp method (not the perforated-patch method) was applied only in four series of experiments in which a series of compounds were delivered from the pipette solution into the interior of the tested pyramidal neuron. These compounds

Please cite this article in press as: Kurowski P et al. Muscarinic receptor control of pyramidal neuron membrane potential in the medial prefrontal cortex (mPFC) in rats. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.07.023

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included: (1) normal guinea-pig IgG (catalog number: sc2711, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), (2) an antibody against Nav1.9 channels (catalog number: AGP-030, Alomone Labs, Jerusalem, Israel), (3) gallein or (4) GRK2i. IgG, the antibody against Nav1.9 channels and GRK2i were diluted in the pipette solution. A stock solution of gallein was prepared in DMSO. The concentration of DMSO in the pipette solution with gallein did not exceed 0.01%. In these experiments, in addition to the tested compound, the pipette solution contained: potassium gluconate (110 mM); KCl (20 mM); HEPES (10 mM); EGTA (0.5 mM); MgCl2 (2 mM); NaCl (5 mM); ATP2Na (2 mM) and GTPNa (0.4 mM); gramicidin was not included. The open-tip pipette resistance was 3–4 MX. After giga-seal formation, the membrane was disrupted by suction, and the tested compound was allowed to diffuse into the cell for 60 min before the effect of carbamoylcholine chloride (CCh) on the membrane potential was examined (compare to Wang et al., 2011). The chemical compounds were purchased from Biotechne (Abingdon, UK), excluding TTX, which was purchased from Latoxan Ltd. (Valence, France); DMSO, gramicidin, choline chloride, N-methyl-D-glucamine, and lithium chloride, which were purchased from Sigma– Aldrich (St. Louis, MO, USA); and picrotoxin, DL-AP4 sodium salt, and pirenzepine dihydrochloride, which were purchased from Abcam Biochemicals (Cambridge, UK). Picrotoxin, DNQX, U-73122, SQ 22536, gallein, 2-APB, and flufenamic acid were dissolved in DMSO. The final concentration of DMSO in the extracellular solution with these compounds was 0.01%. When the compounds were dissolved in DMSO, the control extracellular solution also contained DMSO at a concentration of 0.01%. The remaining compounds were dissolved in extracellular solution. The membrane potential was continuously recorded for 15 min for the control, for 2.5 min for CCh dissolved in the extracellular solution and for 15 min or longer after CCh was washed out. When the effect of the receptor, the transduction system or the ion channel blockers on CCh-dependent depolarization was tested, these substances were delivered for at least 15 min before the CCh application, for 2.5 min during the application and during the washing out of CCh. When the bc transduction system blocker (gallein) was applied extracellularly, this blocker was also delivered to the slice-containing pre-chamber for 2 h before the membrane potentials were recorded (Belkouch et al., 2011). All of the experiments with gallein were performed in the dark. The chemical compounds that were dissolved in the extracellular solution were bath applied by gravity at a flow rate of 2–3 ml/min. The membrane potential recordings were low-pass filtered at 1–3 kHz and digitized at a sampling rate of 3– 10 kHz. The pipettes were fabricated from borosilicate glass capillaries (O.D. = 1.5 mm, I.D. = 0.86 mm; Harvard Apparatus, Edenbridge, UK) using a P-1000 puller (Sutter Instruments, Inc., Novato, CA, USA). The data were analyzed using GraphPad InStat software v3.06 (GraphPad Software, Inc., La Jolla, CA,

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USA). The statistical significance of comparisons between two groups of results was determined using the Mann–Whitney test. The data are presented as the mean ± standard errors of the mean. A dose–response curve (the amplitude of depolarization [y] versus the CCh concentration [x]) was fitted via nonlinear regression to a Hill equation with the following form:

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yðxÞ ¼ 1þ

Amax  nH EC50 ½x

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where Amax is the maximal change in the membrane potential, nH is the Hill coefficient, and EC50 is the half maximal effective concentration.

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Confocal microscopy

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The rats received an overdose of sodium pentobarbital (75 mg/kg, i.p.) and were perfused through the ascending aorta with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS at room temperature. The brains and lower thoracic and lumbar dorsal root ganglia (DRG) were postfixed in situ for 24 h in 4% paraformaldehyde in PBS (at 4 °C). After the brains were postfixed, they were removed from the skulls, and the DRG were dissected. The tissues were exposed to an increasing gradient of sucrose solutions (in PBS) for cryoprotection. Next, serial coronal frozen sections (18- to 20-lm thick) were cut through the cortex, including the infralimbic and prelimbic regions of the mPFC, and through the DRG using a freezing microtome (Leica CM1850UV, Gorlewicz et al., 2009). The mPFC sections were stored in cryoprotectant at 70 °C until they were processed for immunohistochemistry. The cortical slices were covered with 5% goat serum in PBS with 0.01 M Triton X-100 for 45 min to saturate non-specific binding sites. Next, the slices were simultaneously incubated overnight (at 4 °C) in 5% goat serum in PBS with 0.01 M Triton X-100 with two primary antibodies: a chicken anti-MAP-2 antibody (Abcam, catalog number: ab5392, 47 lg/ml) and a guinea-pig anti-NaV1.9 antibody (Alomone Labs, catalog number: AGP-030, 5 lg/ml). Then, the slices were rinsed 5 times for 5 min in PBS with Triton X-100. Next, the slices were incubated for 2 h at room temperature in Alexa FluorÒ 488-conjugated goat anti-chicken IgG (Life Technologies, Warsaw, Poland, catalog number: A-11039), rinsed in PBST 4 times for 5 min each and incubated for 2 h at room temperature in Alexa FluorÒ 568-conjugated goat anti- guinea-pig IgG (Life Technologies, catalog number: A-11075). The DRG slices were transferred to microscope slides immediately after they were cut (SuperFrost Ultra Plus, Menzel-Gla¨ser, Braunschweig, Germany, catalog number: J3800AMNZ). The slides were then stored at 20 °C until they were processed for immunofluorescence using the same protocol used for the brain sections. To assess the antibody specificity for both the brain and DRG staining, two separate modifications of the

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protocol were made: (1) control goat IgG (Abcam, catalog number: ab37373, 5 lg/ml) was used instead of the antiNaV1.9 antibody; and (2) the primary antibody was omitted from the serum solution during the overnight incubation. The slides were imaged using an Olympus Fluoview FV1000 confocal laser-scanning microscope using 10 and 40 lenses and LD 559 nM, HeNe and argon lasers. When comparing the control and antibody staining, the following parameters were held constant: voltage applied to the photomultipier tubes, pinhole size, and percent transmission (Gawlak et al., 2009; Gorlewicz et al., 2009). The images were processed using the Fiji image processing package (Schindelin et al., 2012).

RESULTS

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CCh control of membrane potential

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Application of the broad-spectrum cholinergic agonist CCh (100 lM) to the bath for 2.5 min depolarized the membrane potential. In the majority of cases, repetitive action potentials were fired at the peak of depolarization (Fig. 1Aa). The depolarization was always followed by the recovery of the potential to the control level. Frequently, the depolarization was preceded by a small hyperpolarization (for example, Fig. 1Aa, b, c). When 1 lM TTX was added to the extracellular (bath) solution to block voltage-dependent and TTX-sensitive Na+ currents, the application of CCh (100 lM) evoked

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depolarization without action potentials (Fig. 1Ab, c). In the presence of TTX, depolarization occurred in two forms. In the first, the depolarization had a large amplitude and a plateau, which was followed by an extremely slow recovery. This type was found in 12 (of 29) membrane potential recordings (Fig. 1Ab). In the second form, there was a gradual membrane potential depolarization that was followed by a faster recovery (similar to the example presented in Fig. 1Ac). This type was found in 17 (of 29) recordings. We hypothesized that the large-amplitude and prolonged depolarizations evoked by the application of CCh (Fig. 1Ab) could be caused by secondary activation of the voltagedependent Ca++ currents present in mPFC pyramidal neurons (McKay et al., 2006; Rola et al., 2008; Zhou and Antic, 2012). Indeed, large-amplitude, plateau-like depolarizations were never observed in response to CCh application (100 lM) when, in addition to the blockade of voltage-dependent Na+ currents by TTX (1 lM), Ca++ currents were eliminated by the inclusion of EGTA (2 mM) in the presence of Ca++ (0.1 mM) in the extracellular solution (Fig. 1Ac). In the subsequent set of experiments, the effects of CCh were tested when voltage-dependent and TTX-sensitive Na+ currents were blocked by TTX (1 lM) and voltage-dependent Ca++ currents were eliminated by EGTA, as demonstrated in Fig. 1Ac (2 mM of EGTA in the presence of 0.1 mM Ca++ ions in the extracellular solution). The effects of different CCh concentrations on the amplitude of the depolarizations were tested. The

Fig. 1. Effects of CCh on the membrane potential of mPFC pyramidal neurons. (Aa) Effects of CCh (100 lM) on the membrane potential. Action potentials were truncated. Inset: Full amplitude of the action potentials (trace with an extended time base). (b) Effect of CCh on the membrane potential in the presence of TTX (1 lM) in the extracellular solution. (c) Effect of CCh on the membrane potential in the presence of TTX (1 lM), EGTA (2 mM) and Ca++ (0.1 mM) in the extracellular solution. The 2.5-min CCh application is marked by a horizontal bar in this figure and in other figures. (Ba) Effects of different concentrations of CCh on the membrane potential. The concentration of CCh is marked in the figure. (b) The relationship between the concentration of CCh in the extracellular solution and the amplitude of CCh-dependent depolarization. Please cite this article in press as: Kurowski P et al. Muscarinic receptor control of pyramidal neuron membrane potential in the medial prefrontal cortex (mPFC) in rats. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.07.023

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amplitude increased when the concentration of CCh was increased from 0.1 lM to 100 lM (Fig. 1Ba, b); the IC50 was 2.8 lM (nH = 1.0). When the perforated-patch method was used to perform the experiments, two groups of results were invariably compared: the mean amplitude of the depolarizations evoked by CCh when applied alone (100 lM; control, 10.0 ± 1.3 mV, n = 33) and the mean amplitude of the depolarizations evoked by CCh (100 lM) in the presence of one of the tested compounds. The peak amplitude of the control CChdependent depolarization occurred 2.1 ± 0.5 (n = 33) min after the 2.5-min CCh delivery (Fig. 1Ac). Therefore, when the CCh-dependent depolarization was abolished in the presence of some of the compounds, the amplitude of the membrane potential was measured 2.1 min after the termination of the delivery of CCh to the bath. If the membrane potential was unaffected by the presence of the tested compound, the CChdependent depolarization was always followed by recovery after the CCh was washed out (as in Fig. 1Ac).

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Pharmacology of membrane potential control by CCh

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The mean resting membrane potential of the pyramidal neurons before CCh application was 69.11 ± 0.52 mV (n = 172). Nicotinic or muscarinic receptor blockers were applied to the bath before, during and after CCh application to identify the cholinergic receptors responsible for the membrane potential changes in the pyramidal neurons. CCh-dependent depolarization was completely abolished in the presence of the M1/M4 muscarinic receptor antagonist pirenzepine dihydrochloride (2 lM, 0.13 ± 0.07 mV, n = 6, Fig. 2Aa, d) or of the selective M1 muscarinic receptor blocker VU 0255035 (5 lM, 0.17 ± 0.42 mV, n = 6, Fig. 2Ab, d). The mean amplitude of the CCh-dependent depolarization did not differ when CCh was applied alone or in the presence of the broad-spectrum nicotinic receptor antagonist mecamylamine hydrochloride (10 lM, 13.7 ± 2.0 mV, n = 5, Fig. 2Ac, d). These results indicate that M1 muscarinic receptors were responsible for the CCh-dependent depolarization of the mPFC pyramidal neurons.

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Identification of the transduction pathway involved in CCh-dependent depolarization Muscarinic receptors may be linked to G-protein subunit Gq/11, phospholipase C and downstream second messengers as well as to the G-protein-dependent adenylyl cyclase/cAMP pathway (Felder, 1995). Therefore, we tested whether these pathways were involved in the muscarinic-dependent depolarization of the pyramidal neurons. Blockers of the transduction pathways were delivered to the bath 15 min before, during and after CCh application. After applying the membranepermeable phospholipase C inhibitor U-73122 (10 lM) or the membrane-permeable protein kinase C inhibitor chelerythrine chloride (5 lM), the mean amplitude of CCh-dependent membrane depolarization was

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10.1 ± 2.9 mV (n = 5) and 9.3 ± 1.2 mV (n = 6), respectively; these mean amplitudes did not differ from the mean amplitude of the control depolarizations evoked by the application of CCh alone (p > 0.05; Fig. 2B). Additionally, application of the membrane-permeable adenylyl cyclase inhibitor SQ 22536 or the protein kinase A inhibitor H-89 did not modify the CCh-induced depolarization of the pyramidal neurons. The mean depolarization evoked in the presence of SQ 22536 (100 lM) and H-89 (2 lM) was 8.7 ± 1.2 mV (n = 5) and 10.8 ± 2.0 mV (n = 5), respectively, which did not significantly differ from the depolarization evoked by CCh alone (p > 0.05; Fig. 2C). Because the blockers of the common second messenger transduction pathways did not even partially inhibit the M1-dependent depolarization, we considered the possibility that the muscarinic receptors controlled the membrane effectors in a membrane-delimited fashion (involving bc subunits). Strong membrane depolarization is known to disrupt the link between bc subunits and cellular effectors, abolishing the effects of metabotropic receptors on membrane channels. When the link between bc subunits and cellular effectors was disrupted, the effect on Ca++ channels was brief (Kaneko et al., 1999; Dascal, 2001; Rola et al., 2008). To test whether bc subunits might be involved in the transduction process, we applied a current pulse (100 ms, 500 pA) in 10 pyramidal neurons at the peak of CCh-induced depolarization. In five neurons, the membrane potential was repolarized by 11.5 ± 2.3 mV and then returned to the pre-step (depolarized) level immediately after the current pulse; the repolarization lasted 840 ± 132 ms (Fig. 3Aa and inset to Fig. 3Aa). During the washout, the membrane potential returned to a level close to the resting membrane potential (Fig. 3Aa). Current was then injected into the same cell until the membrane was depolarized to the peak level attained during the application of CCh (Fig. 3Ab). At this depolarized level, the current step never evoked cell repolarization (inset to Fig. 3Ab – only a stimulus artifact is observed). In the remaining five neurons, the current step did not change the membrane potential depolarization evoked by either CCh or current injection. This result suggests that in mPFC pyramidal neurons, bc subunits may convey the signal from M1 muscarinic receptors to the effectors responsible for depolarization. To verify this effect, gallein (20 lM), which is a membrane-permeable blocker of the bc subunit transduction pathway (Bonacci et al., 2006; Lehmann et al., 2008; Irannejad and Wedegaertner, 2010; Belkouch et al., 2011; Ukhanov et al., 2011; Kodama and Togari, 2013; Schwetz et al., 2013), was applied before (for 2 h), during and after CCh application. The mean amplitude of the CCh-dependent depolarization evoked in the presence of gallein was markedly lower compared to the CCh-dependent depolarization evoked in the absence of gallein (1.7 ± 0.42 mV, n = 12, p < 0.0001; Fig. 3Ba1, a2). Blockers of the bc subunit transduction pathway were also applied intracellularly. To deliver the compounds to the cell interior, current-clamp recordings were

Please cite this article in press as: Kurowski P et al. Muscarinic receptor control of pyramidal neuron membrane potential in the medial prefrontal cortex (mPFC) in rats. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.07.023

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Fig. 2. The type of cholinergic receptors responsible for CCh-dependent depolarization (A) and the lack of evidence for cytoplasmic second messenger involvement in CCh-dependent depolarization (B, C) in mPFC pyramidal neurons. (A) CCh-dependent depolarization was abolished in the presence of M1/M4 (a. pirenzepine dihydrochloride, 2 lM) and M1 (b. VU 0255035, 5 lM) muscarinic receptor blockers and was not affected in the presence of a nicotinic receptor blocker (c. mecamylamine hydrochloride, 10 lM). (d) Average amplitude of CCh-dependent depolarization evoked by the application of CCh alone (100 lM, control, n = 33), pirenzepine dihydrochloride (2 lM, n = 6), VU 0255035 (5 lM, n = 6) and mecamylamine hydrochloride (10 lM, n = 5). The applications of the cholinergic receptor blockers are marked by horizontal bars. (B) Effects of bath application of a phospholipase inhibitor (U-73122, 10 lM, n = 5) and a kinase C blocker (chelerythrine chloride, 5 lM, n = 6) on the amplitude of CCh-dependent depolarization. A bar indicates the amplitude of the depolarization evoked by the application of CCh alone (100 lM) and is labeled ‘‘control’’ in this figure and in other figures. (C) Effects of an adenylyl cyclase blocker (SQ 22536, 100 lM, n = 5) and a protein kinase A blocker (H-89, 2 lM, n = 5) on CCh-dependent depolarization.

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performed in the ‘‘classical’’ whole-cell configuration after access to the cell was obtained by suction (not via the perforated-patch method). In this condition, the control amplitude of CCh-dependent depolarization was measured without blockers in the pipette solution 1 h after access to the cell was obtained and was 14.7 ± 2.0 mV (n = 10). In the presence of gallein in the pipette solution (50 lM, Kienitz et al., 2014), CCh-dependent depolarization was abolished 1 h after access to the cell interior was obtained. The mean CCh-dependent depolarization was 0.7 ± 0.15 mV (n = 6), which was significantly lower than the amplitude of the control CCh-dependent depolarization measured without gallein in the pipette solution (14.7 ± 2.0 mV, n = 10, p < 0.0001; Fig. 3Bb1, b2). GRK2i, which is Gbc-binding peptide that corresponds to the Gbc-binding domain of GRK2 (G-protein-coupled receptor kinase 2), has been shown to selectively prevent Gbc-mediated signaling (Koch et al., 1994; Stott et al., 2015). We tested the effect of intracellular application of GRK2i for 1 h on CCh-dependent depolarization. In the presence of GRK2i, the CCh-related depolarization was 3.7 ± 1.0 mV (n = 5), which was significantly lower than the amplitude of the control CCh-dependent depolarization evoked without GRK2i in the pipette solution (14.7 ± 2.0 mV, n = 10, p < 0.0004; Fig. 3Bc1, c2).

Identification of the cellular effector responsible for CCh-dependent depolarization

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In mPFC pyramidal neurons, CCh-dependent depolarization may be evoked through the closing of constitutively active inward-rectifying K+ channels (Kir channels, Carr and Surmeier, 2007). The inhibition of Kir currents with Ba++ ions (200 lM, Hibino et al., 2010) did not change the amplitude of CCh-induced depolarization (8.8 ± 1.7 mV, n = 6) compared with the depolarization evoked by the application of CCh alone (p > 0.05, Fig. 4A). Therefore, Kir currents were not involved in the CCh-dependent depolarization. Gulledge and Stuart (2005) found that mPFC pyramidal neurons were hyperpolarized during phasic, short-lasting CCh application due to the activation of Ca++-dependent SK potassium channels. However, in our study, the depolarization evoked by the prolonged application of CCh was not modified in the presence of the selective SK channel blocker apamin (100 nM; 8.4 ± 2.7 mV, n = 7, Fig. 4A), suggesting that SK-dependent hyperpolarization did not interfere with the CCh-dependent depolarization analyzed in our study. We tested the possibility that the CCh-induced depolarization depended on the presence of Na+ ions in the extracellular solution. Accordingly, Na+ ions in the extracellular solution were replaced by an equimolar concentration of choline chloride (n = 5) or

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Fig. 3. Effect of the current step, gallein and GRK2i on CCh-dependent depolarization. (Aa) Depolarization evoked by bath application of CCh (CCh, 100 lM, horizontal bar), shown at a timescale of 5 min. Inset: effect of a rectangular current step applied at the peak of the CCh-dependent depolarization on the membrane potential, shown with the expanded timescale (horizontal calibration, 2 s). (b) Effect of the rectangular current step on the membrane potential in the absence of CCh in the extracellular solution, shown with the expanded timescale (2 s, inset). The cell membrane potential was depolarized to the maximum level attained after CCh application (see a). The current steps (100 ms, 500 pA) are marked in the figure. The recordings in a and b were obtained from the same pyramidal neuron. (Ba1) Effect of bath application of the bc subunit blocker gallein (gallein, 20 lM) on the membrane potential recorded before, during and after the application of CCh (CCh, 100 lM). (a2) The mean amplitude of CChdependent depolarization in the absence (control) and presence of gallein (gallein, 20 lM) in the bath. (b1) Effect of the presence of gallein (gallein, 50 lM) in the intracellular solution on the membrane potential recorded before, during and after the application of CCh (CCh, 100 lM). (b2) The mean amplitude of CCh-dependent depolarization in the absence (control) and presence of gallein (gallein, 50 lM) in the intracellular solution. (c1) Effect of the presence of GRK2i (GRK2i, 10 lM) in the intracellular solution on the membrane potential recorded before, during and after the application of CCh (CCh, 100 lM). (c2) The mean amplitude of CCh-dependent depolarization in the absence (control) and presence of GRK2i (GRK2i, 10 lM) in the intracellular solution. The calibration in Bb1 applies to Ba1, Bb1 and Bc1. The horizontal bars mark the application of the compounds.

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N-methyl-D-glucamine (NMDG, n = 3). In the absence of Na+ ions, CCh-dependent depolarization was abolished (Fig. 4Ba), and 2.1 min after the start of CCh washout (the time point at which maximal CCh-dependent depolarization occurred under control conditions), the membrane potential was 1.09 ± 0.58 mV (n = 8, Fig. 4Bb). The result described above indicates that the CChinduced depolarization depends on the inhibition of constitutively active Na+-dependent K+ currents or on the activation of inward Na+ currents. If this is the case, CCh-induced inhibition of the Na+-dependent K+ currents would be abolished under conditions of Na+-free extracellular solution because in this condition, Na+-dependent K+ currents are inactivated. mPFC pyramidal neurons have been shown to express Na+-dependent K+ currents, which are controlled by muscarinic receptors (Santi et al., 2006). A common test for the involvement of Na+-dependent K+ currents is to

measure the effect of replacing Na+ ions with Li+ ions in the extracellular solution, which leads to the replacement of Na+ ions with Li+ ions in the cytoplasm. In the presence of Li+ ions, Na+-dependent K+ currents are inactivated, as in the absence of Na+ ions (Dryer et al., 1989; Bischoff et al., 1998; Kaczmarek, 2013). We found that following the replacement of Na+ ions with Li+ ions, the CCh-dependent depolarization was 9.2 ± 1.1 mV (n = 5) and did not differ from the control depolarization (p > 0.05; Fig. 4C). Hence, Na+-dependent K+ currents could not be responsible for the CCh-dependent depolarization. Next, we tested the possibility that the CCh-dependent depolarization was due to the activation of an inward Na+ current that was TTX-resistant and that could be activated from the level of the resting membrane potential, as suggested previously by Haj-Dahmane and Andrade (1996). Numerous known inward Na+ currents meet these criteria, including

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Fig. 4. Membrane effector responsible for CCh-dependent depolarization in mPFC pyramidal neurons. (A) Effects of blockers of Kir channels (Ba++, 200 lM) and Ca++-dependent SK potassium channels (apamin, 100 nM) on the amplitude of CCh-dependent depolarization. (Ba) Original membrane potential recordings before, during and after CCh application (100 lM, black horizontal bar) when NaCl in the extracellular solution was replaced with choline chloride (choline chloride). (b) The average amplitude of CCh-dependent depolarization in the presence of Na+ ions (control) and after the replacement of Na+ ions in the extracellular solution with choline chloride or NMDG (choline chloride/NMDG). (C) Effects of Li+ ions (130 mM NaCl in the extracellular solution replaced by 130 mM LiCl), non-selective cation current blockers (2-APB, 200 lM; flufenamic acid, FFA, 200 lM) or a transient receptor potential channel blocker (SKF 96365, 50 lM) on the amplitude of CCh-dependent depolarization. (D) The effects of an Ih channel blocker (ZD 7288, 50 lM) and a blocker of the Na+/Ca++ exchanger (benzamil, 20 lM) on the amplitude of CCh-dependent depolarization.

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Ca++-activated non-selective cation currents in hippocampal neurons (Fisahn et al., 2002), non-selective cationic currents in prefrontal cortical neurons (HajDahmane and Andrade, 1996) and a group of transient receptor potential (TRP) channel currents in the entorhinal cortex (Zhang et al., 2012). We tested the effects of 2-APB (Lievremont et al., 2005) and flufenamic acid, which block non-selective cation currents (Partridge and Valenzuela, 2000), and SKF 96365, which blocks different subtypes of TRP channel currents (Ramsey et al., 2006). When CCh was applied together with 2-APB (200 lM), flufenamic acid (200 lM) or SKF 96365 (50 lM), the amplitude of the CCh-dependent depolarization was 15.1 ± 2.5 mV (n = 6), 14.5 ± 2.0 mV (n = 5), and 12.6 ± 2.3 mV (n = 5), respectively. The depolarizations evoked in the presence of flufenamic acid and SKF 96365 did not differ from the control depolarizations evoked by CCh alone (p > 0.05); however, in the presence of 2-APB, the evoked depolarizations were significantly greater than the control depolarizations evoked by CCh alone (p < 0.05; Fig. 4C). Therefore, the blockers of non-specific cation currents did not suppress the CCh-dependent depolarization; in contrast, 2-APB application augmented the depolarization. Cyclic nucleotide-gated Ih currents are activated during the stimulation of M1 muscarinic receptors (Pian et al., 2007), and these channels are permeable to Na+ and K+ ions (Biel et al., 2009). Therefore, their activation

can evoke cell depolarization due to an increase in inward Na+ currents. However, in the presence of a specific blocker of this current (ZD 7288, 50 lM, Chu et al., 2010), the mean amplitude of the CCh-induced depolarizations (11.0 ± 1.2 mV, n = 8) did not differ from the mean amplitude of the depolarizations evoked by CCh alone (p > 0.05, Fig. 4D). CCh could potentially augment the activity of the Na+/Ca++ exchanger, resulting in a net inward Na+ current, as observed in other cells (Eriksson et al., 2001). However, when the Na+/Ca++ exchanger was blocked by benzamil (20 lM), the amplitude of the CChinduced depolarization was 8.0 ± 2.5 mV (n = 11), which did not differ from the depolarization evoked by CCh alone (p > 0.05; Fig. 4D). Na+ channels are permeable to Li+; however, Li+ ions are not transported by the Na+/Ca++ exchanger. Therefore, the replacement of Na+ with Li+ ions in the extracellular solution would abolish CCh-dependent depolarization if this depolarization were dependent on the activation of the Na+/Ca++ exchanger (Fraser and MacVicar, 1996). Following the replacement of Na+ ions with Li+ ions, the CCh-dependent depolarization did not differ from the control depolarization (p > 0.05; Fig. 4C). These results indicate that the Na+/Ca++ exchanger was not involved in the CCh-evoked depolarization. Because the compounds proposed in the literature that were also tested here were likely not responsible

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for the CCh-dependent depolarization, we continued our search for another membrane effector that could be responsible for the inward Na+ current. The current properties of the Nav1.9 channel closely matched the characteristics of the inward Na+ current supposedly responsible for the CCh-induced depolarization of the mPFC pyramidal neurons. These channels contribute to TTX-insensitive, non-inactivating Na+ currents that have a threshold below the resting membrane potential (DibHajj et al., 2002; Coste et al., 2004, 2007; Liang et al., 2013). Reportedly, Nav1.9 channels are expressed in small DRG neurons (for review Dib-Hajj et al., 2002), in hippocampal pyramidal neurons (Blum et al., 2002), in magnocellular neurosecretory cells of supraoptic nucleus (Black et al., 2014) and ubiquitously throughout the brain (Jeong et al., 2000). Prior to investigating Nav1.9 channel expression in layer V mPFC neurons, we confirmed that Nav1.9 antibodies stained small rat DRG neurons as a positive control (Dib-Hajj et al., 2002). DRG neurons were identified based on labeling for the neuronal marker MAP2

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(green fluorescence in Fig. 5A). As expected, these neurons were also immunopositive for Nav1.9 (red fluorescence, Fig. 5B). Nav1.9 immunoreactivity was also detected in mPFC layer V pyramidal neurons. These neurons were first stained for MAP2 (for example, white arrows, green fluorescence, Fig. 5C) and then with antibodies against Nav1.9 (white arrows, red fluorescence, Fig. 5D). Control experiments with control goat IgG and omission of the primary antibody were also performed on the DRG and mPFC neurons, and significant signal reduction was observed. This finding raised the possibility that Nav1.9 channels might indeed be responsible for the CCh-dependent depolarization of mPFC pyramidal neurons. Chimeric hNav1.9/Kv2.1 currents have previously been demonstrated to be markedly augmented by the tarantula toxin ProTx-I, suggesting that Nav1.9 channel currents are also enhanced in the presence of this toxin (Bosmans et al., 2011; Gilchrist and Bosmans, 2012). The application of ProTx-I (100 nM) alone to the extracellular solution evoked depolarization (8.1 ± 0.6 mV, n = 6; Fig. 6Aa1, Ab, ProTx-I). The application of CCh

Fig. 5. Expression of Nav1.9 channels in the DRG (A, B) and in mPFC pyramidal neurons (C, D). Detection of Nav1.9 channel expression in DRG neurons. DRG neurons were labeled for MAP2 (A, green fluorescence) and Nav1.9 channels (B, red fluorescence). Pyramidal neurons were labeled for MAP2 (C, for example, white arrows, green fluorescence) and Nav1.9 (D, white arrows, red fluorescence). Scale bars: A – 250 lM, C – 50 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Kurowski P et al. Muscarinic receptor control of pyramidal neuron membrane potential in the medial prefrontal cortex (mPFC) in rats. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.07.023

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Fig. 6. Effects of the tarantula toxin ProTx-I and the anti-Nav1.9 antibody on CCh-dependent depolarization. (Aa) Effects of the presence of ProTx-I (100 nM) in the extracellular solution on the membrane potential (1) and amplitude of CCh-dependent depolarization (2). (b) Mean amplitude of the depolarization evoked during the application of CCh alone (control) and ProTx-I alone (ProTx-I) and when CCh was applied during the delivery of ProTx-I (CCh, amplitude marked as ‘‘2’’ in Aa). (B) Effects of the presence of IgG (a, IgG) and anti-Nav1.9 antibodies (b, anti-Nav1.9) in the intracellular solution on CCh-dependent depolarization. The mean amplitude of CCh-dependent depolarization in the presence of IgG (IgG) and anti-Nav1.9 (anti-Nav1.9) antibodies in the intracellular solution (c). The calibration applies to Aa and B. 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680

in the presence of ProTx-I evoked additional depolarization that was greater than the level attained during the application of ProTx-I alone (Fig. 6Aa2, 20.2 ± 2.6 mV for CCh + ProTx-1, n = 6). The amplitude of this depolarization (Fig. 6Aa2, Ab, CCh) was significantly larger than the amplitude of the depolarization evoked by CCh alone (Fig. 6Ab, control, p < 0.0001). This result indicates that ProTx-I evokes depolarization and augments CCh-dependent depolarization in mPFC pyramidal neurons. Next, recordings were performed in the whole-cell configuration after the cell membrane was disrupted via suction instead of using the perforated-patch method. In this experimental condition, after the pyramidal neurons were loaded with normal guinea-pig IgG (4 lg/ml) for 60 min, the application of CCh evoked depolarization in every tested neuron (Fig. 6Ba, c, 14.6 ± 2.5, n = 7). However, after 60 min of cell ‘‘dialysis’’ with the Nav1.9 antibody that was dissolved in the pipette solution (4 lg/ml, anti-Nav1.9), the CCh-dependent depolarization was nearly abolished, and the maximum amplitude of the depolarization was significantly lower than that of the CCh-dependent depolarization measured in the presence of IgG (Fig. 6Bb, c, 0.58 ± 0.29 mV, n = 7, p < 0.001).

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Hyperpolarization evoked in pyramidal neurons

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As indicated above, small-amplitude hyperpolarizations (0.72 ± 0.14 mV, n = 33, Fig. 1Aa, b, c) were

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observed prior to the onset of the CCh-evoked depolarizations. Slight hyperpolarizations were also present after the CCh-dependent depolarization was suppressed, e.g., in the presence of a selective M1 receptor blocker (VU 0255035, 5 lM, 0.86 ± 0.17 mV, n = 7, Fig. 2Ab) and after removing Na+ ions from the extracellular solution (1.06 ± 0.51 mV, n = 8, Fig. 4Bab). In the presence of the M1/M4 blocker (pirenzepine dihydrochloride, 2 lM), neither CCh-dependent depolarization nor the preceding hyperpolarization were observed (Fig. 2Aa). The properties of the CCh-dependent hyperpolarization were not further investigated.

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DISCUSSION

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The results of this study indicate that the depolarization evoked by the stimulation of M1 muscarinic receptors in layer V mPFC pyramidal neurons depends on the opening of Nav1.9 Na+ channels. The signal transduction involved G-protein bc subunits.

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Pharmacology of CCh-dependent depolarization

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CCh activates nicotinic and muscarinic receptors in both pyramidal neurons (Poorthuis et al., 2013) and GABAergic interneurons (Aracri et al., 2010) of the mPFC. In our study, glutamatergic and GABAA receptor blockers were added to the extracellular solution. Moreover, experiments were performed in the presence

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of TTX and in the absence of Ca++ ions in the extracellular solution, which blocked the release of transmitters from axonal endings. Therefore, the pyramidal neurons tested were isolated from any synaptic input. CCh-dependent depolarization was completely abolished in the presence of the M1/M4 muscarinic receptor antagonist pirenzepine dihydrochloride (Moriya et al., 1999) or the recently introduced, highly selective M1 muscarinic receptor blocker VU 0255035 (Sheffler et al., 2009; Shirey et al., 2009; Weaver et al., 2009). Together with the results of earlier studies (Haj-Dahmane and Andrade, 1996, 1998; Carr and Surmeier, 2007; Gulledge and Stuart, 2005; Gulledge et al., 2007, 2009), these results indicate that muscarinic receptors are responsible for CCh-dependent depolarization in mPFC pyramidal neurons. These results are also consistent with the finding that M1 muscarinic receptors are preferentially expressed postsynaptically in the cortex (Wei et al., 1994; Flynn et al., 1997; Krejci and Tucek, 2002; Nathanson, 2008). In the present study, the amplitude of CCh-evoked depolarization was not diminished in the presence of a nicotinic receptor blocker, confirming that the properties of M1 muscarinic-dependent depolarization in mPFC pyramidal neurons were assessed. This result is also compatible with the recent finding of Hedrick and Waters (2015), who demonstrated that the control of layer V mPFC pyramidal neurons by nicotinic receptors is insignificant in mice. The transduction pathway involved in M1-dependent depolarization M1 muscarinic receptors couple to G proteins, which in turn control membrane and cellular effectors through the activation of different transduction systems (Felder, 1995). Use of the perforated-patch method, which enables electrical access to the cell while minimizing the exchange of the intracellular milieu with the pipette solution (Akaike and Harata, 1994), was particularly important in the experiments that tested the involvement of intracellular second messengers in the transduction system. In the present study, there was no indication that the two common transduction systems linked to protein kinase A and protein kinase C (Felder, 1995) were responsible for M1-dependent depolarization. The amplitude of depolarization was not affected by blockers of either adenylyl cyclase and protein kinase A or of phospholipase C and protein kinase C, although these blockers were applied in concentrations that typically evoke effects in mPFC neurons in slices (Wang and O’Donell, 2001; Witkowski et al., 2008; Fu et al., 2010; Huang and Hsu, 2010; Szulczyk et al., 2012; Zhang et al., 2012; Bai et al., 2014; Szulczyk, 2015). The effects of metabotropic receptors on ion channels are frequently irreversible (up to a 20-min washout) when intracellular second messengers are involved in the signal transduction and when the cytoplasmic milieu is left intact. We observed that CCh-dependent depolarization was relatively short-lived; the membrane potential returned to the control level 5–10 min after CCh washout. This timing of metabotropic receptor action suggests the involvement of G-protein bc subunits in the transduction

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system (Kaneko et al., 1999). This transduction system is sometimes called ‘‘membrane-delimited’’ to emphasize that its elements are embedded in the cell membrane. Muscarinic receptors have already been demonstrated to control cellular effectors within the plasma membrane through the release of bc subunits from G proteins (Herlitze et al., 1996; Nemec et al., 1999; Olianas and Onali, 2000). The control of Ca++ and K+ channels by bc subunits has been well documented and described as voltage dependent because a strong depolarizing pulse temporarily abolishes this transduction pathway due to the disconnection of bc subunits from these channels (for review Kaneko et al., 1999; Dascal, 2001). In half of the pyramidal neurons tested, we found that a current step partially and temporarily abolished CCh-dependent depolarization, suggesting bc subunit involvement in the transduction system. In the remaining tested neurons, the current step did not abolish CCh-dependent depolarization. The absence of an effect in some neurons may have been caused by a relatively high access resistance because our recordings were performed using the perforated-patch method. Under these conditions, the current step did not always evoke sufficiently rapid depolarization to disrupt the connection between bc subunits and the structure responsible for depolarization in all of the tested neurons. Recently, gallein, a membrane-permeable compound that inhibits G-protein bc subunit-dependent signaling, was identified (Bonacci et al., 2006; Lehmann et al., 2008; Irannejad et al., 2010; Belkouch et al., 2011; Ukhanov et al., 2011; Kodama and Togari, 2013; Schwetz et al., 2013) and shown to dock to a site on the bc subunit that mediates its interaction with effector proteins. Furthermore, gallein does not interfere with other G-protein-dependent transduction systems (Bonacci et al., 2006; Lehmann et al., 2008). In our study, CCh-dependent depolarization was greatly diminished or abolished in the presence of gallein in the extra- or intracellular solution, respectively, indicating that a bc subunitdependent mechanism is responsible for signal transduction. CCh-dependent depolarization was also attenuated in the presence of GRK2i in the intracellular solution; GRK2i is a peptide analog of the G-protein receptor kinase (GRK2) that acts as a G-protein bc subunit antagonist (Koch et al., 1994; Stott et al., 2015). Overall, CCh-dependent depolarization was not affected by blockers of transduction systems linked to G-protein a subunits, whereas CCh-dependent depolarization was diminished or abolished by inhibitors of bc subunit-dependent transduction systems. These findings suggest that G-protein bc subunits are involved in the transduction of signals from M1 muscarinic receptors and are responsible for CCh-dependent depolarization in mPFC pyramidal neurons.

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Effector responsible for M1-dependent depolarization

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The results of our study and the results of the study of Haj-Dahmane and Andrade (1996) suggest that an inward

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Na+ current is responsible for CCh-dependent depolarization. These results also indicate that activation of the Na+ component of non-selective cation channels (Haj-Dahmane and Andrade, 1996; Fishan et al., 2002), TRP channels (Zhang et al., 2012) or Ih channels (Thuault et al., 2013) was not responsible for CChdependent depolarization. However, in contrast to the expected results, 2-APB, which was applied to block TRP channels, slightly increased the amplitude of CChdependent depolarization. Several types of TRP channels exist. TRPC-type channels are inhibited by 2-APB (Bouhadfane et al., 2013), whereas TRPV types are activated by 2-APB (Xu et al., 2006). Because TRPV channels are also expressed in the mPFC (Fogac¸a et al., 2012), an increase in the amplitude of CCh-dependent depolarization during 2-APB application may depend on TRPV channel activation. The Na+/Ca++ exchanger (Eriksson et al., 2001) was also found to not be responsible for CCh-dependent depolarization. The cellular effector that may be responsible for CCh-dependent depolarization is the Nav1.9 channel. This channel is selectively permeable to Na+ ions and has a voltage threshold close to or below the resting membrane potential. This channel is also TTX resistant and exhibits minimal inactivation over time. Reportedly, Nav1.9 channels are predominantly expressed in small DRG neurons (Dib-Hajj et al., 2002; Coste et al., 2004; Liang et al., 2013; Vanoye et al., 2013), although these channels are also found in the central nervous system (Jeong et al., 2000; Blum et al., 2002; Black et al., 2014). The following arguments suggest that M1-induced depolarization depends on the activation of Nav1.9 channels in mPFC pyramidal neurons: (1) The present study demonstrated that Nav1.9 channels are expressed in mPFC pyramidal neurons. (2) The present study demonstrated that the amplitude of CCh-dependent depolarization doubled in the presence of the tarantula toxin ProTx-I, and other studies have demonstrated that chimeric hNav1.9/Kv2.1 currents are augmented by ProTx-I (Bosmans et al., 2011; Gilchrist and Bosmans, 2012). (3) Intracellular application of antibodies against Nav1.9 channels abolished CCh-dependent depolarization. (4) Previous studies of different cells have demonstrated that the expression of Nav1.9 channels is frequently controlled by G-protein bc subunits (Ma et al., 1997; Mantegazza et al., 2005; Belkouch et al., 2011; Liang et al., 2013; Vanoye et al., 2013).

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CCh-dependent hyperpolarization

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Gulledge and Stuart (2005) observed hyperpolarization in response to phasic (short-lasting) CCh application; however, depolarization was observed during tonic CCh application in mPFC pyramidal neurons. In the present study, prolonged (2.5 min) application of CCh evoked a prominent depolarization in mPFC pyramidal neurons that was preceded by a small (below 1 mV) hyperpolarization. This hyperpolarization was blocked by the M1/M4 receptor blocker and was not affected by the M1 receptor

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blocker, suggesting that this hyperpolarization might depend on M4 muscarinic receptor activation.

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Significance of M1 muscarinic receptor-dependent control of Nav1.9 channels in mPFC pyramidal neurons

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In a wide array of neuropsychiatric disorders, including Alzheimer’s disease, senile dementia and schizophrenia, cognitive function is impaired; this impairment has been linked to reduced cholinergic input to prefrontal cortex neurons. Consistent with this hypothesis, decreased cholinergic muscarinic input to the prefrontal cortex impairs cognitive function, whereas increased input improves cognitive function (Hasselmo and Sarter, 2011). Unfortunately, direct activation of muscarinic (presumably M1) receptors is impractical due to the numerous serious side effects evoked by the activation of these receptors. The results of the present study indicate that M1 muscarinic receptor-dependent depolarization of layer V pyramidal neurons depends on the G-protein bc subunit-dependent transduction system and the activation of Nav1.9 channels, suggesting that clinically beneficial effects similar to those of M1 receptor stimulation can be achieved in pathologies involving cognitive impairment through the stimulation of the bc subunit-dependent transduction system and/or Nav1.9 channels. M1 muscarinic receptor activation may also play a key role in epileptogenesis. Inhibiting (Gigout et al., 2012) or eliminating these receptors in knock-out mice (Hamilton et al., 1997) has been shown to suppress CCh- or pilocarpine-dependent epileptic discharges. Therefore, blockers of Nav1.9 channels could be useful in the treatment of epilepsy.

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Acknowledgments—This study was sponsored by grant nos: NN401584638 and NN301572940. We thank Dr. Ewa Nurowska for the helpful comments regarding the statistical analyses.

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(Accepted 8 July 2015) (Available online xxxx)

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