Nicotine Enhances Firing Activity of Layer 5 Pyramidal Neurons in the Medial Prefrontal Cortex through Inhibition of Kv7 Channels

Abstract

Nicotine enhances attention, working memory and recognition. One of the brain regions associated with these effects of nicotine is the medial prefrontal cortex (mPFC). However, cellular mechanisms that induce the enhancing effects of nicotine remain unclear. To address this issue, we performed whole-cell patch-clamp recordings from mPFC layer 5 pyramidal neurons in slices of C57BL/6J mice. Shortly (approx. 2 min) after bath application of nicotine, the number of action potentials, which were elicited by depolarizing current injection, was increased, and this increase persisted for over 5 min. The effect of nicotine was blocked by the α4β2 nicotinic acetylcholine receptor (nAChR) antagonist dihydro-β-erythroidine, α7 nAChR antagonist methyllycaconitine, or intracellular perfusion with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Additionally, the voltage-dependent potassium 7 (Kv7) channel blocker, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991), as well as nicotine, shortened the spike threshold latency and increased the spike numbers. By contrast, the Kv7 channel opener, retigabine reduced the number of firings, and the addition of nicotine did not increase the spike numbers. These results indicate that nicotine induces long-lasting enhancement of firing activity in mPFC layer 5 pyramidal neurons, which is mediated by the stimulation of the α4β2 and α7 nAChRs and subsequent increase in intracellular Ca²⁺ levels followed by the suppression of the Kv7 channels. The novel effect of nicotine might underlie the nicotine-induced enhancement of attention, working memory and recognition.

INTRODUCTION

Nicotine enhances attention, working memory and recognition,^1–3) and improves symptoms of major neuropsychiatric disorders, such as Alzheimer’s disease, schizophrenia, and attention-deficit hyperactivity disorder.^1,4) These beneficial effects are mediated by nicotinic acetylcholine receptors (nAChRs). The most abundant and widely expressed nAChR subtypes in the brain are the heteromeric α4β2 and the homomeric α7 nAChRs.^5,6) The former has a high affinity for nicotine and desensitizes slowly, while the latter exhibits a low affinity for nicotine and rapid desensitization.^7–10) nAChRs are distributed both presynaptic and postsynaptic sites, regulating neurotransmitter release and neuronal excitability.^5,6,11)

Several brain regions are considered to be the sites of action of nicotine to exert the beneficial effects. One of those brain regions is the medial prefrontal cortex (mPFC), a brain region associated with attention, working memory, decision-making and recognition.^12–15) Both α4β2 and α7 nAChRs are expressed in the mPFC in a layer-specific manner; in layers 2/3, nAChRs are mainly expressed in γ-aminobutyric acid (GABA)-ergic interneurons, whereas in the deeper layers 5/6, both glutamatergic pyramidal neurons and GABAergic interneurons, as well as glutamatergic presynaptic terminals, express the nAChRs.^16–18) It has been reported that nicotine-induced enhancements of attention, working memory and recognition are associated with long-lasting activities of mPFC neurons.^19–22) Considering that the nicotine-induced potentiation of excitatory transmission in mPFC pyramidal neurons readily disappears due to a desensitization of nAChRs,^17,23) it was hypothesized that there exit other mechanism(s) that generates a persistent increase in mPFC neuronal activity. Thus, in the present study, we examined the effect of nicotine on the firing activity of layer 5 pyramidal neurons in the mPFC and investigated the cellular mechanisms underlying the effects of nicotine using whole-cell recordings obtained from mouse brain slices.

MATERIALS AND METHODS

Animals

Male and female C57BL/6J mice (4–6 weeks of age, n = 71) were used in this study. Mice were maintained in a constant ambient temperature (22 ± 2 °C) under a 12-h light/dark cycle with food and water available ad libitum. All experiments were performed with the approval of the Institutional Animal Care and Use Committee at Kanazawa University. All efforts were made to minimize the suffering and number of animals used in this study.

Drugs

DL-2-Amino-5-phosphonopentanoic acid (AP5; Tocris, Bristol, U.K.), (−)-Nicotine (Sigma-Aldrich, St. Louis, MO, U.S.A.), mecamylamine hydrochloride (Mec; Sigma-Aldrich), dihydro-β-erythroidine hydrobromide (DHβE; Tocris), methyllycaconitine citrate (MLA; Sigma-Aldrich), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA; Sigma-Aldrich) and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991; Cayman Chemical, MI, U.S.A.) were dissolved in H₂O and stored at −30 °C. Picrotoxin (Sigma-Aldrich), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Alomone Labs, Jerusalem, Israel) and retigabine (RGB; Alomone Labs) were dissolved in dimethyl sulfoxide (DMSO) and stored at −30 or −80 °C. Stock solutions were diluted with the recording solution (final concentration of DMSO was 0.1% for bath application of picrotoxin, CNQX and RGB).

Slice Preparation, Electrophysiology, and Histology

Mice were anesthetized with isoflurane and decapitated. The brains were submerged in ice-cold modified Ringer’s solution containing (in mM): choline chloride, 125; KCl, 4.0; NaH₂PO₄, 1.25; MgCl₂, 7.0; CaCl₂, 0.5; NaHCO₃, 26; glucose, 20; ascorbate, 1.0; and pyruvate, 3.0; and bubbled with 95% O₂/5% CO₂ (pH 7.4). Coronal slices (250 µm thick) including the mPFC were cut with a microslicer (VT1200S; Leica, Wetzlar, Germany) and incubated at 32–34 °C for 15–30 min in standard Ringer’s solution containing (in mM): NaCl, 125; KCl, 2.5; NaH₂PO₄, 1.25; MgCl₂, 1.0; CaCl₂, 2.0; NaHCO₃, 26; and glucose, 25; and bubbled with 95% O₂/5% CO₂ (pH 7.4). The slices were transferred to standard Ringer’s solution at room temperature, then mounted in a recording chamber on a fluorescence microscope (BX-51WI; Olympus, Tokyo, Japan) equipped with an IR camera (IR-1000; Dage-MTI, Michigan City, IN, U.S.A.), and continuously superfused with standard Ringer’s solution at a flow rate of 2.0–2.5 mL/min.

Data were amplified using a MultiClamp 700B amplifier (Molecular Devices; Foster City, CA, U.S.A.) and stored on a computer using the pClamp10 software (Molecular Devices). Whole-cell voltage-clamp recordings were obtained from layer 5 pyramidal neurons in the mPFC by patch pipettes under visual control. Pipettes were prepared from borosilicate glass capillaries and filled with an internal solution containing (in mM): CsOH, 150; CsCl, 5.0; MgCl₂, 2.0; Na₂ATP, 4.0; Na₃GTP, 0.3; ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10; N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 10; and QX-314, 3.0 (pH 7.3 with gluconic acid). For whole-cell current-clamp recordings, we used an internal solution containing (in mM): KOH, 150; KCl, 10; MgCl₂, 2.0; Na₂ATP, 2.0; Na₃ guanosine 5′-triphosphate (GTP), 0.3; EGTA), 0.2; HEPES), 10 and spermine, 0.1 (pH 7.3 with gluconic acid). To identify and stain the recorded neurons, Alexa Fluor 594 (0.02 mM; Thermo Fisher Scientific, Waltham, MO, U.S.A.) and biocytin (1–3 mg/mL; Sigma-Aldrich) were dissolved in the internal solution. The input resistance was 3–9 MΩ in the Ringer’s solution. All recordings were performed at 32–34 °C.

To record spontaneous excitatory postsynaptic currents (sEPSCs), the membrane potential was voltage-clamped at −70 mV in the presence of the GABA_A receptor antagonist picrotoxin (50 or 100 µM). The frequency and amplitude of sEPSCs were continuously measured before and after nicotine (1 µM) bath application and averaged every 10 s. To evaluate changes in membrane excitability, a series of 400-ms depolarizing current pulses (40 pA steps, 0 to +360 pA) were injected, and the numbers of spikes were counted in the current-clamp recordings. At the start of a recording, the membrane potential was adjusted at approximately −70 mV by current injections in the presence of picrotoxin (50 µM) and the ionotropic glutamate receptor blocker kynurenic acid hydrate (KYNA; 1 or 2 mM, Tokyo chemical industry; Tokyo, Japan). In some experiments, CNQX (10 µM) and AP5 (50 µM) were used instead of KYNA. The numbers of spikes were measured in two time periods during nicotine application; shortly (30–120 s; Nic (S)) and over 300 s (Nic (L)) after initiation of the nicotine bath application. In preliminary examinations, we observed that the effects of nicotine on firing activity of mPFC pyramidal neurons are similar between male and female mice (data not shown). Thus, we pooled data from both sexes in the results. To investigate the mechanisms of the observed nicotine effects, Mec (10 µM), DHβE (1 µM), MLA (100 nM), XE-991 (5 µM), or RGB (5 µM) was bath-applied, or BAPTA (10 mM) was included in the recording pipette. The latency to threshold was measured from the start of the depolarizing current injection to the time point of the spike threshold of the first action potential.

To monitor the membrane input and series resistance, a hyperpolarizing pulse (−5 mV, 30 ms) was applied through the patch-clamp electrode. Recordings in which either parameter was altered by >20% during the course of the recording were excluded from the analysis.

The recorded slices were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) at 4 °C. After three rinses with 0.05 M PBS, the slices were incubated in 0.6% H₂O₂ in methanol for 30 min at room temperature, eliminating endogenous peroxidase activity. After three rinses with 0.05 M PBS, the slices were incubated for 3 h in an avidin-biotin-peroxidase complex using the R.T.U. ABC Reagent kit (Vector Laboratories; Burlingame, CA, U.S.A.). The slices were rinsed in 0.05 M Tris–HCl (pH 7.5) and reacted with 0.05% 3,3′-diaminobenzidine (Nacalai; Kyoto, Japan) solution. When biocytin-filled cells were stained brown, the reaction was stopped by rinsing with 0.05 M PBS. Biocytin-labeled cells were recognized under a microscope equipped with bright-field optics (BZ-9000; Keyence, Osaka, Japan). Layer 5 pyramidal neurons were identified by the following characteristics: 350–550 µm distance of the soma from the midline, pyramidal-shaped soma, and apical dendrites.

Statistical Analyses

Data were expressed as the mean ± standard error of the mean (S.E.M.) and analyzed by one-way repeated measures ANOVA with the Bonferroni post hoc test, two-way repeated measures ANOVA, two-way repeated measures ANOVA with the Holm–Sidak post hoc test, and paired t-test using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, U.S.A.). Differences with p < 0.05 were considered statistically significant.

RESULTS

Nicotine Transiently Enhances Excitatory Synaptic Transmission in mPFC Layer 5 Pyramidal Neurons

Previous studies demonstrated that nicotine increases synaptic transmission through the activation of presynaptic nAChRs^23,24) and this short-lived effect vanishes due to desensitization of the nAChRs.^23,25) Thus, we first confirmed the nicotine effects on excitatory synaptic transmission in mPFC layer 5 pyramidal neurons. Nicotine (1 µM) significantly increased the frequency of sEPSCs approximately 30 s after bath application, and this significant increase continued for about 80 s (Figs. 1A–C). However, nicotine did not affect the sEPSC amplitude (Figs. 1A, B, D). These results indicate that nicotine enhances the excitatory synaptic transmission in mPFC layer 5 pyramidal neurons, but this enhancement quickly decays probably due to a desensitization of nAChRs.

Fig. 1. Nicotine (Nic) Transiently Enhances the Postsynaptic Excitatory Synaptic Transmission in Medial Prefrontal Cortex Layer 5 Pyramidal Neurons

(A) Representative trace showing spontaneous excitatory postsynaptic currents (sEPSCs) before and during bath application of nicotine (1 µM). (B) Expansion traces of the marks in (A) showing sEPSCs before (1) and 60 s (2), 180 s (3) and 280 s (4) after bath application of nicotine. (C, D) Summary graphs showing the effects of nicotine on sEPSC frequency (C) (F_30,150 = 7.57 p < 0.0001, n = 6 from 5 mice) and amplitude (D). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way repeated measures ANOVA followed by the Bonferroni post hoc test).

Nicotine Increases the Firing Activity of mPFC Layer 5 Pyramidal Neurons

We next tested the effects of nicotine on the firing activity of mPFC layer 5 pyramidal neurons with depolarizing current injections during two time periods: shortly (30–120 s; Nic (S)) and over 300 s (Nic (L)) after nicotine bath application (Fig. 2A), corresponding to the time before and after desensitization of the nAChRs, respectively. Our data demonstrate that nicotine significantly increased the number of spikes during the Nic (S) period (Figs. 2B, C). Besides, nicotine still increased the number of spikes during the Nic (L) period, and this increase was significantly larger than that observed during the Nic (S) period (Figs. 2B, C). These results indicate that nicotine increases the firing activity even after the induction of nAChR desensitization in mPFC layer 5 pyramidal neurons.

Fig. 2. Nic Increases the Firing Activity of Layer 5 Pyramidal Neurons in the Medial Prefrontal Cortex Even after Desensitization of Nicotinic Acetylcholine Receptors

(A) Drug application schedule. (B) Representative traces showing firing responses to depolarizing step current injections before (Con, left) and 30–120 s (Nic (S), middle) and 300 s (Nic (L), right) after bath application of nicotine (1 µM). (C) Summary graph showing the effects of nicotine on the number of spikes evoked by depolarizing step currents (interaction, F_16,496 = 7.41, p < 0.0001; current, F_8,248 = 823, p < 0.0001; drug effect, F_2,62 = 32.6, p < 0.0001, n = 32 from 26 mice). ** p < 0.01, **** p < 0.0001 (two-way repeated measures ANOVA followed by the Holm–Sidak post hoc test). KYNA, kynurenic acid; Pic, picrotoxin.

The Nicotine-Induced Increase in Firing Is Mediated by Activation of α4β2 or α7 nAChRs and Subsequent Intracellular Ca²⁺ Increase

We next investigated the neural mechanisms underlying the nicotine-induced enhancement of the firing rate. We first examined the involvement of nAChRs (Fig. 3A). Bath application of Mec (10 µM) did not affect the number of spikes (Figs. 3B, C). Under this condition, nicotine had no significant effect on the number of spikes (Figs. 3B, C). Similarly, in the presence of DHβE (1 µM) or MLA (100 nM), nicotine did not affect the number of spikes (Figs. 3D–G). We confirmed that neither DHβE nor MLA affected the number of spikes (Figs. 3D–G). These results indicate that the nicotine-induced enhancement of firing activity is mediated by α4β2 and α7 nAChRs.

Fig. 3. α4β2 and α7 Nicotinic Acetylcholine Receptors Contribute to the Nic-Induced Excitatory Effects

(A) Drug application schedule. (B) Representative traces showing firing responses to 160 pA (upper) and 320 pA (lower) depolarizing step current injections before (Con, left), after bath application of 10 µM Mec (middle) and 300 s after addition of 1 µM nicotine (Mec + Nic, right). (C, E, G) Summary graphs showing the effects of nicotine on the number of spikes evoked by depolarizing step currents in the presence of Mec (interaction, F_16,80 = 0.615, p = 0.863; current, F_8,40 = 73.7, p < 0.0001; drug effect, F_2,10 = 2.15, p = 0.167, n = 6 from 6 mice) (C), DHβE (interaction, F_16,128 = 1.19, p = 0.287; current, F_8,64 = 242, p < 0.0001; drug effect, F_2,16 = 1.76, p = 0.204, n = 9 from 7 mice) (E), and MLA (interaction, F_16,96 = 1.00, p = 0.462; current, F_8,48 = 161, p < 0.0001; drug effect, F_2,12 = 0.0709, p = 0.932, n = 7 from 6 mice) (G). (D) Representative traces showing firing responses to 160 pA (upper) and 320 pA (lower) depolarizing step current injections before (Con, left), after bath application of 1 µM DHβE (middle), and 300 s after addition of 1 µM nicotine (DHβE + Nic, right). (F) Representative traces showing firing responses to 160 pA (upper) and 320 pA (lower) depolarizing step current injections before (Con, left), after bath application of 100 nM MLA (middle), and 300 s after addition of 1 µM nicotine (MLA + Nic, right). AP5, DL-2-Amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DHβE, dihydro-β-erythroidine; KYNA, kynurenic acid; Mec, mecamylamine; MLA, methyllycaconitine; n.s., not significant; Pic, picrotoxin.

Since both α4β2 and α7 nAChRs are known to be permeable to Ca²⁺,^26,27) we next tested the possible involvement of an elevation of intracellular Ca²⁺ levels in the nicotine-induced increase in firing activity. When the internal solution contained the Ca²⁺ chelator BAPTA (10 mM), the increase in spike number following nicotine administration was not observed (Figs. 4A, B), indicating that the increase in intracellular Ca²⁺ concentration plays a critical role in the excitatory effect of nicotine.

Fig. 4. Intracellular Calcium Increase Contributes to the Nic-Induced Excitatory Effects

(A) Representative traces showing firing responses to 160 pA (upper) and 320 pA (lower) depolarizing step current injections before (BAPTA, left) and 300 s after bath application of 1 µM nicotine (BAPTA + Nic, right) recorded with a BAPTA-containing electrode. (B) Summary graph showing the effects of nicotine on the number of spikes evoked by depolarizing step currents in the presence of BAPTA (interaction, F_16,80 = 3.07, p = 0.000500; current, F_8,40 = 110, p < 0.0001; drug effect, F_2,10 = 0.00230, p = 0.998, n = 6 from 3 mice). n.s., not significant. BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N,′N′-tetraacetic acid.

The Nicotine-Induced Increase in Firing Is Mediated by Inhibition of Kv7 Channels

To further investigate the mechanisms of nicotine-induced increase in excitability, we focused on the timing of action potential initiation. Nicotine significantly shortened the latency of the threshold membrane potential for spikes (Figs. 5A, D). Because a previous study showed that the Kv7 blocker XE-991 reduces firing latency,²⁸⁾ we examined the involvement of Kv7 channels in the nicotine-induced reduction of firing latency. Bath application of XE-991 (5 µM) significantly shortened the latency (Figs. 5B, E). Moreover, in the presence of XE-991, the addition of nicotine did not further shorten the threshold latency (Figs. 5B, E). On the other hand, bath application of the Kv7 channel opener RGB (5 µM) did not affect the threshold latency (Figs. 5C, F), and in the presence of RGB, the addition of nicotine did not change the threshold latency (Figs. 5C, F). These results indicate the involvement of Kv7 channels in the nicotine-induced shortening of the spike threshold latency.

Fig. 5. Kv7 Channels Contribute to the Nic-Induced Reduction in Threshold Latency

(A–C) Representative traces showing the first spike induction before (Con) and after bath application of nicotine (1 µM) (A), 5 µM XE-991 and XE-991 with nicotine (XE-991+ Nic) (B), and 5 µM retigabine (RGB) and RGB with nicotine (RGB + Nic) (C). (D–F) Summary graphs showing the effect of nicotine alone (t₄ = 3.27, p = 0.0310, n = 5 from 5 mice), * p < 0.05 (paired t-test) (D), in the presence of XE-991 (F_2,8 = 9.236, p = 0.0083, n = 5 from 4 mice), * p < 0.05 (one-way repeated measures ANOVA followed by the Bonferroni post hoc test) (E), and in the presence of RGB (F_2,8 = 0.0810, p > 0.999, n = 6 from 4 mice) (F) on the latency to threshold. XE-991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride; RGB, Retigabine.

Next, we examined whether Kv7 channels are associated with the nicotine-induced enhancement of firing activity (Fig. 6A). Bath application of XE-991 significantly increased the number of spikes (Figs. 6B, C), and in the presence of XE-991, the nicotine-induced increase in spike number was not observed (Figs. 6B, C). On the other hand, bath application of RGB significantly decreased the number of spikes (Figs. 6D, E), and in the presence of RGB, the nicotine-induced increase in spike number was also not observed (Figs. 6D, E). These findings indicate that the nicotine-induced excitatory effect on membrane excitability is mediated by the inhibition of Kv7 channels.

Fig. 6. Kv7 Channels Contribute to the Nic-Induced Excitatory Effects

(A) Drug application schedule. (B) Representative traces showing firing responses to 160 pA (upper) and 320 pA (lower) depolarizing step current injections before (Con, left), after bath application of 5 µM XE-991 (middle), and 300 s after addition of 1 µM nicotine (XE-991 + Nic, right). (C) Summary graph showing the effects of nicotine on the number of spikes evoked by depolarizing step currents in the presence of XE-991 (interaction, F_16,64 = 5.41, p < 0.0001; current, F_8,32 = 206, p < 0.0001; drug effect, F_2,8 = 11.5, p = 0.00440, n = 5 from 4 mice). (D) Representative traces showing firing responses to 160 pA (upper) and 320 pA (lower) depolarizing step current injections before (Con, left), after bath application of 5 µM retigabine (RGB; middle), and 300 s after addition of 1 µM nicotine (RGB + Nic, right). (E) Summary graph showing the effects of nicotine on the number of spikes evoked by depolarizing step currents in the presence of RGB (interaction, F_24,120 = 15.4, p < 0.0001; current, F_8,40 = 298, p < 0.0001; drug effect, F_3,15 = 30.5, p < 0.0001, n = 6 from 4 mice). * p < 0.05, ** p < 0.01, **** p < 0.0001 (two-way repeated measures ANOVA followed by the Holm–Sidak post hoc test). KYNA, kynurenic acid; n.s., not significant; Pic, picrotoxin. XE-991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride; RGB, Retigabine.

DISCUSSION

The main findings of the present study are as follows: (1) bath application of nicotine induced long-lasting enhancement of the firing activity of mPFC layer 5 pyramidal neurons, and this effect was blocked by Mec, DHβE and MLA, as well as intracellular perfusion of BAPTA; (2) bath application of XE-991, as well as nicotine, shortened the spike threshold latency and increased the number of firings, and the addition of nicotine to XE-991 did not further affect the latency and the number of firings; and (3) bath application of RGB significantly reduced the number of firings, and additional application of nicotine did not increase the number of firings. Together, these results suggest that the stimulation of α4β2 and α7 nAChRs and subsequent increase in intracellular Ca²⁺ levels, which leads to the activation of mPFC pyramidal neurons through the suppression of Kv7 channels.

Our present data and previous studies demonstrated that the nicotine-induced enhancement of excitatory synaptic transmission, probably mediated by the stimulation of α4β2 nAChRs expressed in presynaptic terminals,^16,24) rapidly disappears due to desensitization.^8,24) On the other hand, our current-clamp recordings revealed that bath application of nicotine increases the firing activity over 5 min, implying that this effect of nicotine persists after desensitization of nAChRs. To the best of our knowledge, this is the first report demonstrating the long-lasting modulation of the intrinsic activity of mPFC layer 5 pyramidal neurons by nicotine, although similar persistent activation has been observed in dopaminergic neurons of the ventral tegmental area.²⁹⁾ Given that we recorded the change in firing activity in the presence of KYNA, the nicotine-induced enhancement of the firing may not be due to a secondary response through the activation of ionotropic glutamate receptors, which, in the absence of KYNA, would have been activated by accelerated glutamate release caused by stimulation of presynaptically expressed nAChRs.

A previous study reported the specific expression pattern of nAChRs with an absence of α4β2 nAChRs in mPFC layer 5 pyramidal neurons.¹⁶⁾ On the other hand, we found that DHβE and MLA suppressed the nicotine-induced increase in firing activity. Additionally, Zolles et al. demonstrated that, using voltage-clamp recordings, acetylcholine (ACh)-induced inward currents were inhibited by DHβE and MLA in layer 5 pyramidal neurons of rat frontal cortex.³⁰⁾ Thus, although the discrepancy is not clear at present, it is likely that functionally expressed α4β2 and α7 nAChRs may mediate the nicotine-induced increase in firing activity of mPFC layer 5 pyramidal neurons. It should be noted that DHβE antagonizes not only α4β2 nAChRs but also other subtypes such as α4β4 nAChRs.³¹⁾ Thus, we could not exclude the possible involvement of nAChR subtypes other than α4β2 in the nicotine-induced excitatory effects in mPFC layer 5 pyramidal neurons.

Because firing activity is modulated by a variety of ion channels, including potassium channels, and we observed that the latency to spike threshold is shortened by nicotine, it was hypothesized that voltage-dependent potassium channels might be involved in the nicotine-induced increase in excitability of mPFC layer 5 pyramidal neurons. Kv7, also known as KCNQ, channels are broadly expressed in the brain, including the mPFC.³²⁾ Specifically, Kv7 channels are localized on proximal axon and somatodendritic regions of mPFC layer 5 pyramidal neurons.^28,33) We found that bath application of XE-991 significantly shortened the latency to spike threshold and increased the number of spikes elicited by current injection, consistent with a previous study in rat mPFC layer 5 pyramidal neurons.²⁸⁾ Importantly, the additional application of nicotine after XE-991 administration did not further shorten the spike threshold latency nor did it increase firing activity, suggesting that XE-991 and nicotine may both suppress the same Kv7 channels. On the other hand, RGB greatly reduced the number of spikes in mPFC layer 5 pyramidal neurons, and the addition of nicotine did not further increase the spike numbers. These results suggest that the nicotine-induced increase in firing activity in layer 5 pyramidal neurons may be at least partly mediated by suppression of Kv7 channels and that the opening of Kv7 channels by RGB inhibits the nicotine-induced increase in excitability.

Previous electrophysiological and immunohistochemical studies indicated that α4β2 nAChRs and Kv7 channels are expressed in mPFC layer 5 pyramidal neurons.^34,35) Thus, although we did not confirm the co-expression of these receptors and channels in the same neurons, together with our present results, it is likely that α4β2 nAChRs and Kv7 channels may be co-expressed in the same mPFC layer 5 pyramidal neurons. Future studies would be necessary to address this issue.

At present, the cellular mechanisms underlying the nicotine-induced suppression of Kv7 channels remain unclear. Given that the nicotine-induced increase in firing activity was not observed when the recording was performed with a BAPTA-containing electrode, elevated intracellular Ca²⁺ levels may be critical. This is supported by previous studies indicating that an intracellular Ca²⁺ increase directly or indirectly suppresses Kv7 channel activity.^36–38) Thus, a nAChR-mediated Ca²⁺ rise and subsequent activation of a signaling pathway might underlie the increased activity of mPFC layer 5 pyramidal neurons.

Although we observed a persistent increase in the firing activity of mPFC layer 5 pyramidal neurons by nicotine, it remains unclear how this increase contributes to the nicotine-induced behavioral changes such as enhancement of recognition memory and attention.^2,39) One possible mechanism is the establishment of spike-timing-dependent plasticity (STDP),⁴⁰⁾ in which the membrane potentials of postsynaptic neurons are critical for the induction of plasticity. Because of the increased firings of layer 5 pyramidal neurons, the probability of spike occurrence immediately after excitatory synaptic transmission might increase. Additionally, the nicotine-induced increase in glutamate release might also contribute to the induction of STDP, although this nicotine effect cannot be very long due to the desensitization of nAChRs. These concomitant effects of nicotine may modulate the probability of STDP induction.

In summary, the current results revealed that nicotine induces a persistent increase in excitability of mPFC layer 5 pyramidal neurons via α4β2 and α7 nAChR-mediated Ca²⁺ influx and suppression of Kv7 channels. This novel effect might underlie the nicotine-induced enhancement of attention, working memory and recognition.

Acknowledgments

This study was supported by Grant-in-Aid for Scientific Research (C) (18K06520 to K.K.) from the Japan Society for the Promotion of Science and Smoking Research Foundation (K.K.).

Conflict of Interest

The authors declare no conflict of interest.

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