Cell-type specific modulation of intrinsic firing ... - Semantic Scholar

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Articles in PresS. J Neurophysiol (August 29, 2007). doi:10.1152/jn.00033.2007 1

Cell-type specific modulation of intrinsic firing properties and

subthreshold

membrane

oscillations

by

the

M(Kv7)-current in neurons of the entorhinal cortex

Running head: Modulation of the M(Kv7)-current in the entorhinal cortex

Motoharu Yoshida* and Angel Alonso†

Department of Neurology and Neurosurgery, Montreal Neurological Institute and McGill University, Montreal, Québec H3A 2B4, Canada

*Corresponding author: Email: [email protected] Present address: Center for Memory and Brain, Boston University, 2 Cummington Street, Boston MA, 02215, United States Phone: 617-358-2769 Fax: 617-358-3296

†

In memoriam, July 6, 2005

Copyright © 2007 by the American Physiological Society.

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ABSTRACT The M-current (current through Kv7 channels) is a low-threshold non-inactivating potassium current that is suppressed by muscarinic agonists. Recent studies have shown its role in spike burst generation and intrinsic subthreshold theta resonance, both of which are important for memory function. However, little is known about its role in principal cells of the entorhinal cortex (EC). In this study, using whole-cell patch recording techniques in a rat EC slice preparation, we have examined the effects of the M-current blockers linopirdine and XE991 on the membrane dynamics of principal cells in the EC. When the M-current was blocked, layer II non-stellate cells (non-SCs) and layer III cells switched from tonic discharge to intermittent firing mode, during which layer II non-SCs showed high-frequency short-duration spike bursts due to increased fast spike after-depolarization (ADP). When three spikes were elicited at 50 Hz, these two types of cells reacted with a slow ADP that drove delayed firing. In contrast, layer II stellate cells (SCs) and layer V cells never displayed intermittent firing, bursting behavior and delayed firing. Under the M-current block, intrinsic excitability increased significantly in layer III and V cells but not in layer II SCs and non-SCs. The M-current block also had contrasting effects on the subthreshold excitability, greatly suppressing the subthreshold membrane potential oscillations in layer V cells but not in layer II SCs. The modulation of the M-current, therefore, shifts the firing behavior, intrinsic excitability and subthreshold membrane potential oscillations of EC principal cells in a cell-type dependent manner.

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INTRODUCTION The M-current is a voltage-gated potassium current suppressed by muscarinic agonists that was first described in frog sympathetic neurons (Brown and Adams 1980). Since then, the M-current has been shown to be present in many other cell types including neurons in the central nervous system (reviewed by Brown 1988). It is now known that M-channels belong to the Kv7 (KCNQ) gene family, are mainly composed of Kv7.2 and Kv7.3 subunits in an hetero-multimetric complex (Kv7.2/Kv7.3; Wang et al. 1998) with additional contributions from Kv7.2 homomers and Kv7.5/Kv7.3 heteromers (Hadley et al. 2003; Shah et al. 2002), and are ubiquitous in the brain (Cooper et al. 2001). The low-threshold (below -60 mV), slowly activating and deactivating, and non-inactivating properties of the M-current are suggestive of a role in regulating neural excitability. Using bullfrog sympathetic neurons, Adams et al. (1982a, b) demonstrated that the M-current helps “clamp” the membrane potential near rest, due to its subthreshold activation and persistent nature. Suppression of the M-current by muscarinic agonists or by selective blockers of the M-channels, such as linopirdine (Aiken et al. 1995; Lamas et al. 1997; Costa and Brown 1997; Schnee and Brown 1998) and XE991 (Zaczek et al. 1998) causes an increase in intrinsic excitability. Spike frequency adaptation was reduced in hippocampal pyramidal cells (Cole and Nicoll 1983; Madison and Nicoll 1984; Aiken et al. 1995; Peters et al. 2005), neocortical neurons (McCormick and Williamson 1989), and superior cervical ganglion cells (Wang et al. 1998; but see Romero et al. 2004 and Miles et al. 2005) with M-current suppression. Reduced spike after-hyperpolarization (AHP) in hippocampal CA1 pyramidal neurons (Storm 1989; Gu et al. 2005), superior cervical ganglion (Wang et al. 1998), and rat ventral tegmental

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area dopamine neurons (Koyama and Appel 2006) has also been demonstrated. Two lines of recent studies have shown that the M-current controls membrane dynamics of neurons in addition to its classical role in the control of excitability. Yue and Yaari (2004) have shown that the suppression of the M-current by linopirdine shifts the firing mode of the hippocampal CA1 pyramidal cells from regular firing to burst firing by augmenting the spike after-depolarization (ADP). Hu et al. (2002) and Peters et al. (2005) have shown that suppression of the M-current in hippocampal pyramidal cells reduces intrinsic subthreshold theta resonance. Spike bursts and subthreshold membrane potential oscillations are believed to be important for synaptic plasticity (Magee and Johnston 1997; Thomas et al. 1998) and network oscillation (Buzsáki 2002; Fransén et al. 2004). These studies suggest the importance of the M-current not only in excitability control but also in brain functions such as memory. However, the role of the M-current in the entorhinal cortex (EC) has been investigated to a relatively lesser degree. The EC, located in the temporal lobe between the hippocampus and the cortical mantle, is important for aspects of memory function (Scoville and Milner 1957; Squire and Zola-Morgan 1991; Leonard et al. 1995; Suzuki et al. 1997), including associative memory (Buckmaster et al. 2004) and spatial memory (Steffenach et al. 2005). Principal neurons in the EC receive cholinergic projections from the basal forebrain (Alonso et al. 1996) and cholinergic modulation is crucial for tuning the temporal lobe to mnemonic function (Hasselmo 1999). Being suppressed by cholinergic activation, the M-current could play an important role in the cholinergic regulation of mnemonic function. EC principal cells have layer and cell-type specific electrophysiological phenotypes (Alonso and Klink 1993; Dickson et al. 1997; Hamam et al. 2000), as well as dynamic properties such as subthreshold membrane potential

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oscillations (Alonso and Llinas 1989), delayed firing (Klink and Alonso 1997) and persistent firing (Egorov et al. 2002), all of which are demonstrated to be important for memory function (Fransén et al. 1999; Fransén et al. 2002; McGaughy et al. 2005). Modulation of the dynamic properties of neurons through the M-current might thus be crucial in tuning the activity of the EC for memory function. Moreover, anatomical connections between the other cortical regions and the EC (Insausti et al, 1987; Burwell and Amaral, 1998a; Burwell and Amaral, 1998b; Insausti et al, 1997), and between the EC and the hippocampus (Schwartz and Coleman, 1981; Witter and Amaral, 1991; Tamamaki and Nojyo, 1993; van Groen et al, 1986; Van Groen and Lopes da Silva, 1986; Naber et al, 2001) are restricted to specific layers of the EC. This suggests that layer specific modulation might be important. Using whole-cell patch recording techniques in an EC slice preparation, we examined the effects of the M-current blocker linopirdine (10 µM) and XE991 (5 and 10 µM) on the firing behavior, intrinsic excitability, and subthreshold membrane potential oscillations of stellate cells (SCs) and non-stellate cells (non-SCs) from layer II, and of cells from layers III and V. We found that blockade of the M-current changed the firing pattern of the layer II non-SCs and layer III neurons from regular to intermittent firing. While layer II non-SCs fired in high-frequency short-duration bursts nested in long-duration clustered firing, layer III cells fired regularly in a long-duration cluster during the intermittent firing mode. When three consecutive spikes were evoked at 50 Hz, a slow ADP developed in these two types of neurons resulting in delayed firing. As for the intrinsic excitability, spike frequency adaptation decreased significantly in layer III and V cells but not in layer II cells. Subthreshold membrane potential oscillations in the layer V cells were greatly suppressed while those in layer II SCs remained intact. These results suggest that the M-current shapes the firing behavior, excitability

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and subthreshold membrane potential oscillations in the EC in a cell type specific manner. Some of the results of this paper have been reported in abstract form (Yoshida and Alonso 2005).

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METHODS Slice preparation Experimental protocols were approved by the McGill University Animal Care Committee and the Institutional Animal Care and Use Committee at Boston University, and were in compliance with guidelines of the Canadian Council on Animal Care. Long-Evans rats (postnatal days 21 to 27; Charles River, Quebec or Wilmington, MA) were anesthetized with ketamine/xylazine through intraperitoneal injection and intracardially perfused with ice-cold modified ACSF containing (in mM) 110 choline chloride, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 7 MgCl2, 7 glucose, 3 pyruvic acid and 1 ascorbic acid (pH adjusted to 7.4 by saturation with 95% O2 - 5% CO2). The brain was then removed from the cranium and placed in ice-cold modified ACSF. 350 µm-thick horizontal slices of the hippocampal-entorhinal region were cut using a vibratome (Pelco series 1000, Leica VT 1000S or World Precision Instruments Vibroslicer) and then transferred to a holding chamber, where they were kept submerged for over an hour at room temperature before recording. The holding chamber contained an extracellular solution containing (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1.6 CaCl2, 1.8 MgSO4, 10 glucose (pH adjusted to 7.4 by saturation with 95% O2 - 5% CO2).

Recording Whole-cell recordings were obtained while slices were maintained in a submerged recording chamber at 30 ± 1ºC and perfused with extracellular solution containing the glutamatergic and GABAergic ionotropic receptor blockers kynurenic acid (2 mM) and

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picrotoxin (100 µM), respectively. Stock solutions of linopirdine (10 mM, in ethanol), XE991 (5 mM, in water), XE991 (10 mM, in water), and Apamin (300 µM, in water) were prepared and diluted 1000 times in the extracellular solution to make a final concentration of 10 µM, 5 µM, 10 µM and 300 nM, respectively. Patch pipettes were fabricated from borosilicate glass capillaries by means of a Sutter P-97 or P-87 horizontal puller. The patch pipettes were filled with intracellular solution containing (in mM) 120 K-gluconate, 10 HEPES, 0.2 EGTA, 20 KCl, 2 MgCl, 7 phosphocreatine-diTris, 4 Na2ATP and 0.3 TrisGTP (pH adjusted to 7.3 with KOH). The intracellular solution also contained 0.1 % biocytin for the purpose of labeling. When filled with this solution, the patch pipettes had a resistance of 4-8 M . Slices were visualized with an upright microscope (Nikon E600FN), equipped with a ×60 water-immersion objective lens, Nomarski optics, and a Newvicon camera (Dage-MTI NC-70), or with an upright microscope (Zeiss Axioskop 2), equipped with a ×40 water-immersion objective lens, and a near-infrared charge-coupled device (CCD) camera (JAI CV-M50IR). Each layer was visually distinguished and the location of the cell was confirmed by biocytin staining after recording. Layer II SCs and non-SCs were distinguished from their morphology. Tight seals (>1 G ) were formed on cell bodies and the membrane was ruptured with negative pressure. Current-clamp recordings were made with an Axopatch 1D, Axopatch 200B, or Multi Clamp 700B amplifier (Axon Instruments). Signals were filtered at 10 kHz and sampled at 20 kHz using Clampex 9.0 software (Axon Instruments). Drugs were purchased from Sigma and Tocris.

Data analysis

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Clampfit 9.0 (Axon Instruments), IGOR Pro 5.0 (Wavemetrics) and Matlab (MathWorks) were used for data analysis. Input resistance was measured from the voltage deflection in response to a 5 pA hyperpolarizing current pulse injection at a membrane potential of -60 mV. ADP amplitude was measured as the difference between the peak voltage of ADP and the resting potential. In cells with no clear peak of ADP (mainly layer III and layer V cells), peak voltage was measured at the mean time of the ADP peak of layer II cells (8 ms after the onset of current pulse). To measure the ADP duration and area, the onset of ADP was defined as the time of the fast AHP (indicated by an arrow in Fig. 2(aii)), or the time of the largest deflection (indicated by an arrow in Fig. 2(cii)) if the fast AHP was not evident. The duration of ADP was measured as the time from the onset of ADP to the time the membrane potential returned to the resting potential. The ADP area was measured as the integration of the difference between the membrane potential and the resting potential for the duration of ADP. The AHP amplitude was measured as the difference between the potential at negative peak of AHP and the resting potential. In cells where AHP was only observed in the control, which often happened in layer III and V cells, measurement at the time of peak in control was used as the AHP amplitude under M-current block. For each cell, these values were measured using the average of five traces in each condition. To quantify the development of the slow ADP in Fig. 4, the after-potential area was measured as the integration of the difference between the membrane potential and the resting potential (shown by dashed lines in Fig. 4(aii) - (dii)) from the time of ADP onset to 30s after current injection. The after-potential areas measured in five traces were averaged for each cell. ISI histograms (Fig. 6(biv)-(bvi)) were obtained from the same set of membrane potential traces used for the analysis of the intrinsic excitability. Bin size of the histogram was 20 ms.

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Histograms were normalized by the total number of the ISIs. For analysis of the subthreshold membrane potential oscillations, membrane potential was depolarized to just above firing threshold where subthreshold membrane potential oscillations were most rhythmical (Hamam et al., 2000). The membrane potential was kept at this level for at least 20 s (54 s on average). Because spikes occasionally occurred at this membrane potential, we used a short (3.28s) window of membrane potential that does not include spikes to obtain the power spectrum of the subthreshold membrane potential oscillation without interference from spikes. Our Matlab code obtained power spectra from multiple 3.28s windows by sliding the window by 50 ms intervals throughout the recording period. Power spectra were obtained by FFT using a Hanning window. Any window that contained spikes was eliminated. For each window, the area between 1.53 to 3.97 Hz in its power spectrum was calculated. Three non-overlapping windows with the three largest areas were selected automatically. The frequency range (1.53 to 3.97 Hz) was chosen because the peak frequency of the subthreshold membrane potential oscillations fell into this range. Lower traces in the Fig. 7 (i) and (ii) show examples of windows and Fig. 7 (iii) and (iv) show their power spectra. Three areas from three membrane potential windows were averaged for each condition, and average value and standard error among the cells were plotted in the Fig. 7 (v). The significance was evaluated using a repeated measures ANOVA followed by Tukey post hoc test in the Fig. 8. A paired t test was used in the other figures. Significance level < 0.05 (ns: not significant, *: 0.01 U P < 0.05, **: 0.001 U P < 0.01, ***: P < 0.001) was used. Data are expressed as means ± SEM.

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RESULTS Modulation of intrinsic firing patterns In hippocampal CA1 neurons, blockade of the M-current by linopirdine switches their firing pattern from tonic to burst firing (Yue and Yaari 2004). We, therefore, first explored how the M-current block affected the intrinsic firing pattern of the principal neurons in the EC. Principal cells were categorized into four groups throughout this study; (1) layer II SCs, (2) layer II non-SCs, (3) layer III pyramidal cells and (4) layer V cells (including pyramidal, horizontal and polymorphic cells). Layer V cells were not sub-divided into three groups because there is no clear link between morphological characteristics and electrophysiological characteristics (Hamam et al., 2000). All the recordings were done in the presence of glutamatergic and GABAergic ionotropic receptor blockers. The input resistances of the four groups of cells were 119.4 ± 8.1, 146.0 ± 22.3, 477.2 ± 28.3, 435.5 ± 50.2 M , respectively. The resting membrane potential of the four groups of cells varied from -64.8 to -61.9 mV in the control conditions (Table 1) and no cell showed spontaneous firing. To examine their firing pattern in control conditions, cells were depolarized by constant current injection to just above the threshold level (Fig. 1 (ai)-(di)). Minimum current that could cause at least 5 spikes in 15 sec was chosen. When the cells responded with intermittent firing or bursts of spikes, this current caused at most 31 spikes in 15 sec. In some cases, stronger depolarization was also induced (data not shown). After termination of the current injection, we bath applied 10 µM linopirdine. Linopirdine blocks the Kv7 family (Brown et al. 2002), including the Kv7.2/Kv7.3 channel (Wang et al. 1998), from the extracellular side (Lamas et al. 1997; Costa and Brown 1997), and is 10-fold more selective for the M-current over other K+ channels, such

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as the delayed rectifier, transient and Ca2+ activated K+ channels (Lamas et al. 1997; Schnee and Brown 1998). At this concentration, linopirdine blocks 60 - 90 % of the M-current, depending on cell types (Aiken et al. 1995; Lamas et al. 1997; Costa and Brown 1997; Schnee and Brown 1998). The resting membrane potential slowly depolarized for 15-30 min, and the effect of the blockade was clear 30 min after the linopirdine application. The depolarization of the resting membrane potential often caused spontaneous firing. The membrane potentials in linopirdine were measured in between firing of action potentials and varied from -57.5 to -50.6 mV (Table 1). To compare firing patterns among cell types and control conditions, cells were injected with a depolarizing or hyperpolarizing constant current to maintain the membrane potential just above the threshold level (Fig. 1 (aii)-(dii)) and at higher levels (data not shown). In control conditions, all layer II SCs (n = 8) showed subthreshold membrane potential oscillations (Fig. 1(ai)) with an increase in depolarizing current injection, and fired single action potentials (except one cell that showed doublets) at peaks of subthreshold membrane potential oscillations at just above the threshold level. The average membrane potential between spikes measured as -53.1 ± 0.9 mV. Firing often clustered as shown previously (Alonso and Klink 1993). This mixed mode of subthreshold membrane potential oscillations and firing was observed up to -51.8 ± 1.4 mV (n = 6). Further depolarization caused tonic firing, and subthreshold membrane potential oscillations no longer co-existed with spikes. In linopirdine, the subthreshold membrane potential oscillations and clustered firing remained intact in all eight cells at just above the threshold level (-53.9 ± 0.8 mV; Fig. 1(aii)). The mixed mode was observed up to -52.6 ± 1.0 mV (n = 6). Further depolarization caused tonic firing and no cells switched to spike burst mode. In control conditions, all layer II non-SCs (n = 12) fired regularly, with no prominent

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subthreshold membrane potential oscillations, at just above the threshold level (-53.6 ± 0.8 mV; Fig. 1(bi)) and at more depolarized levels (n = 10). In linopirdine, 75 % (9 out of 12) of layer II non-SCs switched to burst firing at just above the threshold level (-53.0 ± 0.8 mV; Fig. 1(bii)). They tended to generate intermittent firing with long-duration (5.6 ± 0.6 s) clustered firing which consisted of short-duration high-frequency bursts as shown in the inset in Fig. 1(bii). The high-frequency burst was most often a doublet but sometimes consisted of more than three spikes. The highest intra-burst frequency was 45 ± 11 Hz. This nested bursting pattern was observed at membrane potentials up to -50.2 ± 2.1 mV (n = 4), and the high-frequency bursts alone (without intermittent firing) were observed with further depolarization. The remaining (3 out of 12) layer II non-SCs fired regularly in linopirdine. In control conditions, all layer III cells (n = 14) showed regular firing at just above the threshold level (-55.2 ± 0.7 mV; Fig. 1(ci)) and at more depolarized levels (n = 8). In linopirdine, 86 % (12 out of 14) of these cells showed intermittent firing with long-duration (4.3 ± 1.3 s) low-frequency clustered firing at just above the threshold level (-54.9 ± 0.9 mV; Fig. 1(cii)). The highest intra-cluster frequency was 9.2 ± 1.9 Hz. Depolarization during the clustered firing was larger than that in layer II non-SCs resulting in a firing pattern similar to that of up and down states observed in slow-wave sleep (Steriade et al.. 1993). In 33 % (4 out of 12) of the cells, this intermittency resulted in a sinusoidal oscillation. While layer II non-SCs fired in high-frequency bursts, layer III cells did not show high-frequency bursts and fired regularly in the depolarized part of intermittent firing. The intermittent firing of these cells were observed at membrane potentials up to -51.2 ± 0.6 mV (n = 8) and showed tonic firing with further depolarization. The remaining layer III cells (2 out of 14) fired regularly in linopirdine.

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In control conditions, 53 % (9 out of 17) of the layer V cells had clear subthreshold membrane potential oscillations at just above the threshold level (-52.0 ± 0.8 mV). They often fired an action potential at the peaks of the subthreshold membrane potential oscillations (Fig. 1(di)). This mixed mode of subthreshold membrane potential oscillations and spikes was observed up to -45.1 ± 1.0 mV (n = 5) and further depolarization caused tonic firing. The other cells (8 out of 17) fired regularly without subthreshold membrane potential oscillations. In linopirdine, subthreshold membrane potential oscillations were greatly suppressed in all cells (n = 9). 88 % (15 out of the 17) of the cells fired regularly (Fig. 1(dii)) and the rest showed an intermittent firing pattern similar to those of the layer III cells at just above the threshold level (-51.9 ± 0.7 mV). However, none of them showed sinusoidal membrane oscillations. In summary, the M-current block depolarized the membrane potential of all groups of cells. Depolarization was larger (> 10 mV) in the layer III and layer V cells that had larger (> 400 M ) input resistances compared to the layer II cells that had much smaller (< 150 M ) input resistances, suggesting that input resistances of different groups of cells caused the different degree of depolarization. The firing patterns of layer II non-SCs and layer III cells switched from regular to intermittent firing. In addition, while layer III cells fired regularly in the depolarized part of the intermittent firing, layer II non-SCs fired in high-frequency bursts. Finally, layer V cells showed a great suppression of subthreshold membrane potential oscillations. These data suggest that the modulation of the M-current shaped the intrinsic firing pattern of the EC principal neurons in multiple, possibly different ways. To verify that these results are not due to run-down of the M-channels during whole cell recording, we performed control experiments in five cells in all cell groups. In the experiments with linopirdine (above), control recordings were started right after rupturing of

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the membrane and completed usually in 15 min. Linopirdine was applied right after the completion of the control recordings and recordings in linopirdine were started 30 min after the onset of the linopirdine application. Recordings in linopirdine were, therefore, conducted 45 min after the rupturing of the membrane. To perform the control experiments without linopirdine with the same time course as in the linopirdine study, we conducted the first recordings right after the membrane rupturing (corresponding to the control recording) and the second recordings at 45 min after the rupturing of the membrane (corresponding to the recordings in linopirdine). We compared the firing pattern and membrane potential obtained from the first recordings (right after rupturing) and the second recordings (45 min after rupturing). In contrast to the above results in linopirdine, no cell group in these control experiments showed a significant change of membrane potential (data not shown) and no cell showed spontaneous firing. All cell types fired in the same manner as in the control and no cell switched to intermittent or burst firing.

Modulation of single spike after-potential The M-current block enhances ADP (Yue and Yaari 2004) and decreases AHP (Storm 1989; Wang et al. 1998; Gu et al. 2005; Koyama and Appel 2006). Particularly in CA1 pyramidal cells, enhanced ADP reaches the firing threshold and leads to burst firing (Yue and Yaari 2004). We therefore observed single-spike after-potentials from each group of EC cells to investigate their contribution to the burst firing observed above. Short duration (1 ms) depolarizing current pulses, with an amplitude just sufficient to elicit one action potential, were applied in control (grey traces in Fig. 2(ai)-(di)) and in linopirdine (30 min; black traces

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in Fig. 2(ai)-(di)). In order to quantitatively measure and compare ADP and AHP among the cell groups, all recordings were done at a membrane potential of -60 mV. In layer II SCs (n = 7), responses from spikes were very similar in the control conditions and in linopirdine in 4 s traces (Fig. 2(ai)). However, as shown in the magnified fast ADP part (Fig. 2(aii)), ADP amplitude increased significantly with linopirdine (Fig. 2(aiii)). In one of the 7 cells, which showed a doublet in the previous section, the ADP reached spike threshold and one action potential was elicited on top of the ADP, even in the control conditions. The ADP amplitude was not sufficient to reach spike threshold in linopirdine in the other 6 cells. The decay phase of the ADP in linopirdine was almost identical to, or in some cells even faster than, that in the control conditions (Fig. 2(aii)). On average, neither the ADP duration nor the ADP area changed significantly with linopirdine (Fig. 2(aiv) and (av)). The AHP amplitude also did not change significantly with linopirdine (Fig. 2(avi)). In layer II non-SCs (n = 9), the ADP amplitude in linopirdine also increased significantly (Fig. 2(bi)-(biii)). Unlike in layer II SCs, repolarization after the peak of the ADP was usually slower in linopirdine than in control (Fig. 2(bii) and (biv)). This caused an increase in the ADP area (Fig. 2(bv)). The AHP amplitude decreased significantly with linopirdine (Fig. 2(bvi)). In spite of what we expected, the increase in ADP amplitude was not sufficient to reach spike threshold, unlike the data for CA1 pyramidal cells (Yue and Yaari 2004). The shape of the ADP of layer III cells was different from that of layer II cells (Fig. 2(ci) and (cii), n = 11). The ADP amplitude, measured at the average time of the layer II ADP peak, showed the largest increase among all cell types with linopirdine application (Fig. 2(ciii)). 45 % (5 out of 11) of layer III cells did not have AHP, even in the control condition. Lack of an AHP resulted in the longest ADP duration and largest ADP area in control among all cell types (Fig.

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2(civ) and (cv); note use of different scales compared to other cell types). Application of linopirdine further slowed the repolarization, resulting in an even larger ADP reaching 3 s in duration (Fig. 2(ci) and (civ)). The increase in the ADP area (Fig. 2(cv); 9.91 mV·s on average) was the largest among all four cell groups. With the application of linopirdine, the AHP became a large ADP in all cells (Fig. 2(cvi)). The AHP amplitudes of the 5 cells with no AHP in control were measured at the average time of AHP in the other 6 cells. Layer V cells showed significant increases in ADP amplitude, ADP duration, and ADP area (Fig. 2(di)-(dv)). However, the ADP duration and ADP area were much smaller than those of layer III cells in linopirdine, being comparable to those of layer III cells in control conditions. AHP amplitude decreased significantly, often turning into an ADP (Fig. 2(dvi)). The increase of ADP and decrease of AHP were not observed in the control study conducted without linopirdine (data not shown). In fact, some of the parameters showed average changes in control experiments that were in the opposite direction from that caused by linopirdine. These results indicate that the increase of ADP and decrease of AHP were not caused by run-down of the M-current. What is the mechanism for the smaller AHP and larger ADP with blocking of the M-current? One possibility is that the lack of an M-current activated by an action potential reduced the repolarization drive, resulting in a larger ADP. However, the time constant for activation of the M-current is slow, ranging from 6 to 50 ms, depending on the membrane potential (Brown and Adams 1980; Wang et al. 1998), suggesting that the duration of an action potential is too short for full activation. To get some insight into this, we elicited single spikes from -80 mV and -60 mV in layer V cells (n = 6). We found that the ADP did not increase in size with the application of linopirdine when the spike was elicited from -80 mV

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(Fig. 3(a)). ADP duration and ADP area did not change significantly at -80 mV (Fig. 3(b) and (c)). Similar results were also observed in layer III cells (data not shown). If one action potential can activate the M-current, it should be possible to detect an increase in ADP with the application of linopirdine even at -80 mV because the shape of the action potential does not greatly vary at -80 mV and -60 mV. Therefore, the lack of increase in ADP at -80 mV suggests that a single action potential was not able to activate the M-current. The ADP increase and AHP decrease observed at -60 mV are therefore likely attributable to a lack of M-current activation during the ADP as previously proposed (Yue and Yaari 2006) and not during the action potential. To summarize, all cell types showed increases in fast ADP amplitudes and layer III cells showed a long-lasting, large-amplitude ADP in linopirdine. However, despite our above mentioned expectation that enhanced ADP reaches the firing threshold and leads to burst firing, the ADP did not reach spike threshold in any of the cell types not showing burst activity.

Modulation of the after-potential elicited by three spikes To further examine the contribution of the ADP to intermittent firing and burst firing, we elicited three spikes at a more depolarized level (Fig. 4). Using constant current injection, the membrane potential was kept just below the threshold level both in control and in linopirdine. Three 3 ms depolarizing current pulses, with an amplitude sufficient to elicit one spike per pulse, were applied at 50 Hz as shown at the bottom of Fig. 4. In layer II SCs, each of the three spikes elicited an ADP, which became an AHP after the third spike (Fig. 4(ai) and (aii)). The increase of the fast ADP in linopirdine was not enough to

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trigger a spike on top of the ADP, except for one cell that showed a doublet in control. Traces in control and in linopirdine (30 min) were very similar (Fig. 4(aii)), showing almost no effect of the M-current block after three spikes. The total after-potential area for 30 s after stimulation was almost zero, both in control and in linopirdine (Fig. 4(aiii); n = 8), because the membrane potential was close to the resting level after a short AHP (Fig. 4(aii)). In 25 % (3 out of 12) of layer II non-SCs, the increase in fast ADP in linopirdine was large enough to reach spike threshold (indicated by an arrow in Fig. 4(bi)). These 3 cells were the cells that showed the three highest intra burst frequencies (80.8 Hz on average) among all layer II non-SCs when their firing pattern was analyzed in the previous section (Fig. 1). This suggests that the increased fast ADP is the mechanism for the high-frequency burst activity of the layer II non-SCs. The above mentioned 3 and the other 4 layer II non-SCs (7 out of 12; 58% of layer II non-SCs) showed another period of firing more than 500 ms after the stimulation with the application of linopirdine (Fig. 4(bii)). We call this firing “delayed firing”. Interestingly, the delayed firing resembled the depolarized part of the intermittent firing observed in Fig. 1(bii). Although the duration of delayed firing was usually shorter than that of the depolarization, the firing pattern during delayed firing consisted of doublets and triplets just as observed in Fig. 1(b) (Fig. 4(bii) inset). Delayed firing was observed even in control conditions in one of the 7 cells that showed delayed firing in linopirdine. 3 of these 7 cells and 2 other layer II non-SCs (5 out of 12; 41 % of layer II non-SCs) showed a slow ADP which peaked more than 500 ms after stimulation which corresponds well to the time course of the delayed activity. The after-potential area, which was nearly zero in control conditions, increased significantly, due to the increase in slow ADP and delayed firing with the application of linopirdine (Fig. 4(biii); n =

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12). In all layer III cells (n = 11), the fast ADP was not sufficient to elicit firing. Layer III cells had a small, slow ADP even in the control condition (Fig. 4(cii) grey trace) and in 17 % (2 out of 12) of layer III cells the slow ADP was enough to elicit delayed firing. This slow ADP increased and was enough to cause delayed firing in 83 % (10 out of 12) of cells in linopirdine (Fig. 4(cii) black trace). In contrast to the layer II non-SCs, delayed firing did not consist of high-frequency bursts, but of regular firing. The after-potential area increased significantly in linopirdine and was by far the largest among all cell types (Fig. 4(ciii); n = 11; note different scaling). This slow and large depolarization, with regular firing on top of it, resembled the depolarized part of the intermittent firing observed in Fig. 1 (cii). In all layer V cells (n = 11), the fast ADP was not enough to cause action potentials and there was no slow ADP (Fig. 4(di) and (dii)). They had the largest AHP (in area) among all cell types and the AHP decreased with the application of linopirdine, resulting in a significant increase in the after-potential area (Fig. 4(diii); n = 11). In summary, these data indicate that high-frequency bursts in layer II non-SCs can be caused by the increased fast ADP, and the ability of layer II non-SCs and layer III cells to induce slow depolarization might contribute to the formation of the intermittent firing pattern of these cells. In the control studies conducted without linopirdine, no cells showed high-frequency bursts and the slow ADP after three spikes did not develop in any of the cell types. No cell type thus showed a change in after-potential area (data not shown). We next examined the spike number dependency and voltage dependency of the delayed firing in layer III cells. Fig. 5(a) illustrates delayed firing elicited by three spikes from -60 mV in linopirdine (30 min). Increasing the spike number to ten produced a larger negative peak

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between fast and slow ADP, and decreased the amount of delayed firing (Fig. 5(b)). With 20 spikes, delayed firing did not occur in 83 % (5 out of 6) of cells tested, causing only an AHP (Fig. 5(c)). Further increase in the number of elicited spikes caused a larger AHP and no ADP in all 6 cells tested. In Fig. 5(d) and (e), three spikes were elicited from hyperpolarized membrane potentials. The slow ADP was smaller at -65 mV (Fig. 5(d)) and vanished at -70 mV (Fig. 5(e)), showing that the slow ADP is membrane voltage dependent. In the other four cells that caused delayed firing at above -60 mV, hyperpolarizing the membrane potential to -60 mV was enough to stop delayed firing (data not shown). These results suggest that there is an optimal number (3 to 5) of spikes and an optimal membrane potential for eliciting delayed firing.

Intrinsic excitability The role of the M-current in the control of intrinsic excitability of neurons has been an issue of great interest since its discovery. In this study, the effect of the M-current suppression on intrinsic excitability was investigated and compared among the four groups of neurons in the EC (Fig. 6). Neurons were held at -60 mV both in control and in linopirdine (30 min), and a 1s depolarizing current was injected. The amplitude of the current was chosen to elicit at least 8 spikes (ranging from 8 to 15) in the control recordings. The layer II SCs showed similar firing patterns both in control and in linopirdine under current injections (Fig. 6(ai) and (aii)). The number of spikes during the current injections did not increase significantly (Fig. 6(aiii), n = 7). All layer II non-SCs fired tonically in the control (Fig. 6(bi)) and 75 % (6 out of 8) of them switched to high-frequency burst firing in linopirdine (Fig. 6(bii)). The increase in the number

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of spikes was not significant (Fig. 6(biii); n = 8), which could be due to changes in firing patterns. Both of the two cells that did not switch to high-frequency burst firing showed an increase in the number of spikes (8.5 in control and 11 in linopirdine on average). Figure 6(biv) and (bv) show inter-spike interval (ISI) histograms of the 6 layer II non-SCs whose firing pattern switched to high-frequency burst firing in linopirdine. The ISI histogram shows a peak at 100-120 ms in the control condition where the cells were firing tonically (Fig. 6(biv)). After application of linopirdine, The ISI histogram shows a sharp peak at 20-40 ms which corresponds to the intra-burst ISI and smaller peak at 100-120 ms which corresponds to the inter-burst ISI (Fig. 6(bv)). Figure 6(bvi) shows ISI histograms of all 8 layer II non-SCs in the control (open circles) and in linopirdine (filled circles). The layer III cells showed significantly larger depolarization due to the current injection under linopirdine (Fig. 6(ci) and (cii)). The number of spikes increased significantly (Fig. 6(ciii); n = 6), showing the greatest increase in excitability among the four groups. The layer V cells also showed a significant increase in number of spikes (Fig. 6(d)). These results suggest that an increase in intrinsic excitability depends on the cell type in the EC. In control studies conducted without linopirdine, no cell type showed increased intrinsic excitability. In fact, layer V cells showed a decrease of the number of spikes (data not shown).

Roles of the M-current in subthreshold membrane potential oscillations Layer II SCs and layer V cells in the EC show subthreshold membrane potential oscillations. Subthreshold membrane potential oscillations in layer II SCs are known to be

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induced by the interaction between the persistent low-threshold Na+ current (INaP) and the hyperpolarization-activated inward current (Ih; Klink and Alonso 1993; Dickson et al. 2000). In layer V cells, while the involvement of INaP to the subthreshold membrane potential oscillations is clear (Agrawal et al. 2001), the counterpart that is necessary for the generation of subthreshold membrane potential oscillations has not yet been clarified. As some layer V cells that show no sign of Ih still show robust subthreshold membrane potential oscillations (Hamam et al. 2000), the counterpart has been assumed to be the M-current (Hamam et al. 2002). In this series of experiments, the effects of the M-current blockade on subthreshold membrane potential oscillations were analyzed quantitatively. Fig. 7(ai)-(aii) shows subthreshold membrane potential oscillations of the layer II SCs in the control (2.6 ± 0.1 Hz) and 30 min (2.5 ± 0.2 Hz) after linopirdine application, respectively. The membrane potentials were kept constant by injection of a constant current. The subthreshold membrane potential oscillations were clearly seen even 30 min after the application of linopirdine, suggesting that subthreshold membrane potential oscillations in layer II SCs indeed do not depend on the M-current. Power spectrums (Fig. 7(aiii)-(aiv)) were obtained from short (3.28 s) windows of the membrane potential that corresponded to the section between arrows (Fig. 7(ai)-(aii)) in each condition. The peak at 3.4 Hz, which is the main frequency component of the subthreshold membrane potential oscillations for this cell, was kept intact even 30 min after the application of linopirdine. To quantitatively analyze the subthreshold membrane potential oscillations, we obtained three 3.28 s windows of membrane potential traces whose power spectrum had the three largest areas around the peak (1.53 to 3.97 Hz). Three windows were chosen automatically by a Matlab code in each condition and the areas of the three power spectra were averaged (See Methods for detail). The averaged

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area around the peak of the power spectrum did not decrease significantly with linopirdine (Fig. 7(av); n = 6). In contrast, the subthreshold membrane potential oscillations observed in the layer V cells in control (2.4 ± 0.1 Hz) significantly decreased in amplitude after linopirdine application (Fig. 7(bi)-(bii)). The sharp peak in the power spectrum was greatly reduced at 30 min after application of linopirdine (Fig. 7(biii)-(biv)). The area around the peak of the power spectrum decreased significantly 30 min (n = 7) after linopirdine application (Fig. 7(bv)). These results strongly suggest that the subthreshold membrane potential oscillations in layer V cells depend on the M-current. Control studies without linopirdine were conducted in three layer V cells. Subthreshold membrane potential oscillations were not diminished and remained intact in all three cells, even 45 min after onset of the recordings. Areas of power spectrum were: Control: 0.32 ± 0.16 mV2, 45 min: 0.31 ± 0.15 mV2.

Effect of XE991 and SK channel blocker It is reported that linopirdine moderately inhibits the IAHP which could result from small-conductance calcium-activated potassium (SK) current (Schnee and Brown 1998). We, therefore, tested XE991, an M-current blocker which is more specific to M-channels, along with an SK channel blocker apamin, to confirm that the results obtained with linopirdine were not caused simply because of the inhibition of the SK current. Using layer V cells, we first tested the single-spike protocol used in Fig. 2. Bath application of apamin (300 nM) for 15 min did not cause a significant change in any of the measurements of ADP and AHP in all layer V cells (Fig. 8(a), compare black and blue traces;

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n = 9). We then bath applied XE991 (10 µM) and Apamin (300 nM) at the same time and waited for 30 min. This increased all measurements of ADP (amplitude, duration and area) and decreased AHP amplitudes significantly (Fig. 8(a), compare blue and red traces ; n = 9). We encountered two cells where the ADP was large enough to reach the firing threshold and caused a high frequency burst when XE991 and Apamin were applied at the same time (Fig. 8(b)). These results indicate these two cells had a larger effect of SK current and M-current block on the size of ADP. However, since the response was qualitatively different from other 9 cells, these two cells were not included in the statistical analysis (Fig. 8 (aiii) – (avi), (ciii) and (div)). We next tested the three-spike protocol used in Fig. 4. While application of apamin did not increase the after-potential area significantly, further application of XE991 along with apamin increased the after-potential area significantly (Fig. 8(c); n = 9). Intrinsic excitability was measured in the same way as in Fig. 6. While application of apamin did not increase the number of spikes significantly, further application of XE991 along with apamin increased the number of spikes significantly (Fig. 8 (d); n = 9). To further show that intermittent firing and delayed firing of layer III cells are not dependent on weak selectivity of linopirdine, we tested XE991 in four layer III cells. We applied 5 µM XE991 in two cells and 10 µM XE991 in the other two cells for 30 min. Both of the two cells in 5 µM and 10 µM XE991 caused intermittent firing (Fig. 9(a) and (b)), developed slow ADP and showed delayed firing (Fig. 9(c)). These experiments strongly suggest that the increase of ADP, decrease of AHP, development of slow ADP and intermittent firing patterns that were observed with linopirdine, were not due to inhibition of ionic currents other than the M-current.

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DISCUSSION We investigated the effects of M-current modulation on the principal cells in the EC using linopirdine and XE991. While some consider that Kv7.2 homomers are part of the M-current (Shah et al. 2002; Brown et al. 2007), others consider that the Kv7.2 homomers are not included in the M-current (Schwarz et al. 2006). Slowly activated and deactivating K+ current (Iks) in nodes of Ranvier is encoded by Kv7.2 homomers and is suppressed by both linopirdine and XE991 with lower affinities compared to those for Kv7.2/Kv7.3 heteromers. It is therefore possible that the block of Kv7.2 homomers (nodal Iks) is partly contributing to the result obtained in this paper. We have shown that blockade of the M-current causes intermittent firing in layer II non-SCs and layer III cells. While layer II non-SCs showed high-frequency burst firing during intermittent firing, layer III cells showed regular firing during intermittent firing (Fig. 1). The single spike after-potential at -60 mV did not cause burst firing in any cell type, while layer III cells showed large, slow ADPs (Fig. 2). When spikes were elicited at depolarized potentials, the increased fast ADP in layer II non-SCs was large enough to cause burst firing, suggesting a contribution of fast ADP to high-frequency bursting (Fig. 4). The after-potential after three spikes also revealed delayed firing in layer II non-SCs and layer III cells, suggesting that the modulation of the M-current played a role in their delayed firing activity. The increase in excitability was most pronounced in the layer III cells (Fig. 6). The subthreshold membrane potential oscillations in layer V cells were greatly suppressed, while those in the layer II SCs remained intact after blockade of the M-current (Fig. 7). The M-current thus shaped the firing

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behavior, intrinsic excitability and subthreshold membrane potential oscillations of principal cells in the EC in a cell-type specific manner. In this section, we discuss how these modifications would contribute to the physiological functions of the EC.

Induction of burst firing by M-current suppression: In the present work, the layer II non-SCs showed high-frequency (up to 98 Hz) burst firing (Fig. 1 (bii)). Although ADP did not reach the spike threshold when we tested cells at a membrane potential of -60 mV (Fig. 2(b)), firing at the peak of the ADP was observed at the depolarized level in three cells (Fig. 4(bi)). This indicates that ADP in EC layer II non-SCs is indeed large enough to reach firing threshold at depolarized levels. Alonso and García-Austt (1987) observed firing patterns of EC neurons during theta rhythm activity. Interestingly, they showed two classes of rhythmic cells in the layer II. The class I rhythmic cells showed strong modulation to the theta rhythm and a small tendency to burst. The class II rhythmic cells showed weak modulation to the theta rhythm and a strong tendency to burst. These two classes of cells are implicative of SCs and non-SCs, respectively. In our data, the SCs showed intact subthreshold membrane potential oscillations and no burst firing, and the non-SCs showed no clear subthreshold membrane potential oscillations, and burst firing, under the blockade of the M-current. These different tendencies in regard to burst firing also agree with a previous observation by Klink and Alonso (1997). They showed, in an EC slice preparation, that only non-SCs show burst firing with a bath application of carbachol. As mentioned above, the layer II non-SCs project mainly to the dentate gyrus (DG) and hippocampal CA3 regions through the perforant pathway (Schwartz and Coleman 1981; Witter and Amaral 1991; Tamamaki and Nojyo 1993). Since spike bursts facilitate synaptic

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plasticity (Magee and Johnston 1997; Thomas et al. 1998), high-frequency bursts of layer II non-SCs may help to not only drive neurons in the DG and CA3, but to also store memories during cholinergic activation.

ADP increase and AHP decrease during M-current suppression: The spike ADP results from currents through T, R and L-type voltage-gated Ca2+ channels, which are mainly distributed in distal dendrites (Wong and Prince 1981; White et al. 1989; Magee and Carruth 1999; Golding et al. 1999; Metz et al. 2005), and persistent Na+ channels, which are mainly distributed proximal to soma (Azouz et al. 1996; Yue et al. 2005). Although it is not clear which of the two types of currents contributes most to the ADP observed in neurons in the EC, both of them have been shown to be under the control of K+ currents. Suppression of A-type K+ currents at distal dendrites increases Ca2+ mediated ADP (Magee and Carruth 1999) and suppression of the M-current at the soma increases Na+ mediated ADP (Yue and Yaari 2006), and both of these cause burst firing. It has also been shown that under A-type K+ current suppression, the M-current contributes to the enhancement of Ca2+ spike generation in dendrites, suggesting that the M-current can modulate both somatic and dendritic sources of the ADP (Yue and Yaari 2006). In the present study, the application of linopirdine increased the ADP and decreased the AHP except in the case of layer II SCs (Fig. 2). We further suggested that the increase in ADP is not due to the lack of M-current that is activated by an action potential (Fig. 3). The time constant for activation of the M-current (6 to 50 ms depending on the membrane potential; Brown and Adams 1980; Wang et al. 1998) is too long for an action potential to activate the M-current. However, when an action potential is elicited at -60 mV, although the action

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potential itself lasts for only a few milliseconds, the ADP lasts for tens of milliseconds, keeping the membrane potential well above the threshold for the M-current activation. Thus, the ADP itself, whether it has dendritic or somatic origin, would activate the M-current in normal conditions. Once activated by the ADP, the M-current takes time to deactivate, and during this time it probably contributes to the AHP. Block of the M-current, would thus, “unleash” development of the ADP (Yue and Yaari 2006) and decrease the contribution to the AHP.

Ionic currents underlying delayed firing: In the present study, layer II non-SCs and layer III cells showed delayed firing (Fig. 4(b) and (c)). Delayed firing was observed previously in layer II SCs and non-SCs during cholinergic activation with carbachol (Klink and Alonso 1997; Magistretti et al. 2004). It is believed to play an important role in delayed matching tasks and may be the underlying mechanism for sustained spiking activity (Fransén et al. 2002). Delayed firing is caused by depolarization via a non-specific cation current (INCM) that is activated by the muscarinic receptor (Shalinsky et al. 2002). The INCM has transient tail and sustained plateau components (Magistretti et al. 2004). The tail part of this current is responsible for the transient depolarization during the delayed firing and is sensitive to Ca2+ influx from the voltage-gated Ca2+ channels. The time course of the tail current fits well with that of delayed firings observed in this study. The INCM in layer II cells has many analogies with currents mediated by transient receptor potential (TRP) channels (Shalinsky et al. 2002), and a recent study in EC layer V cells suggests that the INCM is mediated by TRP channels (Tahvildari et al. 2004). Many TRP channels display constitutive activity (Pedersen et al. 2005) suggesting that some INCM channels are active without muscarinic activation. Based on this, we propose

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that the delayed firing in our experiments were caused by the tail component of constitutive INCM. Three spikes elicited at 50 Hz caused larger Ca2+ influx than a single spike and induced INCM tail. Indeed, some of the layer II non-SCs and layer III cells had a small slow ADP even before applying linopirdine. Application of linopirdine, which eliminated the “clamping effect”, enabled the relatively small and constitutively active INCM tail current to drive delayed firing. Carbachol induced persistent firing in layer V cells in the medial EC (Egorov et al. 2002), and layer III cells in the lateral EC (Tahvildari and Alonso 2005) due to INCM. Persistent firing is believed to be crucial for mnemonic activities of the EC (McGaughy et al. 2005). In the present study, extended periods of persistent firing were never observed, even though different numbers of elicited spikes were tested (Fig. 5(a)-(c)). Rather, the increased number of spikes caused a larger AHP and less delayed firing. This shows that the INCM current which is constitutively active, is significantly smaller than the one during muscarinic activation, resulting in domination by the AHP current. In Fig. 5(d) and (e), we have shown voltage dependency of the delayed firing. Delayed firing and the slow ADP were not observed at hyperpolarized membrane potentials. Very similar voltage dependency was observed in layer V cells in carbachol (Egorov et al. 2002), where stimulation causing persistent firing at -60 mV, caused only delayed firing at around -70 mV and no depolarization at all at around -80 mV. This observation thus supports the idea that delayed firing is caused by INCM. Nevertheless, suppression of the M-current during cholinergic activation would help INCM to depolarize the membrane potential to trigger delayed firing in layer II cells, or to initiate and maintain (see below) persistent activity in layer III cells.

Modulation of excitability: Effects of the M-current on intrinsic excitability have been

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investigated since early studies by Adams et al. (1982a, b). Increased excitability was often measured as reduced spike frequency adaptation (Cole and Nicoll 1983, Madison and Nicoll 1984, McCormick and Williamson 1989, Aiken et al. 1995; Peters et al. 2005, Wang et al. 1998) while in some preparations, reduction of spike frequency adaptation was not observed as a result of M-current blockade (Romero et al. 2004; Miles et al. 2005). In the EC, while layer III and V cells showed a clear reduction of spike frequency adaptation, layer II SCs and non-SCs did not show a significant change (Fig. 6). Layer II SCs in particular showed the least dependency on the M-current, with no significant change in their firing patterns (Fig. 1(a)), ADP area and AHP amplitude (Fig. 2(a)), delayed firing properties (Fig. 4(a)) or ability to produce subthreshold membrane potential oscillations (Fig. 7). One of the characteristics of the layer II SCs is their huge Ih conductance. When the membrane potential is hyperpolarized, Ih is activated and the resulting inward current depolarizes the membrane. On the other hand, when the membrane is depolarized, Ih is deactivated and the reduced inward current hyperpolarizes the membrane. Ih therefore also contributes to the “clamping effect”. Layer II SCs, having the largest Ih conductance among all cell types compared here, would still have a substantial “clamping effect”, even under the blockade of the M-current, resulting in the smallest contribution of the M-current on excitability. Layer III cells, which have the least Ih conductance (in the soma), showed the largest increase in excitability (Fig. 6(c)). Mutations of M-channel (Kv7.2 and Kv7.3) genes lead to benign familial neonatal convulsions (BFNC), a dominantly inherited epilepsy (Biervert et al. 1998; Singh et al. 1998; Charlier et al. 1998). The finding that hyperexcitability in BFNC seems to be caused by only a 25% reduction of the M-current (Schroeder et al. 1998) revealed the significant contribution of the M-current in the maintenance of proper excitability. The EC is often the source of temporal

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lobe epilepsy (TLE; Bartolomei et al. 2005) and hyper-excitability during epilepsy causes neuronal loss. Within the EC, neuronal loss is preponderant in layer III after TLE while layer II cells are relatively resistant (Du et al. 1993; Du et al. 1995; Scharfman 2000; Schwarcz and Witter 2002). Interestingly, our results showed that the increase in excitability was greatest in layer III cells and smallest in layer II cells after linopirdine treatment (Fig. 6). Furthermore, while the membrane potential of layer III and layer V cells depolarized about 12 mV, those in layer II cells depolarized less than 6 mV in linopirdine. This consistency of a pattern, linking cell loss in TLE and sensitivity of the intrinsic excitability to the M-current block suggests that hyper-excitability, resulting from the suppression of M-current during TLE, could contribute to the cell loss in layer III. Increased excitability would also contribute to persistent firing of EC cells (Egorov et al. 2002; Tahvildari and Alonso 2005) under muscarinic activation. During persistent firing, membrane potentials of neurons are kept depolarized at supra-threshold level. In this study a significant increase in excitability was observed at this level under the M-current block. Increased excitability would greatly assist INCM in keeping the membrane potential above the threshold. The M-current would thus help both the initiation and maintenance of the persistent firing of neurons in the EC. Indeed, layer III and V cells, which showed a significant increase in excitability, are the neurons that show persistent firing.

Modulation of subthreshold membrane potential oscillations: Underlying ionic currents for subthreshold membrane potential oscillations vary in different neuronal populations (Alonso and Llinas 1989; Wang 1993; Gutfreund et al. 1995; Pape et al. 1998). The subthreshold membrane potential oscillations in layer II SCs has been shown to be generated by

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INaP and Ih (Klink and Alonso 1993; Dickson et al. 2000). As for layer V cells, Agrawal et al. (2001) have shown that subthreshold membrane potential oscillations depend on INaP. However, the other current that interacts with INaP remained unknown. The observation that there are layer V cells that have no sign of Ih (hyperpolarization induced sag) and still show robust subthreshold membrane potential oscillations (Hamam et al. 2000), suggested that the mechanism for subthreshold membrane potential oscillations of layer V cells is different to that for layer II SCs. Gutfreund et al. (1995) have shown that subthreshold membrane potential oscillations in guinea-pig frontal cortex neurons can be replicated by INaP and a non-inactivating potassium current with the kinetics of the M-current, suggesting that this is also the case in the EC layer V cells (Hamam et al. 2002). Our results clearly show that blockade of the M-current did not affect subthreshold membrane potential oscillations in layer II SCs, but suppressed the subthreshold membrane potential oscillations in layer V cells (Fig. 7), showing that subthreshold membrane potential oscillations in layer V cells indeed depend on the M-current. That cholinergic modulation differently affects the subthreshold membrane potential oscillations in layer II and layer V cells is interesting because cholinergic modulation is believed to set the functional state of the entorhinal-hippocampal network. A high acetylcholine level promotes encoding of information in the hippocampus while a low acetylcholine level promotes consolidation of information from the hippocampus into neocortical regions (Hasselmo 1999). In the encoding phase, the EC layer II serves as the main source of information to the hippocampus through the perforant pathway. In this phase, theta rhythm, crucial for the temporal coding and synaptic modification (Buzsáki 2002), is observed in the EC and the hippocampus. In the EC, neurons in the superficial layers (layers II and III) fire in synchrony with the theta rhythm, while most of the neurons from deep layers

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(layers V and VI) do not (Alonso and García-Austt 1987; Chrobak and Buzsáki 1994; Frank et al. 2001). For the generation of the theta rhythm, input from septal fibers plays a crucial role. Intrinsic oscillatory properties of the EC cells, however, may also contribute to the generation of the theta rhythm, as is believed to be the case in the hippocampus (Buzsáki 2002). If this is true, the subthreshold membrane potential potential oscillations which remain intact in layer II SCs, and vanishe in layer V cells under suppression of the M current, will contribute to the layer-dependent properties of the theta rhythm activity during mnemonic processes. In conclusion, modulation of the M-current has the potential to help shape the dynamical properties of neurons in the EC, such as burst firing patterns, delayed firing, persistent firing and oscillation properties, all of which are important components of memory function. This tuning, performed in a layer and cell-type specific manner, is in keeping with the transition of the entorhinal-hippocampal area to the encoding stage. This study suggests that the M-current is therefore not simply a controller of excitability, but is a key component that tunes the dynamics of neurons for mnemonic function.

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ACKNOWLEDGMENTS In memory of Professor Angel A. Alonso. We thank Professor Daniel Johnston and Professor Michael Hasselmo for their insightful advice and great support on this work, and critical reading of the manuscript. We also thank Professor Philippe Séguéla and Dr. Antonio Reboreda for their useful comments and critical reading of the manuscript. Linguistic help was provided by Dinah Tabizel.

Current address for Motoharu Yoshida: Center for Memory and Brain, Boston University, 2 Cummington Street, Boston MA, 02215, United States

GRANTS This work was supported by NIH grant 01MH061492 and CIHR grant MOP-10914.

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differential electroresponsiveness of medial entorhinal cortex layer II neurons. J Neurophysiol 70: 144-157, 1993. Klink R and Alonso A. Muscarinic modulation of the oscillatory and repetitive firing properties of entorhinal cortex layer II neurons. J Neurophysiol 77: 1813-1828, 1997. Koyama S and Appel SB. Characterization of M-current in Ventral Tegmental Area Dopamine Neurons. J Neurophysiol doi:10.1152, 2006. Lamas JA, Selyanko AA, and Brown DA. Effects of a cognition-enhancer, linopirdine (DuP 996), on M-type potassium currents (IK(M)) and some other voltage- and ligand-gated membrane currents in rat sympathetic neurons. Eur J Neurosci 9: 605-616, 1997. Leonard BW, Amaral DG, Squire LR, and Zola-Morgan S. Transient memory impairment in monkeys with bilateral lesions of the entorhinal cortex. J Neurosci 15: 5637-5659, 1995. Madison DV and Nicoll RA. Control of the repetitive discharge of rat CA 1 pyramidal neurones in vitro. J Physiol 354: 319-331, 1984. Magee JC and Carruth M. Dendritic voltage-gated ion channels regulate the action potential firing mode of hippocampal CA1 pyramidal neurons. J Neurophysiol 82: 1895-1901, 1999. Magee JC and Johnston D. A synaptically controlled, associative signal for Hebbian plasticity in hippocampal neurons. Science 275: 209-213, 1997. Magistretti J, Ma L, Shalinsky MH, Lin W, Klink R, and Alonso A. Spike patterning by Ca2+-dependent regulation of a muscarinic cation current in entorhinal cortex layer II neurons. J Neurophysiol 92: 1644-1657, 2004. McCormick DA and Williamson A. Convergence and divergence of neurotransmitter

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action in human cerebral cortex. Proc Natl Acad Sci U S A 86: 8098-8102, 1989. McGaughy J, Koene RA, Eichenbaum H, and Hasselmo ME. Cholinergic deafferentation of the entorhinal cortex in rats impairs encoding of novel but not familiar stimuli in a delayed nonmatch-to-sample task. J Neurosci 25: 10273-10281, 2005. Metz AE, Jarsky T, Martina M, and Spruston N. R-type calcium channels contribute to afterdepolarization and bursting in hippocampal CA1 pyramidal neurons. J Neurosci 25: 5763-5773, 2005. Miles GB, Dai Y, and Brownstone RM. Mechanisms underlying the early phase of spike frequency adaptation in mouse spinal motoneurones. J Physiol 566: 519-532, 2005. Naber PA, Lopes da Silva FH and Witter MP. Reciprocal connections between the entorhinal cortex and hippocampal fields CA1 and the subiculum are in register with the projections from CA1 to the subiculum. Hippocampus 11:99-104, 2001. Pape HC, Pare D, and Driesang RB. Two types of intrinsic oscillations in neurons of the lateral and basolateral nuclei of the amygdala. J Neurophysiol 79: 205-216, 1998. Pedersen SF, Owsianik G, and Nilius B. TRP channels: an overview. Cell Calcium 38: 233-252, 2005. Peters HC, Hu H, Pongs O, Storm JF, and Isbrandt D. Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nat Neurosci 8: 51-60, 2005. Romero M, Reboreda A, Sanchez E, and Lamas JA. Newly developed blockers of the M-current do not reduce spike frequency adaptation in cultured mouse sympathetic neurons. Eur J Neurosci, 19: 2693-2702, 2004. Scharfman HE. Epileptogenesis in the parahippocampal region. Parallels with the

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dentate gyrus. Ann N Y Acad Sci 911: 305-27, 2000. Schnee ME and Brown BS. Selectivity of linopirdine (DuP 996), a neurotransmitter release enhancer, in blocking voltage-dependent and calcium-activated potassium currents in hippocampal neurons. J Pharmacol Exp Ther 286: 709-717, 1998. Schroeder BC, Kubisch C, Stein V, and Jentsch TJ. Moderate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy. Nature 396: 687-690, 1998. Schwarcz R and Witter MP. Memory impairment in temporal lobe epilepsy: the role of entorhinal lesions. Epilepsy Res 50: 161-177, 2002. Schwartz SP and Coleman PD. Neurons of origin of the perforant path. Exp Neurol 74: 305-312, 1981. Scoville WB and Milner B. Loss of recent memory after bilateral hippocampal lesions. J Neurol Neurosurg Psychiatry 20: 11-21, 1957. Shah MM, Mistry M, Marsh SJ, Brown DA and Delmas P. Molecular correlates of the M-current in cultured rat hippocampal neurons. J Physiol. 544:29-37, 2002. Shalinsky MH, Magistretti J, Ma L, and Alonso AA. Muscarinic activation of a cation current and associated current noise in entorhinal-cortex layer-II neurons. J Neurophysiol 88: 1197-1211, 2002. Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, Ronen GM, Bjerre I, Quattlebaum T, Murphy JV, McHarg ML, Gagnon D, Rosales TO, Peiffer A, Anderson VE, and Leppert M. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet 18: 25-29, 1998. Squire LR and Zola-Morgan S. The medial temporal lobe memory system. Science 253:

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1380-1386, 1991. Storm JF. An after-hyperpolarization of medium duration in rat hippocampal pyramidal cells. J Physiol 409: 171-190, 1989. Steffenach HA, Witter M, Moser MB, and Moser EI. Spatial memory in the rat requires the dorsolateral band of the entorhinal cortex. Neuron 45: 301-313, 2005. Steriade M, Nunez A, and Amzica F. A novel slow (< 1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components. J Neurosci 13: 3252-3265, 1993. Suzuki WA, Miller EK, and Desimone R. Object and place memory in the macaque entorhinal cortex. J Neurophysiol 78: 1062-1081, 1997. Tahvildari B and Alonso A. Switching-on and -off persistent activity in entorhinal cortex layer III pyramidal cells; an intrinsic neuronal toggle mechanism. Society for Neuroscience Program No. 737.7, 2005. Tahvildari B, Egorov A, Klink R, and Alonso A. Persistent activity in the entorhinal cortex neurons, TRP channels and intracellular calcium stores. Society for Neuroscience Program No. 516.3, 2004. Tamamaki N and Nojyo Y. Projection of the entorhinal layer II neurons in the rat as revealed by intracellular pressure-injection of neurobiotin. Hippocampus 3: 471-480, 1993. Thomas MJ, Watabe AM, Moody TD, Makhinson M, and O'Dell TJ. Postsynaptic complex spike bursting enables the induction of LTP by theta frequency synaptic stimulation. J Neurosci 18: 7118-7126, 1998. Van Groen T and Lopes da Silva FH. Organization of the reciprocal connections between the subiculum and the entorhinal cortex in the cat: II. An electrophysiological study.

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J Comp Neurol 251:111-120, 1986. van Groen T, van Haren FJ, Witter MP and Groenewegen HJ. The organization of the reciprocal connections between the subiculum and the entorhinal cortex in the cat: I. A neuroanatomical tracing study. J Comp Neurol 250:485-497, 1986. Wang HS, Pan Z, Shi W, Brown BS, Wymore RS, Cohen IS, Dixon JE, and McKinnon D. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science 282: 1890-1893, 1988. Wang XJ. Ionic basis for intrinsic 40 Hz neuronal oscillations. Neuroreport 5: 221-224, 1993. White G, Lovinger DM, and Weight FF. Transient low-threshold Ca2+ current triggers burst firing through an afterdepolarizing potential in an adult mammalian neuron. Proc Natl Acad Sci U S A 86: 6802-6806, 1989. Witter MP and Amaral DG. Entorhinal cortex of the monkey: V. Projections to the dentate gyrus, hippocampus, and subicular complex. J Comp Neurol 307: 437-459, 1991. Wong RK and Prince DA. Afterpotential generation in hippocampal pyramidal cells. J Neurophysiol 45: 86-97, 1981. Yoshida M and Alonso A. Differential role of the M-current on the oscillatory and bursting behavior of principal cells from layers II, III and V of the entorhinal cortex. Society for Neuroscience Program No. 971.5, 2005. Yue C, Remy S, Su H, Beck H, and Yaari Y. Proximal persistent Na+ channels drive spike afterdepolarizations and associated bursting in adult CA1 pyramidal cells. J Neurosci 25: 9704-9720, 2005. Yue C and Yaari Y. KCNQ/M channels control spike afterdepolarization and burst

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generation in hippocampal neurons. J Neurosci 24: 4614-4624, 2004. Yue C and Yaari Y. Axo-somatic and apical dendritic Kv7/M channels differentially regulate the intrinsic excitability of adult rat CA1 pyramidal cells. J Neurophysiol 95: 3480-3495, 2006. Zaczek R, Chorvat RJ, Saye JA, Pierdomenico ME, Maciag CM, Logue AR, Fisher BN, Rominger DH, and Earl RA. Two new potent neurotransmitter release enhancers, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone

and

10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone: comparison to linopirdine. J Pharmacol Exp Ther 285: 724-730, 1998.

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FIGURE LEGENDS Fig. 1. Modulation of intrinsic firing patterns by linopirdine. The firing pattern of (a) layer II SCs, (b) layer II non-SCs, (c) layer III cells and (d) layer V cells were compared (i) in control and (ii) in linopirdine (10 µM, 30 min). Cells were depolarized by constant current injection to just above the threshold. Rightmost traces in (ii) are magnifications of the underlined traces. Firing patterns of layer II SCs were least affected by the M-current block (aii). One example of long-duration clustered firing consisting of multiple high-frequency short-duration burst firing during intermittent firing is shown in (bii). Layer III cells showed intermittent firing consisting of regular firing (cii). Subthreshold membrane potential oscillations and clustering were not seen in layer V cells in linopirdine (dii). SC: stellate cell.

Fig. 2. Modulation of the single spike after-potential by linopirdine. (a) layer II SCs, (b) layer II non-SCs, (c) layer III cells and (d) layer V cells. (i) 1 ms depolarizing current pulse (shown at bottom) was applied to elicit one spike at the membrane potential of -60 mV in control (grey traces) and in linopirdine (30 min; black traces). (ii) Magnification of the fast ADP in (i). (iii) ADP amplitude, (iv) ADP duration, (v) ADP area and (vi) AHP amplitude in control (open) and linopirdine (filled). Long-lasting ADP with large amplitude was observed in layer III cells (ci). Note different scales for ADP duration (civ) and ADP area (cv). Amplitudes of injected 1 ms current in control/linopirdine were 1.8/1.7, 1.7/2.2, 1.6/2.0 and 1.6/1.8 nA in (a) to (d), respectively. Arrows in (aii) and (cii) show onset of ADP (see Methods).

Fig. 3. Comparison of ADPs at -60 and -80 mV in layer V cells. (a) One spike was elicited

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by 1 ms depolarizing current pulse at the membrane potential of -60 mV and -80 mV in control (grey) and in linopirdine (30 min; black). Note that at -80 mV control and linopirdine traces are almost identical. (b) ADP duration. Increase was seen only at -60 mV. (c) ADP area. Increase was significant only at -60 mV. Amplitudes of injected 1 ms current in control/linopirdine were 1.2/1.2 and 2.8/2.8 nA at -60 mV and -80 mV, respectively.

Fig. 4. After-potential elicited by three spikes. (a) layer II SCs, (b) layer II non-SCs, (c) layer III cells and (d) layer V cells. (i) Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms (bottom trace) in control (grey) and linopirdine (black). The membrane potential was kept just below the threshold level. (ii) Full scale version of the trace in (i). (iii) After-potential area. Sum of ADP and AHP for the period of 30 s after stimulation is shown. Layer II non-SCs showed a spike on top of the fast ADP (bi) and slow depolarization induced delayed firing (bii). Magnification of delayed firing shows that it consists of multiple high-frequency short-duration burst firing (bii, in dotted lines). In layer III cells, huge slow ADPs drove delayed firing consisting of regular firing (cii). Note different scale for after-potential area in (ciii). Amplitudes of injected 3 ms current in control/linopirdine were 1.0/1.0, 1.0/0.7, 0.6/0.6 and 1.0/1.0 nA in (a) to (d), respectively.

Fig. 5. Spike number and voltage dependency of slow ADP in layer III cells. (a) Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms at a membrane potential of -60 mV (as in Fig. 4). Clear delayed firing is seen. (b) Ten spikes were elicited by applying ten 3 ms current pulses at a membrane potential of -60 mV. Note prominent AHP and reduced slow ADP duration. (c) Twenty spikes were elicited by applying twenty 3 ms current

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pulses at a membrane potential of -60 mV. Slow ADP disappeared. (d) Three spikes were elicited at a membrane potential of -65 mV. Amplitude of the slow ADP was not enough to elicit delayed firing. (e) Three spikes were elicited at a membrane potential of -70 mV. Slow ADP is no longer seen. Amplitudes of injected 3 ms current were 2.0 nA in all cases.

Fig. 6. Modulation of intrinsic excitability by linopirdine. (a) layer II SCs, (b) layer II non-SCs, (c) layer III cells, and (d) layer V cells. 1 s current injection, with an amplitude that elicited a minimum of eight spikes in control, was applied both in control and linopirdine (30 min). (i) Trace in control. (ii) Trace in linopirdine. (iii) Number of spikes elicited during current injection. (iv) and (v) Inter-spike interval (ISI) histogram of the 6 layer II non-SCs that showed high-frequency bursts in linopirdine. (iv) shows ISI histogram in control and (v) shows ISI histogram in linopirdine. (vi) ISI histogram of all 8 layer II non-SCs. Open circles and filled circles show ISI histogram in control and in linopirdine, respectively. Note high-frequency burst firing in layer II non-SCs (bii). Number of spikes increased the most in layer III cells (ciii). Amplitudes of injected current were 0.13, 0.18, 0.09 and 0.18 nA in (a) to (d), respectively.

Fig. 7. Modulation of subthreshold membrane potential oscillations in layer II SCs and layer V cells. (a) Layer II SCs. (b) Layer V cells. Subthreshold membrane potential oscillations were compared in (i) control and (ii) linopirdine 30 min. Lower traces show magnification in between arrows. (iii)-(iv) Power spectrum obtained from magnified traces in (i)-(ii), respectively. (v) Area of power spectrum around the peak (1.53 to 3.97 Hz) in control (open) and linopirdine 30 min (filled). Power spectra were obtained from three 3.28 s membrane

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potential traces that had the largest area in the power spectrum between 1.53 to 3.97 Hz in each cell in each condition (see Methods). While layer II SC subthreshold membrane potential oscillations were not suppressed, layer V subthreshold membrane potential oscillations were clearly suppressed by linopirdine.

Fig. 8. Effect of XE991 and SK-channel blocker on layer V cells. (a) Single spike after-potential. (ai) 1 ms depolarizing current pulse (shown at bottom) was applied to elicit one spike at the membrane potential of -60 mV in control (black trace), apamin (15 min; blue trace), and apamin and XE991 (30 min; red trace). (aii) Magnification of the fast ADP in (ai). (aiii) ADP amplitude (repeated measures ANOVA, P < 0.01), (aiv) ADP duration (repeated measures ANOVA, P < 0.01), (av) ADP area (repeated measures ANOVA, P < 0.001) and (avi) AHP amplitude (repeated measures ANOVA, P < 0.001) in control (open), apamin (grey) and apamin and XE991 (filled). Note the larger ADP and smaller AHP by application of XE991. (b) Example of cells responding with high-frequency bursts to the single spike protocol. (bi) 1 ms depolarizing current pulse (shown at bottom) caused high-frequency bursts with apamin and EX991 (30 min: red trace) in two out of eleven cells. (bii) Magnification of the fast ADP in (bi). (c) After-potential elicited by three spikes. (ci) Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms (bottom trace). (cii) Full scale version of the trace. (ciii) After-potential area (repeated measures ANOVA, P < 0.01). (d) Intrinsic excitability. (di) Trace in control. (dii) Trace in apamin. (diii) Trace in apamin and XE991. (div) Number of spikes elicited during current injection (repeated measures ANOVA, P < 0.001). Note the significant increase in number of spikes by application of XE991. Significance obtained by Tukey post hoc test is shown in the figure.

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Fig. 9. Effect of XE991 on layer III cells. (a) Firing pattern in control. (b) Firing pattern in XE991. (c) After-potential elicited by three spikes in control (grey trace) and in XE991 (black). Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms (bottom trace). XE991 caused intermittent firing and delayed firing.

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TABLE Table 1. Summary of membrane potentials with linopirdine. Layer II SCs

Layer II non-SCs

Layer III

Layer V

Control

-62.1 ± 0.9

-61.9 ±1.0

-64.8 ± 0.9

-63.0 ± 0.9

Lino 15 min

-55.2 ± 0.6 **

-58.9 ± 1.2

-55.9 ± 2.8 *

-51.5 ± 1.2 ***

Lino 30 min

-56.1 ± 1.4 ***

-57.5 ± 2.0 *

-52.8 ± 1.7 ***

-50.6 ± 1.2 ***

Lino: linopirdine.

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Fig. 1. Modulation of intrinsic firing patterns by linopirdine. The firing pattern of (a) layer II SCs, (b) layer II non-SCs, (c) layer III cells and (d) layer V cells were compared (i) in control and (ii) in linopirdine (10 M, 30 min). Cells were depolarized by constant current injection to just above the threshold. Rightmost traces in (ii) are magnifications of the underlined traces. Firing patterns of layer II SCs were least affected by the Mcurrent block (aii). One example of long-duration burst firing consisting of multiple highfrequency short-duration burst firing is shown in (bii). Layer III cells showed longduration burst firing consisting of regular firing (cii). Subthreshold membrane potential oscillations and clustering were not seen in layer V cells in linopirdine (dii). SC: stellate cell. 190x253mm (600 x 600 DPI)

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Fig. 2. Modulation of the single spike after-potential by linopirdine. (a) layer II SCs, (b) layer II non-SCs, (c) layer III cells and (d) layer V cells. (i) 1 ms depolarizing current pulse (shown at bottom) was applied to elicit one spike at the membrane potential of -60 mV in control (grey traces) and in linopirdine (30 min; black traces). (ii) Magnification of the fast ADP in (i). (iii) ADP amplitude, (iv) ADP duration, (v) ADP area and (vi) AHP amplitude in control (open) and linopirdine (filled). Long-lasting ADP with large amplitude was observed in layer III cells (ci). Note different scales for ADP duration (civ) and ADP area (cv). Amplitudes of injected 1 ms current in control/linopirdine were 1.8/1.7, 1.7/2.2, 1.6/2.0 and 1.6/1.8 nA in (a) to (d), respectively. Arrows in (aii) and (cii) show onset of ADP (see Methods). 190x253mm (600 x 600 DPI)

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Fig. 3. Comparison of ADPs at -60 and -80 mV in layer V cells. (a) One spike was elicited by 1 ms depolarizing current pulse at the membrane potential of -60 mV and -80 mV in control (grey) and in linopirdine (30 min; black). Note that at -80 mV control and linopirdine traces are almost identical. (b) ADP duration. Increase was seen only at -60 mV. (c) ADP area. Increase was significant only at -60 mV. Amplitudes of injected 1 ms current in control/linopirdine were 1.2/1.2 and 2.8/2.8 nA at -60 mV and -80 mV, respectively. 190x253mm (600 x 600 DPI)

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Fig. 4. After-potential elicited by three spikes. (a) layer II SCs, (b) layer II non-SCs, (c) layer III cells and (d) layer V cells. (i) Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms (bottom trace) in control (grey) and linopirdine (black). The membrane potential was kept just below the threshold level. (ii) Full scale version of the trace in (i). (iii) After-potential area. Sum of ADP and AHP for the period of 30 s after stimulation is shown. Layer II non-SCs showed a spike on top of the fast ADP (bi) and slow depolarization induced delayed firing (bii). Magnification of delayed firing shows that it consists of multiple high-frequency short-duration burst firing (bii, in dotted lines). In layer III cells, huge slow ADPs drove delayed firing consisting of regular firing (cii). Note different scale for after-potential area in (ciii). Amplitudes of injected 3 ms current in control/linopirdine were 1.0/1.0, 1.0/0.7, 0.6/0.6 and 1.0/1.0 nA in (a) to (d), respectively.

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190x253mm (600 x 600 DPI)

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Fig. 5. Spike number and voltage dependency of slow ADP in layer III cells. (a) Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms at a membrane potential of -60 mV (as in Fig. 4). Clear delayed firing is seen. (b) Ten spikes were elicited by applying ten 3 ms current pulses at a membrane potential of -60 mV. Note prominent AHP and reduced slow ADP duration. (c) Twenty spikes were elicited by applying twenty 3 ms current pulses at a membrane potential of -60 mV. Slow ADP disappeared. (d) Three spikes were elicited at a membrane potential of -65 mV. Amplitude of the slow ADP was not enough to elicit delayed firing. (e) Three spikes were elicited at a membrane potential of -70 mV. Slow ADP is no longer seen. Amplitudes of injected 3 ms current were 2.0 nA in all cases. 190x253mm (600 x 600 DPI)

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Fig. 6. Modulation of intrinsic excitability by linopirdine. (a) layer II SCs, (b) layer II nonSCs, (c) layer III cells, and (d) layer V cells. 1 s current injection, with an amplitude that elicited a minimum of eight spikes in control, was applied both in control and linopirdine (30 min). (i) Trace in control. (ii) Trace in linopirdine. (iii) Number of spikes elicited during current injection. (iv) and (v) Inter-spike interval (ISI) histogram of the 6 layer II non-SCs that showed high-frequency bursts in linopirdine. (iv) shows ISI histogram in control and (v) shows ISI histogram in linopirdine. (vi) ISI histogram of all 8 layer II non-SCs. Open circles and filled circles show ISI histogram in control and in linopirdine, respectively. Note high-frequency burst firing in layer II non-SCs (bii). Number of spikes increased the most in layer III cells (ciii). Amplitudes of injected current were 0.13, 0.18, 0.09 and 0.18 nA in (a) to (d), respectively. 190x253mm (600 x 600 DPI)

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Fig. 7. Modulation of subthreshold membrane potential oscillations in layer II SCs and layer V cells. (a) Layer II SCs. (b) Layer V cells. Subthreshold membrane potential oscillations were compared in (i) control and (ii) linopirdine 30 min. Lower traces show magnification in between arrows. (iii)-(iv) Power spectrum obtained from magnified traces in (i)-(ii), respectively. (v) Area of power spectrum around the peak (1.53 to 3.97 Hz) in control (open) and linopirdine 30 min (filled). Power spectra were obtained from three 3.28 s membrane potential traces that had the largest area in the power spectrum between 1.53 to 3.97 Hz in each cell in each condition (see Methods). While layer II SC subthreshold membrane potential oscillations were not suppressed, layer V subthreshold membrane potential oscillations were clearly suppressed by linopirdine. 190x253mm (600 x 600 DPI)

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Fig. 8. Effect of XE991 and SK-channel blocker on layer V cells. (a) Single spike afterpotential. (ai) 1 ms depolarizing current pulse (shown at bottom) was applied to elicit one spike at the membrane potential of -60 mV in control (black trace), apamin (15 min; blue trace), and apamin and XE991 (30 min; red trace). (aii) Magnification of the fast ADP in (ai). (aiii) ADP amplitude (repeated measures ANOVA, P < 0.01), (aiv) ADP duration (repeated measures ANOVA, P < 0.01), (av) ADP area (repeated measures ANOVA, P < 0.001) and (avi) AHP amplitude (repeated measures ANOVA, P < 0.001) in control (open), apamin (grey) and apamin and XE991 (filled). Note the larger ADP and smaller AHP by application of XE991. (b) Example of cells responding with high-frequency bursts to the single spike protocol. (bi) 1 ms depolarizing current pulse (shown at bottom) caused high-frequency bursts with apamin and EX991 (30 min: red trace) in two out of eleven cells. (bii) Magnification of the fast ADP in (bi). (c) After-potential elicited by

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three spikes. (ci) Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms (bottom trace). (cii) Full scale version of the trace. (ciii) After-potential area (repeated measures ANOVA, P < 0.01). (d) Intrinsic excitability. (di) Trace in control. (dii) Trace in apamin. (diii) Trace in apamin and XE991. (div) Number of spikes elicited during current injection (repeated measures ANOVA, P < 0.001). Note the significant increase in number of spikes by application of XE991. Significance obtained by Tukey post hoc test is shown in the figure. 190x253mm (600 x 600 DPI)

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Fig. 9. Effect of XE991 on layer III cells. (a) Firing pattern in control. (b) Firing pattern in XE991. (c) After-potential elicited by three spikes in control (grey trace) and in XE991 (black). Three spikes were elicited by three 3 ms current pulses with an interval of 20 ms (bottom trace). XE991 caused burst firing and delayed firing. 190x253mm (600 x 600 DPI)