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1 J Physiol (2014) pp The Journal of Physiology Neuroscience Complementary functions of SK and Kv7/M potassium channels in excitability control and synaptic integration in rat hippocampal dentate granule cells Pedro Mateos-Aparicio, Ricardo Murphy and Johan F. Storm Institute of Medical Basic Sciences, Department of Physiology, University of Oslo, Oslo, Norway Key points Previous studies showed that different firing patterns of hippocampal dentate granule cells (DGCs) can trigger different network responses. However, the intrinsic DGC mechanisms controlling their excitability, spike patterns and synaptic integration in DGCs, remain poorly understood. SK and Kv7/M channels play important roles controlling neuronal integration and excitability, but their specific roles vary between cell types. Both channel types are expressed in DGCs, but their roles are unclear. We found that SK channels are the main generators of medium afterhyperpolarizations in DGCs, thus causing negative feedback regulation of spiking (spike frequency adaptation) and calcium influx. In contrast, Kv7/M perform subthreshold and feed-forward control of input resistance, postsynaptic integration, action potential threshold and excitability, thus weakening EPSP spike coupling. Thus, in DGCs, the SK and Kv7/M channels seem to perform complementary functions in postsynaptic integration and excitability control. This may have important consequences for dentate network physiology. Abstract The dentate granule cells (DGCs) form the most numerous neuron population of the hippocampal memory system, and its gateway for cortical input. Yet, we have only limited knowledge of the intrinsic membrane properties that shape their responses. Since SK and Kv7/M potassium channels are key mechanisms of neuronal spiking and excitability control, afterhyperpolarizations (AHPs) and synaptic integration, we studied their functions in DGCs. The specific SK channel blockers apamin or scyllatoxin increased spike frequency (excitability), reduced early spike frequency adaptation, fully blocked the medium-duration AHP (mahp) after a single spike or spike train, and increased postsynaptic EPSP summation after spiking, but had no effect on input resistance (R input ) or spike threshold. In contrast, blockade of Kv7/M channels by XE991 increased R input, lowered the spike threshold, and increased excitability, postsynaptic EPSP summation, and EPSP spike coupling, but only slightly reduced mahp after spike trains (and not after single spikes). The SK and Kv7/M channel openers 1-EBIO and retigabine, respectively, had effects opposite to the blockers. Computational modelling reproduced many of these effects. We conclude that SK and Kv7/M channels have complementary roles in DGCs. These mechanisms may be important for the dentate network function, as CA3 neurons can be activated or inhibition recruited depending on DGC firing rate. DOI: /jphysiol

2 670 P. Mateos-Aparicio and others J Physiol (Received 5 November 2013; accepted after revision 16 December 2013; first published online 23 December 2013) Corresponding author J. F. Storm: Department of Physiology, IMB, University of Oslo, PB 1104 Blindern, 0317 Oslo, Norway. j.f.storm@medisin.uio.no Abbreviations αepscs, artificial excitatory postsynaptic currents; ADP, afterdepolarization; AHP, afterhyperpolarization; AIS, axon initial segment; AP, action potential; DG, dentate gyrus; DGC, dentate granule cell; fahp, fast afterhyperpolarization; g M,M-conductance;g SK, SK-conductance; I aahp, apamin-sensitive afterhyperpolarization current; I h, H-current; I M, M-current; I mahp, mahp-current; I sahp, sahp-current; ISI, interspike interval; I SK, SK-current; LTP, long-term potentiation; mahp, medium afterhyperpolarization; MEC, medial entorhinal cortex; MF, mossy fibre; NMDA-R, NMDA receptor; PP, perforant path; R input, input resistance; RMP, resting membrane potential; sadp, slow afterdepolarization; sahp, slow afterhyperpolarization; STDP, spike timing-dependent plasticity; V half, voltage for half-maximal conductance, τ, time constant. Introduction The dentate gyrus (DG) is the gateway of the hippocampal memory system, receiving cortical input from all sensory modalities, for forming episodic and spatial memories (Scharfman, 2007). Its granule cells (DGCs) constitute the most numerous hippocampal cell population. During spatial exploration and memory tasks, the DGCs fire sparsely within multiple place fields (Jung & McNaughton, 1993) and perform pattern separation (Leutgeb et al. 2007). Thus, the dispersal of cortical input onto the numerous, sparsely firing DGCs, which in turn connect sparsely to CA3, favours segregation of input entering the CA3 auto-associative network (Treves et al. 2008). The DG is also where long-term potentiation (LTP) was first discovered (Lømo, 1968; Bliss & Lømo 1973), and its output synapses are the site of a distinct form of LTP (Nicoll & Schmitz, 2005). Despite the central role of the DGCs in the hippocampal memory system, their intrinsic membrane properties have only been partly determined and are often neglected in studies of hippocampal functions. In particular, afterhyperpolarizations (AHPs) and their underlying conductances following single action potentials (APs) or spike bursts have been shown to be important for controlling intrinsic neuronal excitability, firing frequency and pattern, including spike frequency adaptation, in a variety of central neurons (Kernell, 1968; Madison & Nicoll, 1982, 1984; Pedarzani et al. 1998; Lancaster et al. 2001; Sah & Faber, 2002; Vogalis et al. 2003; Peters et al. 2005). Many vertebrate neurons typically show three AHP components: a fast AHP (fahp, lasting 2 10 ms), a medium AHP (mahp) lasting ms, and a slow AHP (sahp) lasting 1 5 s (Storm, 1987, 1990; Bean, 2007). Whereas the enigmatic nature of the sahp has received considerable attention in recent years (Tzingounis & Nicoll, 2008; Andrade et al. 2012), the mahp of DGCs remains poorly characterized. The mechanisms controlling both early and late spike frequency adaptation have particular relevance in DGCs, since there is evidence that different DGC discharge frequencies can (1) differentially impact the output of CA3 pyramidal cells and interneurons through the strong mossy fibre (MF) synapses (Henze et al. 2002), (2) recruit different network-related mechanisms (Mori et al. 2004, 2007), and (3) trigger somatic protein synthesis and subsequent transport along the axon to stabilize MF-LTP (Barnes et al. 2010). In CA1 pyramidal cells, the mahp is caused by dual voltage-dependent mechanisms (Storm, 1989, 1990): the M-current (I M, Kv7/M channels) generates the mahp at depolarized membrane potentials, whereas I h (HCN channels) causes a similarly looking mahp at more negative potentials. Later, small-conductance calcium-activated potassium (SK) channels were found to underlie the mahp in many other brain regions and neuron types (for review, see: Bond et al. 1999; Sah & Faber 2002; Vogalis et al. 2003), while convergent evidence has confirmed that the mahp of CA1 pyramidal cells is generated by the dual I M and I h mechanisms with little or no contribution from SK channels (Storm, 1989, 1990; Williamson & Alger, 1990; Gu et al. 2005, 2008; Peters et al. 2005). These findings suggest that different channel types generate AHPs of medium duration (mahp; ms) in different cell types. Among the candidate AHP mechanisms, there is prior evidence for the expression of both SK and Kv7/M channels in DGCs. Thus, a small apamin-sensitive SK current, I SK, was identified in rat (Sailer et al. 2002) and human DGCs (Beck et al. 1997), in agreement with the low or moderate levels of SK1 3 mrna (Stocker & Pedarzani, 2000) and SK1 3 protein (Sailer et al. 2002, 2004) in the DG. There are low levels of Kv7.2 and Kv7.3 immunostaining in the granule cell layer, whereas the MF axons of the DGCs show strong expression of these Kv7/M channel subunits (Cooper et al. 2001; Devaux et al. 2004; Klinger et al. 2011). Furthermore, it was reported that Kv7/M channels generate about 50% of the so-called mahp and sahp currents (I mahp, I sahp ) in mouse DGCs, suggesting that I M contributes to their mahp (Storm 1989; Tzingounis & Nicoll, 2008). However, to our knowledge, the mechanisms underlying the mahp in DGCs have so far not been directly examined. Thus,

3 J Physiol SK and Kv7/M channels underlying mahp and spiking control in DGCs 671 although the roles of perisomatic Kv7/M channels in generating mahps, subthreshold resonance and control of neural excitability have been described in CA1 pyramidal cells (Storm, 1989; Hu et al. 2002; Yue & Yaari, 2004, 2006; Gu et al. 2005; Hu et al. 2007; Shah et al. 2008), little is known about their function in DGCs. Therefore, we have now examined the functions of SK and Kv7/M channels in rat DGCs, including their roles in AHPs, adaptation, spike threshold regulation, and synaptic integration, by combining whole-cell patch clamp recordings, pharmacological tests and modelling. Surprisingly, we find that SK channels are the main generator of the mahp and early spike frequency adaptation, although this previously appeared unlikely based on the comparatively small I SK amplitude in this cell type (Sailer et al. 2002). This result is intriguing also because blockade or knock-out of SK channels seems to facilitate hippocampus-dependent learning and memory (Messier et al. 1991; Deschaux et al. 1997; Ikonen & Riekkinen, 1999; Stackman et al. 2002; Deschaux & Bizot, 2005; Hammond et al. 2006; Brennan et al. 2008). We also find complementary effects of Kv7/M and SK channels on synaptic integration. Two preliminary reports of these results have already been presented in abstract form (Mateos-Aparicio et al. 2010a,b). Methods Ethical approval All procedures were approved by the responsible veterinarian of the Institute, in accordance with the statute regulating animal experimentation (Norwegian Ministry of Agriculture, 1996). The experiments reported in this work comply with the policies and regulations of The Journal of Physiology (Drummond, 2009). The animals were deeply anaesthetized with isoflurane inhalation before rapid decapitation. Hippocampal slice preparation Horizontal hippocampal slices ( μm) were cut from young male Wistar rats (3 5 weeks of age). Following decapitation, the brain was quickly removed and submerged in ice-cold artificial cerebrospinal fluid (ACSF) with the following content (in mm): 87 NaCl, 75 sucrose, 25 NaHCO 3,16D-glucose, 2.5 KCl, 1.25 NaH 2 PO 4, 7 MgCl 2 and 0.5 CaCl 2. Slices were prepared using a vibratome (either Vibratome, MO, USA or Leica VT1200S, Heidelberg, Germany) and incubated for 30 min at 35 CintheACSF solution mentioned above. Before recording, slices were stored at room temperature and then transferred to the recording chamber superfused with ACSF: 125 NaCl, 25 NaHCO 3,25D-glucose, 1 MgCl 2, 2.5 KCl, 1.25 NaH 2 PO 4 and 1.6 CaCl 2. All solutions were saturated with 95% O 2 and 5% CO 2. Electrophysiological recordings Somatic whole-cell patch clamp recordings were obtained from DGCs visually identified under infrared differential interference contrast (IR-DIC) video microscopy. All recordings were made in the suprapyramidal blade of the DGC layer. Slices were superfused with ACSF (2 3 ml min 1 ) and the temperature was maintained between 31 and 33 C (less than 0.5 C variationduring each recording). Patch pipettes (4 7 M ) were pulled from borosilicate glass tubing (Sutter Instruments, CA, USA) and filled with a solution containing (in mm): 120 KMeSO 4, 10 KCl, 10 phosphocreatine disodium salt,10hepes,10inositol,4mgatpand0.3nagtp, adjusted to ph 7.2 with KOH, and with an osmolarity of mosmol l 1. For somatic current clamp recordings, a Dagan BVC 700A amplifier (Dagan Corporation, MN, USA) was used, and signals were low-pass filtered at 5 or 10 khz ( 3dB) and digitized at 10 or 20 khz, respectively. In order to block spontaneous synaptic transmission, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μm), DL-2-amino-5-phosphonopentanoic acid (DL-AP5, 50 μm) and gabazine (5 μm)wereroutinely added to the ACSF during current clamp experiments. In some experiments, the adenylyl cyclase activator forskolin (50 μm) was added to the bath in order to block the sahp and measure the mahp in isolation. Whole-cell voltage clamp recordings were obtained using an Axopatch 1-D amplifier (Axon Instruments, CA, USA), filtered at 5 khz ( 3dB),anddigitizedat10kHz.TorecordSK-mediated currents in relative isolation, TTX (1 μm) and TEA (5 mm) were routinely added to the ACSF, blocking Na + and some K + channels (including BK, Kv7/M, and delayed rectifier channels), respectively (Sailer et al. 2002). The cell was voltage clamped at a holding potential of 50 mv, and currents were elicited by a brief (100 ms) depolarizing voltage step to +10 mv once every 60 s. Under these conditions, the depolarizing step triggered a robust, unclamped calcium spike followed by a brief inward tail current and slower outward tail currents. The access resistance was typically M for current clamp recordings and M for voltage clamp experiments. In current clamp, recordings in which the access resistance changed >25% were discarded. Series resistance compensation was not used in voltage clamp experiments; neurons with >10% variation in series resistance were discarded. Potentials are corrected for the liquid junction potential error (7 mv).

4 672 P. Mateos-Aparicio and others J Physiol Data acquisition, storage and analysis The data were acquired using pclamp 10 (Axon Instruments) and stored in a computer for further analysis with Clampfit 10. Statistical analysis was performed using Origin 8.0 (Microcal). Values are expressed as means ± SEM, two-tailed Student s t test was used for statistical significance (α = 0.05), and the P values are given in the figure legends. During whole-cell current clamp recordings of AHPs, each cell was kept constantly depolarized at 62 mv by injection of a positive holding current in order to unmask the AHPs and measure them at the same potential in all cells. The mahp and sahp values were measured by averaging a time window (30 and 100 ms, respectively) around the peak of each one (20 50 ms and s after a spike train, respectively). Spike frequency was measured by injecting depolarizing pulses (1 s) at different intensities, keeping the background membrane potential at 77 mv. The cell input resistance (R input )wasdeterminedfromthevoltageattheendof 500 ms small hyperpolarizing current pulses (0.1 na), by dividing the steady-state voltage response by the current pulse amplitude (R input = V/ I). For time course analysis, the input resistance was measured by injecting small (0.05 na) hyperpolarizing pulses (1 or 0.5 s long) once every minute, while the cell s membrane potential waskeptdepolarizedat 62mV.Intheexperimentsshown in Figs 9 and 10, we injected (through the somatic patch pipette) trains of artificial excitatory postsynaptic currents ( αepscs ) defined by a double exponential function: f (t) = a((1 exp( t/τ on ))(exp( t/τ off )). In both figures, τ on was set to 0.5 ms and τ off to 10 ms. In voltage clamp, the apamin-sensitive current (I aahp ) was obtained by subtracting traces recorded after apamin application from those recorded before, and the peak of the apamin-sensitive current was calculated by averaging points within a 20 ms time window at the peak of the subtracted trace. Due to the small size of I aahp in the DGCs, digital subtraction was required to distinguish this component from the entire I AHP. Chemicals and drugs Apamin, gabazine and forskolin were purchased from Sigma-Aldrich Norway AS (Oslo, Norway). A different batch of apamin and scyllatoxin (leiurotoxin I) was purchased from Latoxan (Valence, France). DNQX, DL-AP5, 1-EBIO, TTX and XE991 were obtained from Tocris Bioscience (Bristol, UK). Retigabine was obtained from Alomone Labs (Jerusalem, Israel). Potassium methyl sulphate (KMeSO 4 ) was obtained from Fluka Chemie AG (Switzerland) and MP Biomedicals (USA). Other chemicals used in this study were from Sigma-Aldrich Norway AS. All drugs were bath-applied and superfused to the slices (2 3 ml min 1 ). Modelling We used a compartmental model of a DGC based on Aradi & Holmes (1999). Simulations were performed using NEURON 7.2 (Carnevale & Hines, 2006). As described in the online Supporting information (section S1), a number of adjustments to their model were required in order to account for our experimental data. In particular we included an axonal Hodgkin Huxley-type M-conductance (g M ) based on the analysis of voltage clamp data in Main et al. (2000). The model also included a somatic sahp-conductance (g sahp ) but this is set to zero in Fig. 6 to mimic the situation in which the sahp was blocked with forskolin (Fig. 4) or is minimal (Fig. 5). Details of the modelling procedures are included in the online Supporting information. Results In this study, whole-cell patch clamp recordings were obtained from 77 mature DGCs from the suprapyramidal blade of the DG in hippocampal slices from young male rats (3 5 weeks postnatal). The mean resting membrane potential (RMP) was 82.4 ± 0.5 mv. Because neurons in the DGC layer are known to be heterogeneous, all of the DGCs used in this study were identified using the criteria defined by Lübkeet al. (1998). In particular, the DGC layer contains subpopulations of neurons at different stages of maturation (Liu et al. 2000), due to ongoing neurogenesis of DGCs throughout the life of the animal (van Praag et al. 2002). These maturational stages can be distinguished, among other parameters, by the R input, which is known to be markedly higher in young than in mature neurons, as well as by the RMP (Liu et al. 2000; but see Schmidt-Hieber et al. 2004) and AP threshold (Schmidt-Hieber et al. 2004). Therefore, to record from a uniform population of mature DGCs, we selected for this study only cells having RMP more negative than 75 mv, AP threshold between 45 and 50 mv, stable AP amplitude higher than 70 mv, and R input < 350 M. SK channels contribute to the mahp but not to the sahp in DG granule cells SK channels are known to show cumulative activation during and after a train of APs, due to accumulation of calcium in the cytoplasm (Pennefather et al. 1985; Lancaster & Adams, 1986; Sah & Faber, 2002). To compare the mahp and sahp amplitudes following a constant amount of Ca 2+ influx, the cells were kept depolarized at 62 mv by steady current injection, and spike trains of

5 J Physiol SK and Kv7/M channels underlying mahp and spiking control in DGCs spikes were evoked by applying depolarizing current steps (100 ms duration). Under these conditions, all of the recorded DGCs (n = 54) showed a fahp after each spike during the train, as well as the classical sequence of AHPs after each spike train: a mahp followed by a sahp. In order to test whether SK channels contribute to the mahp or sahp we bath-applied apamin (100 nm), a bee venom toxin that selectively blocks SK channels. Figure 1A shows typical recordings from a rat DGC under control conditions and after bath application of 100 nm apamin. The mahp was strongly reduced after apamin application (Fig. 1A, Overlay,andFig.1B), while the sahp was not significantly affected (Fig. 1C). When the depolarizing current pulse was kept constant, apamin caused an increase in spike frequency, indicating that SK channels control excitability (see online Supporting information Fig. S2A, Apamin). To keep the evoked number of APs constant, for comparison of AHPs, we reduced the current pulse amplitude after apamin application (the mean ± SEM was reduced from 0.25 ± 0.03 na to 0.18 ± 0.03 na; Fig. 1A,bottomtraces; n = 8, P < 0.01), and always recorded the mahp from the holding potential of 62 mv. Under these conditions, the peak amplitude of the mahp was rapidly suppressed by more than 80% by apamin (from 4.0 ± 0.6 mv to 0.6 ± 0.2 mv; Fig. 1B; n = 8, P < 0.001). In contrast to its effect on the mahp, apamin did not significantly reduce the sahp amplitude, in agreement with results from CA1 pyramidal and other neurons (Lancaster & Nicoll, 1987; Storm, 1989; Stocker et al. 1999; Gu et al. 2005, 2008). Thus, the peak amplitude of the sahp (4.8 ± 1.6 mv in control medium, before apamin) did not change significantly after apamin application (4.2 ± 1.5 mv; Fig. 1A and C; n = 8, P > 0.05). Under control conditions (without apamin in the bath), the sahp amplitude was more variable between cells (range: mv; Fig. 1C, right) than the mahp amplitude (range: mv; Fig. 1B, right). Although non-specific, SK channel-independent effects of 100 nm apamin have to our knowledge not been reported, such effects are conceivable. Therefore, to further test our hypothesis about the roles of SK channels in the mahp and excitability control, we also used a different SK channel blocker: the scorpion toxin scyllatoxin (leiurotoxin I), which has been reported to block SK channels in dorsal vagal neurons (Pedarzani et al. 2000) and CA1 pyramidal neurons (Stocker et al. 1999). Scyllatoxinhasbeenshowntocompetewith 125 I-apamin binding sites in the rat brain (Auguste et al. 1992). In a subset of five DGCs, we recorded the mahp after a train of eight action potentials (Fig. 2A, Control). Bath application of 100 nm scyllatoxin strongly reduced the mahp (Fig. 2A, Scyllatoxin, Overlay). As we saw with apamin, scyllatoxin also increased the spike frequency in response to a constant, depolarizing step current, so we had to reduce the step current to evoke a constant number of spikes (Fig. 2A,overlay,bottom traces).figure 2C (left) shows the average time course of the mahp amplitude before and after application of scyllatoxin. The effect of scyllatoxin was significant for all the five cells tested (Fig. 2C,right;n = 5, P < 0.01). To further elucidate the contribution of SK channels to the mahp and excitability control, we also applied the SK and IK (SK4) channel opener 1-EBIO, a small organic (benzimidazolinone) compound (Pedarzani et al. 2001, 2005). 1-EBIO is known to increase the apparent sensitivity of SK channels to calcium, therefore increasing the amplitude and duration of SK channel-mediated currents in a variety of neurons, including CA1 pyramidal cells (Pedarzani et al. 2001, 2005). Figure 2B shows that 500 μm 1-EBIO enhanced both the amplitude and duration of the mahp (overlay). In the subset of cells that we used for 1-EBIO experiments, the sahp was small or absent (due to the cell-to-cell variation in sahp), thus making the effect on the mahp clearly visible. The mahp amplitude was 4.8 ± 0.2 mv during the control period and increased to 6.9 ± 0.2 mv after bath application of 1-EBIO (Fig. 2D, right;n = 5, P < 0.001). The left panel in Fig. 1D shows the averaged time course of the effect. In addition, it was necessary to apply a stronger depolarizing current step to maintain a constant number of seven spikes within each spike train, showing that 1-EBIO reduced the cell s excitability (Fig. 2B, Overlay, bottom). Overall, the results indicate that, unlike CA1 pyramidal cells (Gu et al. 2005, 2008), SK channels make a major contribution to the mahp in DGCs. The mahp increased with the number of spikes In order to control the exact number and timing of spikes, we applied a train of brief (2 ms) depolarizing pulses within 100 ms, each pulse evoking only a single AP (Supporting information Fig. S1). We thus applied spike trains at different frequencies: 50, 70, 100 and 120 Hz, while maintaining a depolarized background membrane potentialof 62mV(Fig.S1A).Asexpected,andas observed in CA1 pyramidal cells (Lancaster & Adams, 1986), both the mahp and the sahp increased monotonically in amplitude with the number of spikes in the train (Fig. S1A). As shown in the time course plots, apamin again strongly suppressed the mahp in all cases (Fig. S1B), but had no detectable effect on the sahp amplitude (Fig. S1C). The effect of apamin was significant for all four frequencies (Fig. S1D, n = 6, P < 0.001). Following trains of only five spikes, when the mahp and sahp had relatively small amplitudes, a slow afterdepolarization (sadp) appeared after apamin had abolished the mahp ( sadp in Supporting information Fig. S1A). This suggests that the spike train evokes a

6 674 P. Mateos-Aparicio and others J Physiol long-lasting inward tail current that may cause a sadp, but that this effect is normally prevented (masked) by the mahp and sahp. Taken together, these results indicate that SK channels underlie the mahp but not the sahp following spike trains in DGCs. Feedback regulation of Ca 2+ influx and sahp by SK channels We next asked whether SK channels mediate negative feedback regulation of calcium influx in DGCs, by regulating the spike frequency and spike number. As mentioned above, apamin increased the AP frequency and thus spike number in response to a constant depolarizing current pulse, and this is expected to enhance the Ca 2+ influx. Since the sahp is highly calcium sensitive, we used the sahp amplitude as an indicator of the intracellular calcium level (Supporting information Fig. S2A). We used for this particular purpose a subset of cells (n = 6) which had a sahp amplitude ranging from 3.1 to 13.6 mv. Apamin application increased the number of spikes during the pulse (Fig. S2A, Control, Apamin), while the mahp was reduced (although partly masked by the overlapping sahp, Fig. S2A, Overlay, arrow). In parallel, the sahp amplitude increased from 5.7 ± 1.6 mv (control) to 7.4 ± 2.2 mv after apamin (Fig. S2B and C; n = 6, P < 0.05). Apamin often also increased the duration of the sahp (Fig. S2A). These results support the idea that the SK channels mediate Figure 1. Effect of apamin on the mahp and the sahp in DGCs A, typical examples of whole-cell current clamp recordings from a DGC showing repetitive spiking in response to a depolarizing current pulse (100 ms long) before (black) and after (red) bath application of 100 nm apamin. The APs are followed by fahp, mahp and sahp. The background membrane potential prior to stimulation was kept at 62 mv (dashed line) by depolarizing holding current injection, and the current pulse amplitude was adjusted to produce a train of seven APs. The insets show expanded traces from the marked periods (dashed rectangles). Note that apamin suppressed the mahp, but had essentially no effect on the sahp. B, left, time course of the effect of apamin on the mahp peak amplitude (n = 8). The summary graph (right) shows the effect of apamin (n = 8, P < ( )) on the mahp in all cells tested ( ) and the mean value ( ). C shows that apamin had no significant effect on the sahp peak amplitude (n = 8, P > 0.05 (NS)). For the analysis, the mean AHP amplitudes during the last 3 min (1) before apamin application were compared to the last 3 min after full effect of apamin (2).

7 J Physiol SK and Kv7/M channels underlying mahp and spiking control in DGCs 675 negative feedback regulation of Ca 2+ influx in DGCs, and that this can be detected indirectly through modulation of the sahp. To measure the apamin-sensitive current, I aahp,we used somatic whole-cell voltage clamp (see Methods). Following a depolarizing voltage step, there was an outward tail current that showed no obvious early component, but a prominent slow component lasting about 3 s (Fig. S3A, Control), in agreement with previous results (Sailer et al. 2002). Bath application of 100 nm apamin uncovered an early inward transient and delayed the peak of the total outward current (Fig. S3A, Apamin). Digital subtraction of the traces recorded before and after full effect of apamin revealed an early, outward, I aahp, lasting about 200 ms, with a fast rising phase and a slower decay, also in agreement with Sailer et al The apamin-sensitive current peaked within ms after the onset of the depolarizing step (n = 5), with a mean amplitude of 46.9 ± 6.0 pa (Fig. S3C, Subtracted). Fig. S3D shows the time course of the effect of the apamin on the tail current by plotting the subtracted current recorded once every minute after bath application of 100 nm apamin, for all cells tested. The effect of apamin was significant for all cells tested (Fig. S3E; n = 5, P < 0.001). Do Kv7/M channels also contribute to AHPs in DGCs? Results from whole-cell voltage clamp recordings in DGCs from transgenic mice lacking KCNQ2 and 3 subunits indicate that an M-current component contributes about Figure 2. SK-channel blockade with 100 nm scyllatoxin and enhancement with 500 μm 1-EBIO suppressed and increased, respectively, the mahp amplitude in DGCs A, typical examples showing the mahp under control (black) and 100 nm scyllatoxin application (red). The overlay shows the mahp effectively blocked after toxin application. B, the mahp was enhanced by 500 μm 1-EBIO application. Both the mahp amplitude and duration were increased after 1-EBIO application (red) compared to control traces (black). Note the increased positive current step injected to evoke a train of seven APs after 1-EBIO application. C, time course showing the effect of 100 nm scyllatoxin application during the recording time (n = 5, left panel). Summary graph of the five cells tested with this toxin (right panel) showing the comparison between control period (1) and values with full effect (2), measured in the time plot (n = 5, P < 0.01 ( )). D, similar analysis ofthedatainb, showing enhancement of the mahp after 1-EBIO application (n = 5, P < ( )).

8 676 P. Mateos-Aparicio and others J Physiol % of both the AHP-related tail currents I mahp and I sahp (Tzingounis & Nicoll, 2008). However, the relative contribution of the M-current to the mahp and sahp under current clamp conditions might be different, and this has not been examined previously. For example, in CA1 pyramidal cells, a prominent apamin-sensitive I mahp was found following calcium spikes in voltage clamp (Stocker et al. 1999; Sailer et al. 2002), but apamin had little or no effect on the mahp after a normal spike train in current clamp (Storm, 1989; Gu et al. 2005, 2008). In order to examine the mechanisms of mahp and sahp, we applied both the specific Kv7/M channel blocker XE991 (10 μm) and the Kv7/M channel opener retigabine (10 μm) separatelyandrecordedtheireffectsondgcsincurrent clamp (Fig. 3), using the same protocol as shown in Figs 1 and 2. XE991 application was followed by a reduction of both the mahp and sahp amplitudes (Fig. 3A, top traces). Thus, the mahp peak was significantly reduced from 5.5 ± 0.7 mv (control) to 2.6 ± 0.7 mv in XE991 (Fig. 3D, top left; n = 7, P < 0.05). XE991 application also reduced the peak sahp amplitude from 5.2 ± 0.7 mv during the control period to 2.6 ± 0.5 mv in XE991 (Fig. 3C and D,topright;n = 7, P < 0.01). Next, we tested the effects of retigabine. The mahp was significantly increased after retigabine application, from 5.9 ± 0.2 mv (control) to 7.6 ± 0.3mVinretigabine(Fig.3C and D, bottom left; n = 6, P < 0.001). In contrast, the sahp amplitude was significantly reduced, from 4.5 ± 0.8 mv to 2.0 ± 0.4 mv (Fig. 3A, bottom;fig.3c and D bottom right; n = 6, P < 0.01). For comparison, to test whether Figure 3. Kv7/M channel contribution to the mahp and sahp in DGCs A, representative examples showing the effect of Kv7/M channel blockade (top traces) by XE991 (10 μm). Bottom traces show the effect of the Kv7/M channel opener retigabine (10 μm) on both the mahp and sahp. Note the reduction in holding current and depolarizing step after application of XE991 and the increase in the depolarizing step after application of retigabine. B, time plots for both the mahp and sahp amplitudes in a subset of cells (n = 5) recorded under control conditions, to test the stability of AHPs in our experimental conditions. Dashed lines represent the mean values during the control period (first 5 min), in the case of drug application. C, averaged time plots of the experiments shown in A for both the mahp and sahp. D, summary graphs showing the effect of both XE991 (n = 7, P < 0.05 ( ), P < 0.01 ( ), top panels) and retigabine (n = 6, P < ( ), P < 0.01 ( ), bottom panels) on the mahp (left) and the sahp (right).

9 J Physiol SK and Kv7/M channels underlying mahp and spiking control in DGCs 677 the changes observed in Fig. 3C and D were caused by the added blockers or were merely time dependent, we also show (Fig. 3B) the time courses of mahp and sahp amplitudes for five cells that were recorded in the absence of blockers. These plots show that the AHPs were quite stable for a comparable time period under control conditions (n = 5). XE991 and retigabine seemed to have quite clear, opposite effects on the mahp as judged from the time courses (Fig. 3C), and both effects support the conclusion that Kv7/M channels contribute to the mahp. However, the effects of XE991 and retigabine on the sahp seem at first glance contradictory: the apparent reduction by XE991 seems to suggest that Kv7/M channels contribute to the sahp, but retigabine did not produce the expected sahp enhancement but rather the opposite. As discussed below, this apparent paradox may be resolved if retigabine reduces the calcium influx, or if retigabine opens Kv7/M channels already before the sahp occurs, thus causing occlusion or shunting. Thus, it is possible that XE991 and retigabine may have indirect effects on the sahp. The recent proposal that sahps may be generated by different channel types (Andrade et al. 2012) must also be considered. In summary, our results support the conclusion that Kv7/M channels contribute to the mahp of DGCs, although the full effect of this channel type on the mahp may be partly masked by the overlapping sahp. This may lead to an underestimation of the real contribution of Kv7/M channels to the mahp (Gu et al. 2005; Kaczorowski et al. 2007). To prevent the mahp from being contaminated by the sahp, we next studied the mahp in isolation, after suppressing the sahp. SK and KCNQ channel contribution to the isolated mahp As reported for CA1 pyramidal cells (Madison & Nicoll, 1986; Pedarzani & Storm, 1993; Gu et al. 2005), the adenylyl cyclase activator forskolin strongly suppressed the sahp in DGCs(Fig. 4A, Forskolin). We exploited this to study the mahp in isolation (Gu et al. 2005). Application of 100 nm apamin almost fully suppressed the isolated mahp, reducing the peak amplitude from 3.7 ± 0.3 mv (control) to 0.5 ± 0.3 mv in apamin (Fig. 4A, toptraces, and B; n = 5, P < 0.001). Next, we tested the effect of Kv7/M channel blockade on the isolated mahp (Fig. 4A, bottom traces), and found that XE991 (10 μm) reduced the isolated mahp from 5.6 ± 0.5 mv to 4.0 ± 0.3 mv (Fig. 4C; n = 7, P < 0.05). Thus, in the absence of the sahp, XE991 had apparently a weaker effect on the mahp than shown in Fig. 3, probably because of the apparent XE991 effect on the overlapping sahp in Fig. 3A (top traces). In summary, these results suggest that, in contrast to other hippocampal principal neurons, both SK and Kv7/M channels differentially contribute to the mahp in DGCs, with SK channels being the main generators of the mahp in this cell type. SK channels generate the mahp after single action potentials Previous studies have suggested that mahps after single and multiple APs can contribute to the refractory period and control interspike intervals (ISIs) and spike frequency (Lanthorn et al. 1984; Storm, 1989, 1990). In addition, a recent network model of stellate neurons in the medial entorhinal cortex (MEC) suggests that after-spike dynamics such as mahp and ADP may be crucial for both phase precession and the change in scale of grid fields along the dorso-ventral axis of MEC (Navratilova et al. 2012). To study mahps after single APs, we evoked slow repetitive firing ( Hz) by injecting weak, steady depolarizing current at just suprathreshold intensity (Fig. 5A). Each of these APs was followed by a prominent fahp, a mahp (Fig. 5A, Control), and in some cells also an appreciable sahp, as found in CA1 pyramidal cells (Storm, 1987). Bath application of apamin abolished (n = 6) the single spike mahp (Fig. 5A, Apamin, Overlay), suggesting that SK channels activated by Ca 2+ influx from a single AP generate the mahp. In contrast, blockade of Kv7/M channels by XE991 did not significantly (n = 6) reduce the mahps after single, low frequency APs, although there was a slight reduction in the mean mahp amplitude, from 4.4 ± 0.5 mv to 4.1 ± 0.5 mv (Fig. 5A, bottom;n = 6, P > 0.05). Unexpectedly, it thus appears that, whereas SK channels are strongly activated by a single AP, generating the somatic mahp, Kv7/M channels are not or only minimally activated by single spikes in DGCs, in contrast to hippocampal CA1 pyramidal cells, where it is exactly the opposite (Storm, 1989; Gu et al. 2005, 2008). In many neurons, Kv7/M channels are known to be concentrated in the axon and/or axon initial segment (AIS), and can modulate the spike threshold (Shah et al. 2008). Since both immunocytochemical (Cooper et al. 2001) and patch clamp data (Alle et al. 2009) indicate that there is a high density of Kv7/M channels in the MF axons of the DGCs, we wished to test whether they affect the spike threshold of these cells. We used phase plane plots to accurately determine the threshold of evoked low frequency spikes like those shown in Fig. 5A, and found that 10 μm XE991 shifted the AP threshold to more negative potentials, by 3.6 ± 0.2 mv. This indicates that Kv7/M channels regulate the AP threshold in these cells. In contrast, apamin application caused no significant change in the spike threshold (Fig. 5B, top right) nor in the AP waveform (Fig. 5B, top left), indicating that SK channels have little or no influence on these parameters. This is

10 678 P. Mateos-Aparicio and others J Physiol not surprising, since SK channel activation depends on calcium influx, which is thought to mainly occur during APs in DGCs. In conclusion, these results indicate that SK channels are the main generators of mahps after each single action potential during low frequency firing, whereas the Kv7/M channels regulate the spike threshold, but contribute little to the single-spike mahp. Modelling the contribution of SK and Kv7/M channels to the mahp In order to better understand the contributions of SK and Kv7/M channels to the mahp, we constructed a computational compartmental model of a DGC based on the model of Aradi & Holmes, 1999 (see Methods and online Supporting information). Figure 6A and B shows that the DGC model gives a semiquantitative account of the isolated mahp following seven APs (panel A) or one AP (panel B). The continuous black curves show the control situation in which both the SK- and M-conductances (g SK and g M, respectively) are present. Omitting g SK ( SK in the figure legend) essentially abolished the mahp following both seven APs and one AP (red curves in Fig. 6A and B). In contrast, omitting g M ( M ) resulted in only a small reduction in the mahp following seven APs and virtually no effect following one AP (green curves). Because the sahp was not included in the model, these simulation results should be compared with the experimental data obtained in the presence of forskolin (Fig. 4, 7 APs) and the single AP results where Figure 4. Effects of SK and Kv7/M channel blockade in the mahp of dentate gyrus granule cells after sahp suppression by forskolin A, typical traces showing the effect of apamin (100 nm, top traces) on the isolated mahp after sahp suppression by forskolin (50 μm); bottom traces show representative examples of the effect of XE991 (10 μm) on the isolated mahp. B, averaged time plot (n = 5, left panel) showing the effect of apamin on the time course of the isolated mahp. Summary plot (right panel) showing the individual and mean values before and after application of apamin (n = 5, P < ( )). C, similar plots summarizing the effect of XE991 on the isolated mahp (n = 7, P < 0.05 ( )). Note the smaller mahp reduction after Kv7/M channel blockade compared to SK channel blockade.

11 J Physiol SK and Kv7/M channels underlying mahp and spiking control in DGCs 679 Figure 5. Effects of SK and Kv7/M channel blockade on spike threshold and mahp after single APs A, typical examples of single AP elicited by steady depolarization near the spike threshold. In control conditions (black trace), the spontaneous AP was followed by a fahp and mahp. Bath application of 100 nm apamin (red) effectively blocked the mahp, while 10 μm XE991 (green) had little or no effect on the mahp amplitude. Right panel shows individual and averaged values for the mahp under control and apamin (n = 6, P < 0.01 ( )) or XE991 (n = 6, P > 0.05 (NS)) conditions. B, left, overlay of averaged single APs under control (black) and apamin (red) or XE991 (green) conditions. Right, phase plots showing the dv/dt of the AP waveform plotted against the voltage. Upper panel shows that application of apamin (red) did not affect the AP threshold while XE991 (bottom panel, green) had a hyperpolarizing effect.

12 680 P. Mateos-Aparicio and others J Physiol the sahp was minimal (Fig. 5). Figure 6C and D shows the total SK- and M-currents (i.e. integrated over the model neuron). It can be seen that following seven APs, I SK predominates for the first 100 ms and is the main cause of the peak mahp (Fig. 6C). Note that at the end of the current pulse, I SK is quite similar to the peak apamin-sensitive current measured under voltage clamp (Supporting information Fig. S3). I M is initially smaller than I SK but lasts longer and so produces a more prolonged but small AHP (cf. black and green curves in Fig. 6A). I M is hardlyactivated by asingle AP (Fig.6D) and consequently has little effect on the mahp which is essentially due to I SK (cf. black and green curves in Fig. 6B). In the model, the greater effect of g M activation on the mahp following seven APs as compared with one AP reflects a progressive recruitment of proximal axonal Kv7/M channels during the AP train (Supporting information, section S2, Fig. S8A and B). In the above simulations, g SK was inserted into the soma, the granule cell layer dendrites and the proximal dendrites, as described by Aradi & Holmes, However, the spatial distribution of g SK was not critical. Thus, virtually identical mahps could be obtained with g SK inserted uniformly in the dendrites only, provided the total g SK remained the same, or with g SK in the distal dendrites only, with the total SK-conductance increased 3-fold. The latter increase was necessary to compensate for a reduced Ca 2+ influx through voltage-gated Ca 2+ channels associated with AP attenuation; without this adjustment the mahp was greatly reduced (Supporting information Fig. S11A, grey curve). It should also be noted that Kv7/M channel kinetics and the maximum specific conductance (ḡ M = 20 ps μm 2 ) were spatially uniform in the axon. In particular, the voltage for half-maximal conductance (V half )was 38 mv and the activation time constants were 18- to 30-fold higher than those expected from Main et al. (2000). By contrast, the data of Alle et al. (2009) suggest a V half of around 50 mv and considerably lower τ values in MF boutons. These considerations raise the possibility that g M kinetics in the proximal axon are different from those in the rest of the axon, specifically that in the proximal axon g M activates/deactivates more slowly and at a more depolarized voltage than in the distal axon. To investigate this possibility we included a 20 μm-long AIS in the model. Within the AIS g M parameters were as before, but in the rest of the axon V half was set to 50 mv and the activation time constants were set according to the temperature-adjusted data of Main et al. (2000). Suitable adjustments of ḡ M in the AIS and distal axon yielded similar results to those obtained previously. For example, Figure 6. Model simulations of the mahp following seven APs (A and C) or a single AP (B and D) when gsahp = 0 A and B show plots of V(t) with + and indicating presence and absence of the M- and SK-conductances. These plots should be compared with the experimental data in Figs 4 and 5. C and D show plots of the currents I SK (t) andi M (t) (both integrated over the model neuron) for the +M, +SK regime.

13 J Physiol SK and Kv7/M channels underlying mahp and spiking control in DGCs 681 virtually identical results to those in Fig. 6 were obtained with ḡ M = 100 ps μm 2 in the AIS and 10 ps μm 2 in the distal axon and with no change in the injected I pulse. This proposed difference in g M kinetics between the AIS and distal axon represents a prediction of the model that is in principle experimentally testable. In the above simulations g sahp was set to zero. Since the sahp is not the focus of this paper, simulation results for the sahp are presented in the Supporting information, section S3. The main results are: (a) the simulated effect of retigabine on the sahp is consistent with observation; and (b) contrary to the observed effect of XE991, setting g M = 0 causes a slight increase in the sahp following seven APs (see Supporting information Fig. S9C). Accordingly, it is suggested that either the XE991-sensitive component of the sahp is not due to g M or that when contributing to the sahp, g M is operating in a mode different from that associated with the mahp. Differential control of excitability mediated by SK and Kv7/M channels To test how SK and Kv7/M channels control DGC excitability, we evoked repetitive firing by applying 1 s-long depolarizing current pulses starting from a membrane potential of 77 mv, which is close to the RMP of these cells. Apamin clearly enhanced the excitability: it increased the number of spikes (Fig. 7A, red)and increased the slope of the f I plot from 48.2 ± 1.9 Hz na 1 to ± 6.1 Hz na 1 (Fig. 7C, top panel, where the average spike frequency (f) during the 1 s-long pulse is plotted against the injected current intensity (I); n = 6, P < 0.001). Apamin did not, however, change the neuronal R input,as shown in Fig. 7B (red). To see how SK channels affected the spike frequency adaptation, and thus how the SK channel impact changed during a spike train, we plotted the average spike frequency for 100 ms time windows during a 1 s-long current pulse of 0.15 na (Fig. 7D). Apamin significantly increased the spike frequency only within the first 200 ms of the response (for the interval ms: 6.7 ± 0.8 Hz in control, 23.3 ± 0.8 Hz after apamin, n = 6, P < 0.001; for the interval ms: 6.7 ± 0.8 Hz control, 16.6 ± 0.8 Hz apamin, n = 6, P < 0.001), for all cells tested (Fig. 7D, top panel). Conversely, 1-EBIO (500 μm) shifted the slope of the f I plot from 44.6 ± 10.1 Hz na 1 in control conditions to 31.8 ± 8.8 Hz na 1 (Supporting information Fig. S12A and B; n = 5, P = 0.054). 1-EBIO decreased the number of spikes during a 1 s-long current injection of 0.25 na (Fig. S12B, n = 5, P < 0.05), most notably during the first 200 ms of the response (Fig. S12C). Thus, SK channels are important only for the early spike frequency adaptation, but apparently less for the late adaptation under these conditions. However, as discussed below, it is likely that an enhancement of the sahp due to increased Ca 2+ influx caused by enhanced firing in the beginning of the train, partly masked an effect of the SK channels during the last part of the train. Next, we tested the role of Kv7/M channels in excitability control. XE991 (10 μm) also increased the number of spikes (Fig. 7A, green) and thus also the slope of the f I plot from 46.3 ± 2.1 Hz na 1 to 87.4 ± 3.2 Hz na 1 (Fig. 7C,bottom;n = 6, P < 0.001). In contrast to apamin, XE991 also increased the R input measured at subthreshold potentials (Fig. 7B, green). The plot of the spike frequency within 100 ms time windows (Fig. 7D, bottom) revealed several differences compared to SK channel blockade. While apamin selectively and sharply increased the early spike frequency only during the first 200 ms, XE991 produced a uniform and more moderate increase in spike frequency throughout the entire spike train. Thus, unlike apamin, XE991 enhanced also the steady firing rate towards the end of the 1 s-long train. Finally, we examined in detail the early spike frequency adaptation under conditions of constant initial spike frequency (Fig. 8). Using a stimulation protocol similar to the one used in Figs 1 4, we injected 100 ms-long depolarizing current pulses to evoke a train of six action potentials per pulse. After apamin application, we reduced the current pulse intensity in order to obtain the same number of spikes before and during apamin. By further adjusting the strength of the current pulse, we also made sure that the first ISI duration was very similar before and during apamin (Fig. 8A, Overlay; Fig. 8B, top panel, first ISI), in order to ensure that the rate of spike adaptation could be compared under similar conditions. This is important because both calcium-dependent (e.g. SK, BK and sahp channels) and voltage-dependent (e.g. Kv7/M channel activation and Na v channel inactivation) mechanisms of adaptation are spike-frequency dependent. Spike trains should therefore be compared at similar initial spike frequencies to avoid interference between different mechanisms, e.g. an AP frequency-dependent increase in the sahp masking the effects of SK or Kv7/M channel blockade (Storm, 1989; Gu et al. 2005). We found that SK channel blockade by 100 nm apamin reduced the spike frequency adaptation during the pulse-evoked spike train (Fig. 8A, Overlay). Figure 8B (top panel) summarizes the data for all cells tested (n = 8). In order to compare the overall degree of adaptation during the spike train, we fitted a straight line to both control andapaminplotsinfig.8b, and compared the slope (termed adaptation index ; Gu et al. 2005) of the linear fits. Apamin significantly reduced the adaptation index to 67.4% of the value under control conditions (Fig. 8C, red; n = 8, P < 0.05). In contrast, 500 μm 1-EBIO produced the opposite effect to apamin (Supporting information Fig. S12D). Thus, the SK opener significantly increased the adaptation index to 172.1% of the control value (Fig. S12E and F; n = 5, P < 0.01).