Cav1.2 calcium channels modulate the spiking pattern of hippocampal pyramidal cells

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Life Sciences 82 (2008) 41 – 49 www.elsevier.com/locate/lifescie

Cav1.2 calcium channels modulate the spiking pattern of hippocampal pyramidal cells Lubica Lacinova a,⁎, Sven Moosmang b , Nicole Langwieser b , Franz Hofmann b , Thomas Kleppisch b b

a Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, 833 34 Bratislava, Slovakia Institut für Pharmakologie und Toxikologie, Technische Universität München, Biedersteiner Straβe 29, 80802 München, Germany

Received 26 March 2007; accepted 18 October 2007

Abstract Cav1.2 L-type calcium channels support hippocampal synaptic plasticity, likely by facilitating dendritic Ca2+ influx evoked by action potentials (AP) back-propagated from the soma. Ca2+ influx into hippocampal neurons during somatic APs is sufficient to activate signalling pathways associated with late phase LTP. Thus, mechanisms controlling AP firing of hippocampal neurons are of major functional relevance. We examined the excitability of CA1 pyramidal cells using somatic current-clamp recordings in brain slices from control type mice and mice with the Cav1.2 gene inactivated in principal hippocampal neurons. Lack of the Cav1.2 protein did not affect either affect basic characteristics, such as resting membrane potential and input resistance, or parameters of single action potentials (AP) induced by 5 ms depolarising current pulses. However, CA1 hippocampal neurons from control and mutant mice differed in their patterns of AP firing during 500 ms depolarising current pulses: threshold voltage for repetitive firing was shifted significantly by about 5 mV to more depolarised potentials in the mutant mice (p b 0.01), and the latency until firing of the first AP was prolonged (73.2 ± 6.6 ms versus 48.1 ± 7.8 ms in control; p b 0.05). CA1 pyramidal cells from the mutant mice also showed a lowered initial spiking frequency within an AP train. In control cells, isradipine had matching effects, while BayK 8644 facilitated spiking. Our data demonstrate that Cav1.2 channels are involved in regulating the intrinsic excitability of CA1 pyramidal neurons. This cellular mechanism may contribute to the known function of Cav1.2 channels in supporting synaptic plasticity and memory. © 2007 Elsevier Inc. All rights reserved. Keywords: Neuronal excitability; Action potential; Repetitive firing; CA1 pyramidal cells; Cav1.2 channel

Introduction L-type calcium channel-mediated Ca2+ influx regulates a variety of functions in the CNS. For example, it contributes to resting intracellular Ca2+, is involved in neuronal plasticity, supports memory, and controls gene expression (Grover and Teyler, 1990; Bito et al., 1996; Impey et al., 1996; Magee et al., 1996; Graef et al., 1999; Morgan and Teyler, 1999; Borroni et al., 2000; Dolmetsch et al., 2001; Woodside et al., 2004). Two L-type isoforms are generally prevalent throughout the CNS: Cav1.2 and Cav1.3. In the neocortex and hippocampus, the vast majority belong to the Cav1.2 subtype (Hell et al., 1993; Sinnegger-Brauns et al., 2004). Evidence showing its domi⁎ Corresponding author. Tel.: +421 2 5477 2311; fax: +421 2 5477 3666. E-mail address: [email protected] (L. Lacinova). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.10.009

nance on the functional level came recently from two studies using a genetic approach: we have previously shown that mice lacking the Cav1.2 channel in hippocampal pyramidal cells exhibit defects in spatial learning, late phase LTP (L-LTP), CRE-mediated gene expression and related protein synthesis (Moosmang et al., 2005), while no such phenotypes were observed in mice lacking the Cav1.3 isoform (Clark et al., 2003). L-type channel activity supporting LTP and, likely, memory formation may arise from invading back-propagated somatic APs (bAP) (Spruston et al., 1995; Hoffman et al., 1997; Markram et al., 1997; Kampa et al., 2006). Moreover, somatic APs alone are (i) sufficient to activate signalling mechanisms supporting the protein synthesis-dependent late phase of LTP (L-LTP), and (ii) capable of converting decremental early phase LTP into stable L-LTP (Dudek and Fields, 2002). Mechanisms involved in the shaping of AP trains are, therefore, of major

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functional relevance. The role of L-type calcium channels in generating repetitive firing of neurons in the CNS is not clear. Studies with pharmacological tools yielded contradictory results (Moyer et al., 1992; Whittington and Little, 1993; Pineda et al., 1998; Chen et al., 2005) and, importantly, do not allow to discriminate between individual members of the L-type channel family. Here, we examined the intrinsic excitability of CA1 pyramidal cells from control mice and mice with the Cav1.2 gene inactivated in principal hippocampal neurons. Cells from the Cav1.2 mutants showed (i) elevated threshold and latency for inducing repetitive AP firing, and (ii) a reduced spiking frequency. In our hands, the L-type calcium channel blocker isradipine had the same effects on AP firing in hippocampal pyramidal cells from control mice. Moreover, spiking in these cells was facilitated by the L-type calcium channel activator BayK 8644. In summary, these findings demonstrate that Cav1.2 L-type channel isoform modulates the spiking pattern of CA1 pyramidal neurons in a way that may, ultimately, support synaptic plasticity and memory formation. Materials and methods We used a mouse line (Cav1.2HKO) with a conditional inactivation of the CACNA1C gene in the hippocampus based on the Cre/loxP system. For details regarding its generation, recombination pattern and genotyping see elsewhere (Schwab et al., 2000; Moosmang et al., 2005; Goebbels et al., 2006). Previous analyses of these mice have revealed that the Cav1.2 channel protein is still detectable up to day p14 of postnatal development despite the fact that Cre activity in pyramidal neurons of the hippocampus starts from around embryonic day 11.5 (Schwab et al., 2000; Moosmang et al., 2005; Goebbels et al., 2006). Therefore, all animals used in our experiments for comparison of control and Cav1.2HKO mice were at least 10 weeks of age. Hippocampal pyramidal cells of these mice lack the Cav1.2 protein. For experiments with dihydropyridines and with small current ramp, young animals 2–3 weeks of age were used. Animals were handled in accordance with the Directive 86/ 609/ECC. All necessary precaution was taken in order to minimise discomfort and pain. Mice were deeply anaesthetised with ether and decapitated. The brain was removed quickly into oxygenated ice-cold artificial cerebrospinal fluid (ACSF, composition in smM: NaCl, 125; CaCl2, 2; MgCl2, 1; KCl, 3; NaHCO3, 25; NaH2PO4, 1.25; glucose, 10; pH 7.4 with NaOH). Coronal slices (300 μm thick) including the hippocampus were prepared using a vibratome slicer (Microm GmbH, Walldorf, Germany) and transferred into a storage chamber containing ACSF constantly bubbled with 95% O2/5% CO2 where they were allowed to recover for half an hour and stored up to 6 h at room temperature. For electrophysiological recordings, slices were transferred to a recording chamber mounted on upright microscope BX50WI (Olympus, Hamburg, Germany) perfused with ACSF (24 °C) gassed with 95% O2/5% CO2. Recordings were made from CA1 neurons visually identified by infrared

DIC-videomicroscopy (Dodt and Zieglgansberger, 1990) using a high performance vidicon camera C2400-07 (Hamamatsu, Herrsching, Germany). Patch pipettes were pulled from borosilicate glass capillaries, and had a resistance of 3–5 MΩ when filled with the internal solution containing (in mM): Kgluconate, 120; KCl, 20; MgCl2, 2; Na2ATP, 2; Na2GTP, 0.25; HEPES, 10; and pH 7.3 (with KOH) (Jinno et al., 2003). To prepare acutely isolated hippocampal neurons, mouse brain was removed and cut into halves in ice-cold ACSF. Both hippocampi were then removed and cut into 300 μm thick slices using a vibratome slicer (Microm GmbH, Walldorf, Germany). Slices were transferred into 5 ml ACSF constantly bubbled with 100% O2 supplemented with 19 U/ml papain and digested for 90 min at 30 °C. Afterwards slices were washed three times with ACSF and allowed to rest in this solution bubbled with 100% O2 for 10 min up to 5 h at room temperature. Individual slices were triturated just before patch clamp experiment. Trituration solution contained (in mM): NaCl, 125; KCl, 3; MgCl2, 10; HEPES, 10; Na–HEPES, 10; EGTA, 10; CaCl2, 1; kynurenic acid, 2; pH 7.4 (with NaOH). Following trituration, the solution was exchanged for bath solution and cells were allowed to rest for 15 min before patch clamp measurements were started. Bath solution for measurements of calcium current contained (in mM): NaCl, 105; KCl, 3; TEACl, 25; MgCl2, 0.5; CaCl2, 2; HEPES, 10; D-glucose, 10; pH 7.4 (with NaOH). Patch pipettes were filled with the internal solution containing (in mM): CsCl, 135; MgCl2, 2; TEACl, 20; Na2ATP, 3; Na2GTP, 0.4; EGTA, 3; HEPES, 10; pH 7.4 (with CsOH). Unless mentioned otherwise, all chemical were obtained from Sigma. Stock solutions of BayK 8644 (1 mM; Tocris) and (+/−)isradipine (10 mM, Hofmann-LaRoche) were prepared in ethanol and dissolved in ACSF to the final concentration prior to experiment. All recordings from CA1 pyramidal cells were made with an HEKA EPC 9 amplifier (HEKA, Lambrecht/Pfalz, Germany). Data were acquired, processed and analysed using the Pulse/ Pulsefit (HEKA, Lambrecht/Pfalz, Germany) and Origin 7.5 (OriginLab Co., Northampton, MA, USA) software. All values are given as mean ± SEM. Student's t-test for independent samples was used for statistical comparison and verified by ANOVA and GraphPad PRISM® software. A confidence interval of ≥ 95% was considered as significant. Results Previously, we have demonstrated that inactivation of the CACNA1C gene in hippocampal pyramidal cells virtually abolished dihydropyridine-sensitive calcium currents in these cells (Moosmang et al., 2005). Here, we investigated the effect of this loss in Cav1.2 channel-mediated currents on electrophysiological characteristics of these cells such as resting potential, input resistance, the shape of single action potential (AP) and the pattern of repetitive spiking. Resting membrane potential measured as the membrane voltage observed with zero current injected was virtually identical in the two genotypes (− 64.0 ± 1.0 mV in 17 cells from 17 slices from 6 control animals versus − 65.3 ± 0.8 mV in 16

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cells from 16 slices from 6 Cav1.2HKO animals; p = 0.34). Input resistance, Rin, of each cell was estimated similarly as described by Surges et al. (2004) based on the relationship between the amplitude of currents injected and the resulting deflection in membrane potential measured at the end of 800 ms current pulses. The values of Rin obtained for CA1 pyramidal cells in Cav1.2HKO mice were not significantly different from those in control (86.6 ± 3.1 MΩ in 20 cells from 18 slices from 7 Cav1.2HKO animals versus 79.4 ± 2.2 MΩ in 25 cells from 20 slices from 7 control animals; p = 0.11). It has been suggested recently that L-type channels can become activated during the course of a single AP and, thus, participate in shaping its descending phase (Helton et al., 2005; King and Meriney, 2005). We examined whether activity of the Cav1.2 channel isoform contributes to shaping single APs in CA1 pyramidal cells. AP amplitude and half-maximal width were determined based on the time course of single APs observed during the first supra-threshold current pulses (Fig. 1) when short depolarising current pulses (5 ms) of increasing amplitude were injected from a fixed membrane potential of −70 mV. The first derivative of the AP was used to estimate its maximal up-stroke (vmax[ascend]) and down-stroke velocity (vmax[descend]). The

Fig. 1. Example of a single action potential (AP). Single AP activated by 5 ms long depolarising current pulse in a CA1 pyramidal cell from a Cav1.2HKO animal is shown in the top panel together with its first derivative (dV/dt; middle panel) and the second derivative (dV/dt2; bottom panel). The voltage threshold for firing a single AP (Vthr), peak voltage (Vpeak), width of the AP at the halfmaximal amplitude (half-width), maximal ascending (vmax[ascend]) and maximal descending (vmax[descend]) rate of the AP were evaluated for control and the Cav1.2HKO animals as marked.

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threshold membrane potential was measured at the time point coinciding with the local maximum of the second derivative (Fig. 1). A careful analysis of single AP parameters in control and Cav1.2HKO mice revealed that the loss of the Cav1.2 channel isoform had no significant effect on the threshold for eliciting a single AP (−36.4 ± 1.4 mV versus −38.3 ± 1.6 mV for control and Cav1.2HKO mice, respectively), the peak voltage (+57.3 ± 3.1 mV versus + 52.9 ± 2.8 mV for control and Cav1.2HKO mice, respectively) and the width of single AP at its half-maximum (1.18 ± 0.02 ms versus 1.22 ± 0.02 ms for control and Cav1.2HKO mice, respectively). Likewise, vmax[ascend] (287± 11 mV/ms versus 268 ± 12 mV/ms for control and Cav1.2HKO mice, respectively) and vmax[descend] (−81± 5 mV/ms versus −74 ± 4 mV/ms for control and Cav1.2HKO mice, respectively) were not significantly different between the genotypes. Data were obtained from 18 cells in 18 slices from 7 control animals and 19 cells in 17 slices from 7 mutant animals. Trains of somatic APs may activate gene expression relevant for synaptic plasticity (Dudek and Fields, 2002). We induced firings of spike trains by injecting long-lasting depolarising currents (500 ms) of increasing amplitude (Fig. 2A–B). In contrast to the threshold for eliciting single APs, the voltage threshold to evoke repetitive spiking was significantly different between the two genotypes (Table 1). Hippocampal pyramidal neurons from Cav1.2HKO mice required stronger depolarisation to evoke AP train than control neurons as illustrated by the histograms and cumulative probability plots (Fig. 2). On average, the mean threshold to evoke repetitive AP firing was elevated by ∼ 6 mV (p b 0.001). We suggest that Cav1.2 channels becoming activated during long-lasting depolarisation actively support further depolarisation and, thus, specifically facilitate repetitive firing without affecting single AP firing. The initial AP in a train induced by a first supra-threshold pulse started at variable time points after the pulse onset (Fig. 2A–B). Corresponding histograms and cumulative probability plots illustrate a substantial increase of the spiking latency in hippocampal neurons from Cav1.2 HKO mice compared with the control (Fig. 2E–F). Nearly 50% of hippocampal neurons from control mice fired AP trains within 25 ms after the onset of the first supra-threshold depolarising pulse, while none of the cells from the mutant mice did so (Fig. 2F). Mean latencies in CA1 cells were significantly enhanced in neurons from Cav1.2HKO mice compared to neurons from control mice (Table 1). There was also an apparent tendency to a decrease in the number of spikes observed during depolarising current pulses. To prove or disprove this apparent change, we performed a detailed quantitative analysis of the spiking frequencies in recordings of AP trains evoked by four consecutive pulses of increasing amplitude starting with the minimal supra-threshold amplitude (cf. Fig. 3A). All hippocampal CA1 pyramidal cells examined exhibited the characteristic increase of the interval between APs within a train with each consecutive spike. This is reflected in a progressive decrease of the spiking frequency, so-called spike frequency adaptation (Fig. 3A). Stronger depolarisation resulted in faster spiking, i.e. increased frequency. Spike frequency adaptation was preserved under these conditions. Our analysis

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Fig. 2. The voltage threshold and latency of repetitive AP firing. CA1 neurons were held at − 70 mV by injecting a minor hyperpolarising current. AP trains were evoked by injection of 500 ms long depolarising current pulses with amplitude increasing with a step of +50 pA. Representative recordings of the last subthreshold (solid line), first (solid line) and second (dashed line) supra-threshold depolarising current injected are shown for pyramidal neurons from control (A) and Cav1.2HKO (B) mice. Histograms of the threshold potential (Vthreshold) at which a repetitive AP firing was induced are shown in (C). Corresponding plots for the cumulative probability of an AP train to be fired at a given membrane potential are demonstrated in (D). Histograms and cumulative probability plots for the latency until firing of the first AP in a train are shown in panels E and F. Cells from control mice are represented by the open bars and open triangles (17 cells from 15 slices from 6 animals). Cells from Cav1.2HKO are represented by gray bars and filled triangles (16 cells from 14 slices from 6 animals).

revealed a prominent reduction of the initial spiking frequency in CA1 pyramidal cells from Cav1.2HKO mice. Up to five APs at the beginning of a spike train were fired at a rate significantly slower than in neurons from the mutant animals (Fig. 3B–E). Reduction in initial spike frequency was observed for a whole set of supra-threshold depolarising pulses and, typically, corresponded to a decrease by about 30% from the value in control (Fig. 3B–E). Interestingly, this decrease was most pronounced for relatively short AP trains fired near the actual threshold potential indicating a particular functional impact of the Cav1.2 channel under these conditions. Using an alternative

protocol, in which depolarising current pulses were preceded by 500 ms pulses hyperpolarising the membrane to about − 85 mV, the differences in spiking frequency of CA1 pyramidal cells from control and mutant animals became even more pronounced. Under such conditions, the initial firing frequency of CA1 pyramidal cells from Cav1.2HKO mice was decreased by up to 50% compared with the frequency in control (Fig. 3B–E). Taken together these findings suggest an important function of the Cav1.2 channel in modulating initial frequency of repetitive AP firing generated at the soma of hippocampal pyramidal cells.

L. Lacinova et al. / Life Sciences 82 (2008) 41–49 Table 1 Voltage threshold and latency of the start of repetitive firing determined in hippocampal neurons from control (n = 16) and Cav1.2HKO (n = 23) mice

Threshold for repetitive firing Mean latencies of repetitive firing

Control

Cav1.2HKO

− 47.2 ± 0.9 mV 48.1 ± 7.8 ms

− 40.7 ± 1.3 mV⁎⁎⁎ 73.2 ± 6.6 ms⁎

Asterisks mark significant difference evaluated by non-paired Student's t-test. ⁎, p b 0.05; ⁎⁎⁎, p b 0.001.

To strengthen this view pharmacologically, we studied the effect of dihydropyridines (DHP) modulating L-type calcium channel activity on the cellular excitability of CA1 neurons from young control mice using the experimental parameters, which had previously revealed significant differences between control and mutant mice. In line with our previous findings, increasing

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the activity of L-type calcium channels with the DHP BayK 8644 (1 μM) facilitated the firing of AP series (Fig. 4A). This was reflected in a moderate decrease of the voltage threshold for AP spiking (−37.2 ± 0.7 mV in control to − 38.6 ± 1.0 mV with BayK 8644) and of the latency to the first AP (116 ± 13 ms in control to 87 ± 7 ms with BayK 8644) (n = 5). Conversely, the Ltype calcium channel antagonist isradipine suppressed spiking activity (Fig. 4B). Most notable, isradipine (10 μM) decreased the average number of spikes in an AP series (12.4 ± 3.2 in control versus 6.4 ± 1.3 with isradipine) (n = 5). In addition, we observed a slight increase of the latency until the first AP was fired from 77 ± 24 ms in control to 88 ± 25 ms after isradipine was added to the bath solution. Given that CA1 hippocampal pyramidal cells from mice with an inactivation of the CACNA1C gene virtually lack DHPsensitive calcium currents (Fig. 4C–D) (Moosmang et al.,

Fig. 3. Initial frequency of repetitive firing is decreased in CA1 pyramidal cells from Cav1.2HKO mice. In addition to current protocol of the Fig. 2 (○; control animals, 15 slices, 17 cells; ●; 6 Cav1.2HKO animals, 14 slices, 16 cells) a current protocol in which depolarising current pulses were preceded by 500 ms hyperpolarising prepulse causing membrane hyperpolarisation to approximately − 85 mV was used (△; 8 animals, 13 slices, 17 control cells; ▲; 6 animals, 8 slices, 12 Cav1.2HKO cells). Bursting frequency for spike number i within a train of APs was estimated as fi = 1/ti, where ti is the interval between peaks of spikes i and i + 1 (panel A). Spiking frequency following injection of smallest depolarising currents that evoked a train consisting of at least three consecutive APs (1st supra-threshold) is shown in panel B. Frequency of the 3rd AP evoked by the standard protocol was omitted, because the majority of trains consisted of only three APs. Spiking frequencies following injection of 2nd, 3rd and 4th supra-threshold currents are shown in panels C, D and E, respectively. Asterisks indicate a significant decrease of the spiking frequency in hippocampal pyramidal cells from mutant mice compared to the corresponding controls (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).

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Fig. 4. Effects of L-type calcium channel modulators on AP fired during sustained depolarisation and on inward calcium current measured from isolated hippocampal neurons. BayK 8644 facilitated and isradipine suppressed repetitive action potential firing. Cells were held at a membrane potential of − 70 mV by injecting a minor hyperpolarising current. Spiking was induced by 1 s long depolarising current pulses of increasing amplitude (step +50 pA). Panel A shows representative recordings of AP series activated by the first supra-threshold current pulse in control (dashed line) and in the presence of 1 μM BayK 8644 (solid line). Panel B shows representative recordings of AP series activated by the first supra-threshold current pulse in control (dashed line) and in the presence of 10 μM isradipine. For better resolution of initial spikes only the initial 700 ms are shown. Five cells were investigated in each group. Panels C and D show examples of current traces activated by a voltage step from the membrane potential of −80 mV to 0 mV. Sodium and potassium current were blocked by 1 μM TTX and by TEACl, respectively. Black lines represent the current recorded under the control conditions, gray line the current recorded in the presence of 10 μM (+/−)isradipine and dashed lines the current recorded in the presence of 1 μM BayK 8644. Panel C shows recordings from hippocampal neuron isolated from control mouse. Panel D shows recordings from hippocampal neuron isolated from Cav1.2HKO animal.

2005), these results collectively support our conclusion that the Cav1.2 channel modulates repetitive AP firing in these neurons. The differences in AP firing described above may arise from the lack of slowly activating Cav1.2 channel-mediated currents in the mutant mice and with isradipine. In control, these currents might shape AP firing by actively supporting further depolarisation. To generally prove this concept, we compared series of APs evoked by two similar protocols in the same CA1 pyramidal cell: (1) a current-clamp step depolarising the cell membrane to a potential near the threshold and (2) the same current-clamp step paired with a small ramp to simulate a slowly activating inward current (Fig. 5). Fitting well with our concept, we observed that even a small current ramp of + 5 pA increased the number of spikes while decreasing the latency for AP firing under conditions when intracellular Ca2+ was buffered. Discussion Studies regarding cellular mechanisms for storage of memories in the brain have focussed on synaptic plasticity,

namely long-term potentiation (LTP) (cf. Bliss and Lomo, 1973). On the other hand, information processing also depends on mechanisms modifying the output of postsynaptic neurons (e.g. AP trains) in response to an excitatory synaptic input (cf. Debanne et al., 2003). An important means of such type of information processing in hippocampal pyramidal cell represents their spiking pattern (Lisman, 1997; Harris et al., 2001). Given the high relevance of the hippocampus for the formation of episodic memories, we sought to further elucidate the role of the Cav1.2 channel for repetitive AP firing of CA1 pyramidal cells. Generation of repetitive action potential spikes following prolonged injection of a depolarising current requires activation of an additional sustained current actively promoting further depolarisation of hippocampal pyramidal cells. Such current may be partially carried by slowly-inactivating voltagedependent calcium channels (VDCC) (Stewart and Wong, 1993; Taube, 1993; Jung et al., 2001). Investigation of the role of L-type VDCC isoforms is obstructed by shortage of specific pharmacological tools. To overcome this problem, and in view of the fact that the Cav1.2 channel is the member of the L-type

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Fig. 5. Effect of a small slowly rising depolarising current of action potential (AP) firing in CA1 pyramidal cells. Shown are representative current-clamp recordings from a wild type CA1 neuron. The top panel shows the protocol consisting of a +25 pA current step from resting membrane potential (0 pA current injected) driving the membrane potential slightly above threshold for AP firing followed by a second a +25 pA current step paired with a small depolarising ramp (+5 pA). There was a 15 s interleave between the two parts of the protocol (indicated by the break). The three lower rows illustrate consecutive recordings from the same cell applying the protocol above (30 s pause).

channel family predominantly expressed in hippocampal neurons (Hell et al., 1993; Sinnegger-Brauns et al., 2004), we utilised a transgenic mouse model to perform a comparative analysis of excitability in CA1 pyramidal cells from control and from animals lacking the Cav1.2 channel in principal neurons of the hippocampus and neocortex. We found that basic properties of CA1 pyramidal cells such as resting membrane potential and the input resistance do not critically depend on the function of Cav1.2 channels: both parameters were unaltered in the mutant mice. Further, we analysed parameters of AP including threshold, width, maximal up- and down-stroke velocity. Recently, it has been reported that recombinant neuronal Cav1.2 channels can open rapidly even in response to individual AP-like waveforms (Helton et al., 2005). In our hands, none of the single AP parameters was altered in hippocampal pyramidal cells from the mutant mice. This emphasises that under our conditions the fraction of Cav1.2 channels opening during the course of a single AP was too small to alter its shape detectably. In other words, Ca2+ entry via slowly activating Cav1.2 L-type channels during a single AP is negligible (cf. Mermelstein et al., 2000). However, it remains unclear what impact weakly expressed Cav1.3 channels have on the shape of single APs. The major finding of our study is related to the modulatory role of the Cav1.2 channel for repetitive AP firing of hippocampal CA1 pyramidal cells. Strong excitatory inputs are required to generate trains of APs in this cell type in vivo (Harris et al., 2001). Patterns of strong excitation also cause L-type calcium channelmediated Ca2+ influx promoting hippocampal long-term potentiation (LTP) and spatial memory (Morgan and Teyler, 1999; Woodside et al., 2004). This Ca2+ entry has been recently linked specifically to the Cav1.2 channel subtype (Clark et al., 2003; Moosmang et al., 2005). Previously, we have shown an impairment in spatial learning of mice lacking the Cav1.2 channel

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in hippocampal neurons and that this impairment is likely due to a defect in NMDA receptor-independent LTP. The present study provides additional evidence demonstrating alterations in repetitive AP firing at the soma of CA1 pyramidal cells in these mutants, which may also contribute to the behavioural impairment previously described (cf. Lisman, 1997). Trains of APs were induced by injecting prolonged (500 ms) depolarising currents somatically and showed marked spike frequency adaptation typical for CA1 pyramidal neurons. Under these experimental conditions, the minimal depolarisation from resting potential required to initiate an AP train and the latency until the first AP was fired were elevated in the mutant mice. These alterations likely reflect the function of Cav1.2 channel-mediated inward currents activated during long depolarising pulses (i.e. strong excitatory inputs) for regenerative firing of APs. Furthermore, the lack of the Cav1.2 channel in CA1 pyramidal cells caused a substantial reduction in their initial spiking frequency. This effect was even more pronounced when cells were preconditioned at a more hyperpolarised membrane potential of −85 mV to promote recovery of VDCC from partial inhibition at normal resting membrane potential of CA1 pyramidal cells. These observations suggest in unison a facilitating function of the Cav1.2 channel for repetitive firing. As mentioned above, this function may be due to a depolarising current carried by this channel. On the other hand, Magee et al. (1996) have reported that intracellular calcium concentration of pyramidal cells even near normal resting potential partially depends on L-type channel activity. Given this finding, the loss of a tonic Cav1.2 channel-mediated Ca2+ influx in the mutant mice may contribute indirectly through a Ca2+dependent mechanism to the differences in cell excitability observed here. Arguing against this view, prepulses hyperpolarising hippocampal pyramidal cells to favour the closed state of Cav1.2 channels and to decrease of intracellular calcium concentration even amplified the differences in AP firing between wild type and mutant mice. In addition, AP potential firing in wild type CA1 pyramidal neurons showed differences resembling those observed in our mouse model depending on whether a supra-threshold depolarising current step or the same current step paired with a small current ramp (to mimic a slowly activating calcium current) was used even with intracellular Ca2+ buffered. Additional experiments with DHPs modulating the L-type calcium channel activity validated the findings in our mouse model. Both the activator (BayK 8644) and the blocker (isradipine) of L-type calcium channel showed effects on AP firing compatible with a function of these channels in repetitive AP firing. Thus, BayK 8644 decreased both the voltage threshold and the first latency for firing of an AP series. Blocking L-type calcium channels with isradipine suppressed the number of spikes in an AP series. The latter effect was more pronounced than facilitation of spiking by BayK 8644 may be due to fact that a saturating concentration of isradipine used in our experiments inhibits both Cav1.2 and Cav1.3 in CA1 neurons. Our findings in the Cav1.2HKO mice as well as those obtained with DHP fit well with data from some earlier reports studying the role of L-type calcium channels by means of DHP (Whittington and Little, 1993; Pineda et al., 1998; Chen et al., 2005). For example, the DHP L-type channel blockers nifedipine

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and nimodipine have been shown to slow the spiking frequency in rat neocortical neurons (Pineda et al., 1998) and to suppress intrinsic burst firing in developing rat CA1 pyramidal cells (Chen et al., 2005). According to others, multiple VDCC are involved in shaping of firing patterns, and L-type calcium channels have a minor functional impact compared with R- and T-type channels (Magee and Carruth, 1999; Jung et al., 2001; Metz et al., 2005). Moyer et al. (1992) even reported that nimodipine increases the spiking frequency during prolonged depolarisation of rabbit CA1 pyramidal cells. Besides possible species differences, these discrepancies are likely due to problems associated with the use of DHP L-type channel blockers. Most relevant, DHPs may affect other membrane currents apart from L-type calcium channels (Roger et al., 2004; Takeda et al., 2004; Tanaka et al., 2004; Bett et al., 2006). The role of L-type calcium channels may also have been underestimated owing to the state- and voltagedependent mode of action of DHPs (cf. Helton et al., 2005). Moreover, due to their lack of selectivity for isoforms, alterations observed in the presence of DHP L-type channel blockers cannot be interpreted unambiguously, because it cannot be ruled out that different L-type channel isoforms support mechanisms of functionally opposite impact. Using a transgenic mouse model, we were able to overcome these problems and demonstrate for the first time the function of Cav1.2 channels in promoting repetitive AP firing. We cannot fully rule out that chronic lack of the Cav1.2 channel in CA1 pyramidal cells leads to altered gene expression and causes compensatory adjustments of excitability. However, this view is challenged by findings from the present study and our previous analysis (Moosmang et al., 2005). First, basic electrophysiological parameters in hippocampal neurons in these mice are normal. Second, expression of the Cav1.3 channel protein and the NR1 subunit of the NMDAR in the hippocampus of these mutants was not altered (cf. supplementary information of Moosmang et al., 2005). Conclusion In conclusion, we demonstrated for the first time that the Cav1.2 channel facilitates repetitive AP firing in CA1 pyramidal cells. We suggest that this modulatory function contributes to spatial learning and memory. Acknowledgement This works was supported by grants from the DFG KL 1172, Volkswagen Stiftung, VEGA 2/4009 and APVV-51-027404. References Bett, G.C., Morales, M.J., Strauss, H.C., Rasmusson, R.L., 2006. KChIP2b modulates the affinity and use-dependent block of Kv4.3 by nifedipine. Biochemical and Biophysical Research Communications 340 (4), 1167–1177. Bito, H., Deisseroth, K., Tsien, R.W., 1996. CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87 (7), 1203–1214. Bliss, T.V., Lomo, T., 1973. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. Journal of Physiology (London) 232 (2), 331–356.

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