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HCN1 plasticity in entorhinal cortical (EC) and hippocampal pyramidal cell dendrites is a salient feature of temporal lobe epilepsy (TLE). However, the significance remains undetermined. We demonstrate that adult HCN1 null mice are more susceptible to kainic acid induced seizures. Following termination of these with an anticonvulsant, the mice also developed spontaneous behavioural seizures at a significantly more rapid rate than their wildtype littermates. This greater seizure susceptibility was accompanied by increased spontaneous activity in HCN1−/− EC layer III neurons. Dendritic Ih in these neurons was ablated, too. Consequentially, HCN1−/− dendrites were more excitable, despite having significantly more hyperpolarized resting membrane potentials (RMP). In addition, the integration of excitatory post-synaptic potentials (EPSPs) was enhanced considerably such that at normal RMP, a 50 Hz train of EPSPs produced action potentials in HCN1−/− neurons. As a result of this enhanced pyramidal cell excitability, spontaneous EPSC frequency onto HCN1−/− neurons was considerably greater than that onto wildtypes, causing an imbalance between normal excitatory and inhibitory synaptic activity. These results suggest that dendritic HCN channels are likely to play a critical role in regulating cortical pyramidal cell excitability. Further, these findings suggest that the reduction in dendritic HCN1 subunit expression during epileptogenesis is likely to facilitate the disorder.
Temporal lobe epilepsy (TLE) is the most common, drug-resistant form of the human condition (Engel, 1996; Herman, 2002). There is substantial evidence to suggest that the entorhinal cortex (EC) and hippocampus play pivotal roles in the induction and maintenance of TLE (Spencer and Spencer, 1994). EC layer III pyramidal neurons, particularly, may play a critical role (Du et al., 1993; Jones, 1993; Du et al., 1995; Barbarosie et al., 2000; Avoli et al., 2002; Wu and Leung, 2003; Shah et al., 2004; Dawodu and Thom, 2005; Wozny et al., 2005). Indeed, they have been shown to be spontaneously hyperactive in vivo following the induction of TLE (Shah et al., 2004). Further, their axons (the temporoammonic pathway), which innervate the stratum lacunosum-moleculare (SLM) of CA1 and the molecular layer of the subiculum, have been suggested to provide the major excitatory drive to the hippocampus during chronic TLE (Barbarosie et al., 2000; Avoli et al., 2002; Wu and Leung, 2003; Wozny et al., 2005; Ang et al., 2006). Thus, altered EC layer III pyramidal cell excitability is likely to have a large impact on the development of TLE.
Interestingly, enhanced EC layer III neuronal excitability during TLE is accompanied by a decrease in the hyperpolarization-activated cation current, Ih (Shah et al., 2004). Hyperpolarization-activated Cation Non-selective (HCN) subunits underlie Ih (Robinson and Siegelbaum, 2003). The HCN1 subunit is predominantly expressed in the cortex and hippocampus, where it is primarily located in pyramidal cell dendrites (Lorincz et al., 2002; Notomi and Shigemoto, 2004). Indeed, HCN1 expression is significantly reduced in the EC following TLE (Shah et al., 2004; Powell et al., 2008). Similar HCN1 channel plasticity has also been shown to occur in neocortical and hippocampal neurons in multiple animal models (Brewster et al., 2002; Bender et al., 2003; Dugladze et al., 2007; Jung et al., 2007; Shin et al., 2008; Marcelin et al., 2009) as well as humans (Brewster et al., 2002). This is surprising as the current depolarizes the resting membrane potential (Pape, 1996; Robinson and Siegelbaum, 2003) and hence, a decline in Ih might be expected to reduce excitability. However, Ih inhibition has been suggested to enhance pyramidal cell dendritic excitability by increasing the availability of Ca2+ channels (Tsay et al., 2007) as well as by amplifying the membrane resistance (RN; (Magee, 1998; Stuart and Spruston, 1998), thereby, modifying synaptic signal integration (Magee, 1999). Since Ih,and HCN1, plasticity is a prevalent hallmark of TLE (Chen et al., 2001; Brewster et al., 2002; Bender et al., 2003; Shah et al., 2004; Dugladze et al., 2007; Jung et al., 2007; Dyhrfjeld-Johnsen et al., 2008; Powell et al., 2008; Shin et al., 2008; Marcelin et al., 2009) it is crucial to determine whether alterations in Ih and HCN1 protein expression during TLE are consequential to neural network adaptation or critically influence the disorder. In this study, we have used HCN1 null mice together with in vivo electroencephalographic recordings, in vitro electrophysiological analysis and selective pharmacological tools to address this important question.
HCN1 heterozygote breeding pairs were a kind gift from Prof. E. R. Kandel (Columbia University, USA). Hybrid (HCN1+/−) male and female progeny were maintained on a 129SVEV background and crossed to obtain mixtures of HCN1 null mice, heterozygotes and wildtype littermates (as described previously (Nolan et al., 2003; Nolan et al., 2004)). These mice had been backcrossed for 10 generations. The mouse genotype was determined using PCR. Briefly, genomic DNA was extracted from 0.2 cm tail snips using the HOtShot protocol as detailed in Truett et al. (2000). A PCR reaction was then carried out using TAQ polymerase (Bioline, UK) and the following primers:
The PCR reaction consisted of an initial 2 minutes at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 45 s at 72°C. After the last cycle, the reaction is kept at 72°C for4minandthenheldat10°C. A 359 bp band was observed for mice containing the wildtype allele whereas a 450 bp band wass seen for mice containing the mutant allele. Heterozygotes contained both alleles.
6-8 week old HCN1 null mice and wildtype littermate controls were anaesthetized using a ketamine/xylamine mixture (Sigma, UK) and positioned in a stereotaxic frame. Depth electrodes (Plastics One, USA) were surgically implanted into the EC area using the following stereotaxic co-ordinates: 4.2 mm lateral to lambda and 4.0 mm below the cortical surface with the nosebar set at 3.0 mm and the electrode holder at an angle of 17° posterior to the sagittal plane.
All mice were kept on 12 hr light dark cycles. EEG recordings were obtained 7 days later using a neurolog amplifier. Video recordings were obtained using a wired day/night camera with audio and IR (Nature Cameras Ltd, UK). These together with the EEG data were simultaneously acquired on a computer using the micro1401 analogue to digital converter and spike 2 software (Cambridge Electronic Design Ltd, UK). For baseline EEG, we did 6 hr recordings during light and 6 hr during dark cycle for a minimum period of 5 days. Following kainate injections, 4 hr EEG recordings were made during the light and dark cycles respectively. All recordings were referenced to a frontal surface electrode. Recordings were visually inspected for electrographic seizure activity and interictal spikes as previously defined (Shah et al., 2004). Interictal spikes were defined as high amplitude (at least 3 times baseline amplitude) sharp transients lasting less than 70 ms. Electrographic seizure activity was defined as the appearance of high amplitude (greater than 3 times baseline activity), high frequency, rhythmic activity with an evolution in spike frequency that lasted a minimum of 10 s. In addition, following each experiment, the location of the electrode was confirmed to be in the EC.
Kainic acid (Tocris Ltd, UK) was administered to HCN1 null mice and wildtype littermates to induce Class V seizures (as defined by the Racine scale (Racine, 1972)). These were terminated 1 hr after onset with the use of sodium pentobarbital (SP, 30 mg/kg s.c.; Sigma-Aldrich, UK). Control groups were mice that had been treated SP only (30 mg/kg s.c.). In some mice, EEG recordings were obtained following kainic acid administration. These were quantified as described in (Lehmkuhle et al., 2009). Briefly, EEG raw data was initially band pass filtered in the gamma band range (20-70 Hz) and the ratio of the gamma band power (the square of the average amplitude root mean square (RMS) value) before and after kainic acid induced seizures was calculated and expressed as a percentage. As described in (Williams et al., 2009), the latent period duration was calculated as the time taken for the onset of motor seizures (typically Class III, as defined by Racine (1972). All procedures concerning animals were approved by the UK Home Office.
Entorhinal-hippocampal slices were obtained from 6-9 week old HCN1−/− and HCN1+/+ (wildtype) mice as described previously (see (Shah et al., 2004). Whole-cell and cell-attached recordings were obtained from both the soma and dendrites of EC layer III pyramidal neurons. For recording purposes, slices were placed in a chamber containing external recording solution maintained at 34-36 °C and viewed using an Olympus BX51W1 equipped with differential infra-red optics. The external solution (unless otherwise noted) was supplemented with 0.05 mM APV, 0.01 mM CNQX, 0.01 mM bicuculline and 0.001 mM CGP 55845. The internal recording pipette solution for whole-cell current clamp and EPSC voltage clamp recordings was composed of (in mM): 120 KMeSO4, 20KCl,10HEPES, 2 MgCl2, 0.2 EGTA, 4 Na2-ATP, 0.3 Tris-GTP, 14 Tris-phosphocreatine; pH was adjusted to 7.3 with KOH. To record spontaneous IPSCs, KMeSO4 was replaced with KCl. Further, ZD7288 (15 μM) was added to the intracellular solution when EPSC and IPSC recordings were made. In addition, for cell-attached recordings, the internal pipette solution contained (mM): 120 KCl, 20 TEA Cl, 5 4-AP, 1 BaCl2, 10 HEPES, 1 MgCl2, 2 CaCl2, 0,001 tetradotoxin, 0.1 NiCl2; pH adjusted to 7.3. Pipettes containing any of these internal solutions had resistances of 5 – 12 MΩ. Whole-cell current clamp recordings were obtained using a bridge-mode amplifier (AxoClamp 2B, Molecular Devices, UK), filtered at 10 kHz and sampled at 50 kHz. Series resistance was usually in the order of 10-30 M and was approx. 70% compensated for the whole-cell voltage clamp recordings. Cell-attached recordings were obtained using the Axopatch 200B (Molecular Devices, UK), filtered 2 kHz and sampled at 3.5 kHz. Data were acquired using pClamp 8.2 (Molecular Devices, UK)
αEPSPs were generated by current injection of the order:
where A is the amplitude of the current injected and τ is the rise time constant.. Tungsten electrodes (A-M Systems) were placed in EC layer I to elicit EPSPs. All drugs were bath applied. The effects of ZD7288 (15 μM) occurred within 15 min and recordings were usually made within 25 min of application.
pClamp software was used to analyze whole-cell current clamp and cell-attached voltage clamp recordings.. The RN was calculated from 400 ms hyperpolarizing pulses of 100 pA applied from a holding potential of −70 mV. The αEPSP decay time constants were obtained by fitting the double exponential function:
where τ1 and τ2 represent time constants of the initial and falling phase of the αEPSPs. Since Ih is activated during the falling phase of the αEPSP, only τ2 was used. The summation ratio of EPSPs was calculated as the ratio of the peak of the 5th EPSP to that of the 1st EPSP. Action potential threshold was determined as the point before the first derivative of the trace was no longer equal to zero. For cell-attached recordings, the steady-state current following the 2 s, hyperpolarizing step was used as an indication of the amount of Ih. EPSCs and IPSCs were analyzed using the Mini-analysis program (v6.07, Synpatosoft, USA). Events > 3 pA in amplitude were detected and used for analysis. Decay times and amplitudes of these events were obtained by fitting the averaged EPSC or IPSC with a single exponential equation:
Where I is the current amplitude at any given time (t), A is the peak amplitude of the EPSC or IPSC and τ is the decay time constant. Group data are expressed as mean ± SEM. Statistical significance was determined using either paired or unpaired Student's T tests as appropriate. Statistical significance of p < 0.05 is indicated as * in all figures.
All chemicals were obtained from Sigma-Aldrich (UK) apart from ZD 7288, CGP 55848, CNQX, TTX, bicuculline and APV, which were purchased from Ascent Scientific Ltd (UK). Stock solutions of bicuculline and CGP 55848 were made in DMSO and stored at −20 °C until use. These were then dissolved in the external solution such that the final DMSO concentration was less than 0.1%. Aqueous stock solutions of ZD7288, CNQX, TTX and APV were also kept at −20 °C until use.
To determine how HCN1 subunits influence epileptogenesis, HCN1 null mice were employed. These mice have proved to be useful in exploring the role of HCN1 channels, as expression of HCN2-4 is minimally affected (Nolan et al., 2003; Nolan et al., 2004; Nolan et al., 2007; Tsay et al., 2007). To test the role of HCN1 subunits in influencing seizure susceptibility, depth electrodes were stereotaxically implanted in the EC of adult (7-9 week old) HCN1 null mice and their wildtype littermates (see Methods). Electroencephalographic (EEG) recordings were obtained 7- 10 days later from awake, freely moving mice present in their normal environment. As observed in the hippocampus (Nolan et al., 2004), no epileptiform abnormalities, interictal spikes or spontaneous seizures were detected in the EEG recorded daily from HCN1−/− (n=7) or wildtype (n=8) for 6 hr light and 6 hr dark cycles over a period of 1-3 wks (Fig 1A(i) and 1A(ii); total period of recording from 7 HCN1−/− = 900 hr; total recording time from 8 wt mice = 840 hr), indicating that these mice are not innately epileptic.
To investigate if there were differences in seizure threshold and manifestation of TLE between HCN1−/− and wildtype mice, we employed the commonly used so-called “kainate” model (Ben-Ari and Cossart, 2000; Dudek et al., 2002; White, 2002). In this model, a single episode of Class V seizures (as defined by the Racine Scale (Racine, 1972)) or status epilepticus (SE) is induced in rodents by administering kainic acid and then terminated ~1 hr later with an anticonvulsant such as sodium pentobarbital (see Methods). After a delay of a few weeks, known as the latent period (during which animals appear to be normal), spontaneous overt behavioral seizures occur (defined as the onset of chronic TLE; Ben-Ari and Cossart, 2000; Dudek et al., 2002; White, 2002). This model is widely used as many of the clinical and pathological features of the human disorder (including the latent period) can be reproduced (Ben-Ari and Cossart, 2000; Dudek et al., 2002; White, 2002). As previously demonstrated (He et al., 2004), administration of 20 mg/kg KA intraperitoneally elicited SE in wildtype mice in 43.1 ± 9.6 min (n=8, Fig 1A (v)). All wildtypes treated with 20 mg/kg KA survived the treatment. This concentration, however, caused SE within 5 min in HCN1−/− mice and was lethal (n=3, Fig 1A(vi)). Instead half the amount of KA, 10mg/kg (i.p.), was required to induce SE in a similar time frame (Fig 1A(iv), Fig 1A(vi)) and was not fatal. This lower dose had no effect in wildtype mice for up to 4 hrs (n=4, Fig 1A(iii)). EEG recordings showed that, despite the differences in dose, the intensity of of KA-induced SE in HCN1−/− mice and wildtypes was comparable (% change in gamma band power during SE from baseline of HCN1−/− and wildtype = 143.75 ± 18.75% (n=4) and 168.75 ± 23.66% (n=4) respectively). Since this was the case and the time taken to reach SE no different, we terminated the kainic acid induced- SE in both HCN1−/− and wildtypes with sodium pentobarbital (30 mg/kg s.c.) and used EEG recordings together with video monitoring to determine if there were differences in the latent period duration (see Methods) as well. HCN1−/− mice displayed overt motor convulsions (class III forelimb clonus seizures as defined by (Racine, 1972)) within 72 hr of halting SE (latent period duration = 60.0 ± 7.3 hr, n=4; Fig 1B). Conversely, wildtypes displayed similar spontaneous seizures approx. 2 weeks later (wildtype latent period duration = 386.8 ± 11.1 hr, n=4; Fig 1B). Hence, these mice appeared normal at 72 hr and only interictal spikes were detected in EEG recordings (Fig 1B(ii)). These results, therefore, indicate that that the decrease in HCN1 subunit expression which occurs in animal models and humans following TLE initiation (Brewster et al., 2002; Bender et al., 2003; Shah et al., 2004; Dugladze et al., 2007; Jung et al., 2007; Powell et al., 2008; Shin et al., 2008; Marcelin et al., 2009) is likely to have a substantial impact on the induction (seizure threshold) and expression of the disorder.
Since the EC (Jones, 1993; Spencer and Spencer, 1994; Avoli et al., 2002; Wu and Leung, 2003; Wozny et al., 2005), and EC layer III neurons in particular (Du et al., 1993; Du et al., 1995; Barbarosie and Avoli, 1997; Wu and Leung, 2003; Shah et al., 2004; Wozny et al., 2005; Ang et al., 2006), play a significant role during TLE, it is essential to determine how HCN1 ablation affects EC layer III cellular excitability. However, given that HCN2 subunits are also expressed in the EC, albeit to a much lower level than HCN1 subunits (Notomi and Shigemoto, 2004), it was first important to determine the extent to which Ih is reduced in HCN1−/− neurons. To investigate this, we made cell-attached voltage-clamp recordings from the soma and apical dendrite, approx. 150 μm from the soma (total EC layer III pyramidal neuron apical dendrite length = ~250 μm (Tahvildari and Alonso, 2005)). Ih was activated by applying 3 s hyperpolarizing pulses from −40 mV to −140 mV in the presence of Na+, K+ and Ca2+ channel blockers (see Methods). In agreement with the reported predominant dendritic expression of HCN channels (Notomi and Shigemoto, 2004; Shah et al., 2004), a slowly-activating current with an average steady-state magnitude of 19.25 ± 3.3 pA (n=8, Fig 2A) was observed only in wildtype dendrites. The same protocol elicited no significant current (0.86 ± 1.43 pA, n=6, Fig 2A) from HCN1−/− dendrites. Because western blot analysis has shown that HCN2 levels are unaltered in these mice (Nolan et al., 2003), these results indicate that HCN1 subunits are essential for the generation of Ih in these neurons.
To test how HCN1 deletion affected EC layer III pyramidal cell intrinsic membrane properties and excitability, we made whole-cell current clamp recordings from the soma and dendrites of these neurons. In the absence of both GABA and glutamate receptor blockers, HCN1−/− neurons had significantly more hyperpolarized resting membrane potentials (RMPs) as well as more spontaneous activity than wildtypes (Supp Fig 1). The greater spontaneous post-synaptic potential frequency was detected in HCN1−/− neurons even when the somatic RMP was artificially adjusted to −70 mV (Supp Fig 1), suggesting that EC neural network excitability was enhanced considerably more in HCN1−/− mice compared to that of wildtypes. This may, at least partly, explain why the HCN1−/− mice were more susceptible to kainic acid induced seizures.
The enhanced spontaneous post-synaptic potential frequency could be due to greater action potential driven synaptic release resulting from altered intrinsic excitability of neurons. To assess this, spontaneous post-synaptic potential activity was first suppressed with glutamate and GABA receptor blockers (see Methods). Under these conditions, significantly larger numbers of action potentials could be recorded in HCN1−/− dendrites compared to wildtype neurons when depolarizing current pulses were applied despite a more hyperpolarized RMP (HCN1−/− dendritic RMP = −74.1 ± 0.8 mV, n=17; wildtype dendritic RMP = −68.9 ± 0.9 mV, n=10, p < 0.05; Fig 2B). A comparable effect could be produced by in wildtype dendrites if Ih was suppressed using the inhibitor, ZD7288 (15 μM, a maximal concentration (BoSmith et al., 1993); Fig 3A, C). This inhibitor, however, did not affect HCN1−/− dendrite excitability (Fig 3)
In accordance with the predominant dendritic location of HCN channels (see above), the differences in somatic action potentials elicited in response to depolarizing current steps were much smaller between HCN1−/− and wildtype neurons at their normal RMPs (HCN1−/− somatic RMP = −75.3 ± 0.6 mV, n=33; wildtype somatic RMP = −69.3 ± 0.4 mV, n=22, p < 0.5; Fig 4). If, however, all cells were artificially held at fixed potentials of −70 mV, depolarizing current steps in HCN1−/− neurons produced significantly more action potentials (Fig 4 A, B, E). Application of 15 μM ZD7288 affected wildtype somatic excitability in a similar manner (Supp Fig 2) but had no effect on HCN1−/− somatic excitability (Supp Fig 2). There were also no differences in somatic or dendritic action potential shapes and threshold (measured at the soma, see Methods) between wildtype and HCN1−/− neurons (data not shown).
Why might more action potentials be recorded in dendrites, despite the hyperpolarized RMP? One possible reason might be altered dendritic input resistance (RN; Magee, 1998; Poolos et al., 2002; Shah et al., 2004; Tsay et al., 2007). Indeed, we noticed that hyperpolarizing and depolarizing current steps elicited bigger voltage deflections in HCN1−/− dendrites compared with wildtypes, indicating that the RN had changed (Fig 2B). We, thus, measured RN by applying 400 ms 100 pA hyperpolarizing current pulses from a holding potential of −70 mV (see Methods). Under these conditions, RN was ~200% greater in HCN1−/− dendrites than in wildtypes (dendritic HCN1−/− RN = 188.52 ± 16.04 MΩ, n=16; dendritic wildtype RN = 62.47 ± 8.48 MΩ, n=10, p<0.05; Fig 5A, C). Interestingly, though application of 15 μM ZD7288 had little effect on HCN1−/− dendrites (Fig 5A, D), wildtype dendritic RN was increased by approximately 200% in the presence of the compound (Fig 5A, D). In contrast, somatic RN was only enhanced by ~60% in HCN1−/− neurons compared with wildtypes (somatic HCN1−/− RN= 199.10 ± 16.44 MΩ, n=21; wildtype somatic RN = 124.79 ± 13.47 MΩ, n=19, p<0.05; Fig 5B, C). Further, 15 μM ZD7228 also only augmented wildtype somatic RN by approx. 100% whilst having little effect on HCN1−/− neurons (Fig 5B, D). These results confirm that Ih in EC layer III neurons is predominantly determined by HCN1 subunits. Further, the findings demonstrate that following HCN1 deletion or HCN pharmacological block, EC layer III pyramidal dendrites have enhanced excitability, at least partly, due to substantially increased RN.
Alterations in RN affect the amplitude and shapes of synaptic inputs and thereby their integration (Magee, 2000). Previous studies have shown that pharmacological block of dendritic HCN channels in hippocampal and cortical pyramids critically regulate temporal EPSP integration (Magee, 1999; Williams and Stuart, 2000; Berger et al., 2001; Poolos et al., 2002; Nolan et al., 2004; Shah et al., 2004; Kole et al., 2007; Nolan et al., 2007). In agreement, we found that the amplitude and decay time constant (τ) of single αEPSPs was greater in HCN1−/− dendrites compared with wildtypes (dendritic αEPSP amplitude in HCN1−/− and wildtype = 5.58 ± 2.06 mV (n=10) and 3.65 ± 1.13 mV (n=10, p < 0.05) respectively; Fig 6A). Consequentially, the summation of a 50 Hz train of 5 αEPSPs was enhanced considerably more in HCN1−/− dendrites than in wildtype dendrites (Fig 6B). Somatic αEPSP amplitudes, on the other hand, were no different between HCN1−/− and wildtypes (HCN1−/− and wildtype somatic αEPSP amplitude = 4.05 ± 1.80 (n=11) and 3.42 ± 0.97 (n=11, p = 0.32) respectively). Somatic αEPSP τ, though, was also significantly slower in HCN1−/− neurons than wildtypes (Fig 6A, B). The difference, however, was substantially less than that between HCN1−/− and wildtype dendrites (Fig 6A, B). Consequentially, somatic αEPSPs summated more in HCN1−/− neurons than in wildtypes (Fig 6B) but the effect was smaller than in dendrites (Fig 6A).
To test if the propensity for action potentials to be generated by a train of EPSPs is greater in HCN1−/− neurons compared with wildtypes, we obtained whole-cell current clamp recordings from the soma in the absence of glutamate blockers and minimally stimulated the afferent fibres to their distal dendrites (see Methods). The lack of glutamate inhibitors in the external medium resulted in a substantial increase in non-evoked EPSP frequency in HCN1−/− neurons (see Fig 7). In addition, considerably lowere amplitude extracellular stimulation of distal afferents was required to generate single 1-2 mV EPSPs in HCN1−/− neurons than wildtype neurons. Despite the lower stimulation strength though, a train of 5 stimuli delivered to distal HCN1−/− dendrites generated epileptiform-like activity at normal RMP in these cells (Fig 6C). In contrast, in wildtypes, a train of 5 evoked EPSPs did not summate sufficiently to produce an action potential unless the cell was depolarized above −50 mV (Fig 6C). Interestingly, stimulation of distal HCN1−/− dendrites in the absence of GABAA receptor inhibitors also resulted in trains of action potentials at the normal RMP (n=3, Fig 6C). These results confirm that, like in hippocampal neurons (Nolan et al., 2004; Tsay et al., 2007), HCN1 subunits are fundamental for regulating the temporal summation of EPSPs in EC layer III pyramidal neurons and thus the tendancy for action potentials to occur.
The above results suggest that as a result of the enhanced activity of pyramidal cells, action potential driven excitatory synaptic activity may be altered in HCN1−/− neurons. To test if this were the case, we recorded spontaneous, non-evoked excitatory post-synaptic potentials from EC layer III pyramidal neurons at a fixed potential of −70 mV from HCN1−/− and wildtype soma using the whole-cell voltage clamp technique (as described previously; Cossart et al., 2001; El-Hassar et al., 2007b)). To eliminate effects of post-synaptic HCN channels and to reduce errors due to space-clamp (Williams and Mitchell, 2008), 15 μM ZD7288 was incorporated in the patch pipette. Though the presence of intracellular ZD7288 affected synaptic potential decay in wildtype neurons, it had no effect on their frequency (data not shown). In addition, inclusion of ZD7288 in the patch pipette resulted in the outward holding current of wildtype neurons increasing by 20.45 ± 4.34 mV (n=10), presumably as the RMP of the neurons became more hyperpolarized. In contrast, the outward holding current of HCN1−/− neurons was unaffected by the presence of ZD7288 (n=9). To exclude inhibitory events, these experiments were done in the presence of GABA receptor blockers, bicuculline and CGP 55845. Under these conditions, the HCN1−/− EPSC (EPSC) frequency recorded was enhanced by approximately 4 fold compared with wildtypes (wildtype EPSC frequency = 2.95 ± 0.28, n= 11; HCN1−/− EPSC frequency = 11.08 ± 1.36, n=10, p<0.05; Fig 7A). There were no differences in the amplitude or the kinetics of EPSCs (data not shown).
Since some EC inhibitory neurons may possibly have Ih (Kumar and Buckmaster, 2006), we also investigated if action potential driven inhibitory synaptic potentials (IPSC) frequency was altered by HCN1 deletion (see Methods). Although the IPSC amplitudes and kinetics were unaffected, the IPSC frequency was approx 2 fold greater in HCN1−/− neurons (IPSC frequency = 7.50 ± 2.35, n= 9, Fig 7B) compared with wildtypes (IPSC frequency = 4.65 ± 0.59, n=9, p<0.05, Fig 7B). Intriguingly, despite this the EPSC frequency (11.08 ± 1.36, n=10) was still significantly more enhanced than the IPSC frequency in HCN1−/− neurons (Fig 7). In contrast, in wildtypes, IPSCs occurred substantially more frequently than EPSCs (wildtype EPSC frequency = 2.95 ± 0.28, n= 11, Fig 7) Hence, HCN1 deletion results in disproportionate rise in excitatory synaptic activity. As this is a common feature during epilepsy too (e.g. see (Scimemi et al., 2005; El-Hassar et al., 2007a), this might partly explain why HCN1−/− mice are more susceptible to seizure induction (Fig 1).
Multiple studies have previously shown that altered expression of HCN subunits is associated with temporal lobe epilepsy (TLE; (Brewster et al., 2002; Bender et al., 2003; Shah et al., 2004; Jung et al., 2007; Powell et al., 2008; Shin et al., 2008; Marcelin et al., 2009). In this study, we now demonstrate that a lack of HCN1 subunits is likely to significantly influence seizure threshold as well as the process of TLE. Although in vivo EEG recordings showed that HCN1−/− mice were not inherently epileptic, these mice were significantly more vulnerable to kainic acid induced seizures (Fig 1). Further, following termination of kainic acid-induced seizures, HCN1 null mice developed overt behavioural seizures (chronic epilepsy) at an approximately 6 times faster rate than wildtype littermate controls (Fig 1). In vitro electrophysiological experiments showed that the greater seizure susceptibility of HCN1 null mice could, at least partly, be attributed to the enhanced excitability of EC layer III neurons, caused by lack of dendritic Ih (Fig 2). This is likely to have contributed to increased excitatory synaptic transmission (Fig 7) and consequentially there was an imbalance in excitatory and inhibitory synaptic activity (Fig 7). Hence HCN channels by regulating dendritic intrinsic membrane properties and thereby, pyramidal cell excitability are able to influence cortical neural network activity.
The lack of HCN1 subunits resulted in the ablation of Ih in EC layer III pyramidal neurons (Fig 2), which are known to play a significant part in the progress of TLE (Du et al., 1993; Jones, 1993; Du et al., 1995; Barbarosie and Avoli, 1997; Avoli et al., 2002; Wu and Leung, 2003; Shah et al., 2004; Wozny et al., 2005; Ang et al., 2006). Consistent with this, the RMP was considerably more hyperpolarized and RN significantly greater in HCN1−/− neurons compared with wildtype littermate controls (Fig (Fig22,,4).4). The greater RN would give rise to bigger voltage deflections in response to depolarizing pulses (Fig (Fig22,,4).4). This in conjunction with increased Na+ and Ca2+ channel availability due to the hyperpolarized RMP (Tsay et al., 2007) is likely to explain why more action potentials were elicited in HCN1−/− dendrites than wildtype dendrites in response to the depolarizing current pulses of similar magnitude (Fig 2). This effect was not noticeable at the soma as, in agreement with the predominant dendritic location of HCN1 subunits (Fig (Fig22,,3;3; Notomi and Shigemoto, 2004; Shah et al., 2004), somatic RN was less affected by the loss of HCN1 subunits (Fig 5). In addition, because of the significantly enhanced dendritic RN (Fig 5), EPSP integration in HCN1−/− dendrites was considerably greater than in wildtype dendrites (Fig 6). Consequentially, trains of excitatory synaptic inputs that did not elicit action potentials in wildtype neurons unless they were substantially depolarized, resulted in epileptiform-like activity in HCN1−/− neurons at their normal RMP (Fig 6). Thus, these results, in accordance with those obtained in previous studies (Shah et al., 2004; Tsay et al., 2007), indicate that dendritic Ih acts an electrical shunt, normalizing any significant changes in voltage. In this manner, dendritic Ih in these neurons also contributes to regulating action potential driven output.
Under physiological conditions, experimental evidence suggests that excitatory and inhibitory inputs onto cortical neurons are proportional and balanced, allowing stability of neural network activity (Shu et al., 2003; Haider et al., 2006). Application of ZD7288 has been previously shown to boost action potential firing and thereby recurrent network activity in pre-frontal cortex (Wang et al., 2007). In agreement, our findings also suggest that a loss of HCN1, by increasing the propensity for action potentials to occur in pyramidal cells with any given synaptic input (Fig 6C), would lead to greater neuronal output and hence substantially more synaptic activity. Indeed, action potential driven HCN1−/− EPSC frequency was enhanced 4 fold (Fig 7A). However, this might also be predicted to augment interneuron activity and thereby raise inhibition (Dugladze et al., 2007). Further, some interneurons have also been suggested to have Ih (McBain and Fisahn, 2001; Aponte et al., 2006; Kumar and Buckmaster, 2006; Dugladze et al., 2007) and a loss of Ih may also contribute to greater activity of these (Dugladze et al., 2007). Although we did not test if interneuron activity per se was altered either as a consequence of enhanced synaptic activation or a loss of Ih, our findings suggested that this might be the case as IPSC frequency onto EC layer III pyramidal neurons was amplified (Fig 7B). In spite of this though, the EPSC/IPSC ratio changed from 0.63 in wildtypes to 1.47 in HCN1−/− neurons (Fig 7). Hence, loss of HCN1 subunits leads to a disproportionate increase in excitatory synaptic activity.
We have shown that a lack of HCN1 subunits results in EC layer III pyramidal cell hyperexcitability, enhanced action potential driven spontaneous HCN1−/− EPSC frequency and thus, altered EC neural network activity. Previous studies have also demonstrated that HCN1 deletion results in greater dendritic excitability and EPSP summation in hippocampal pyramidal CA1 neurons (Nolan et al., 2004; Tsay et al., 2007), which are also involved in seizure generation during TLE (Spencer and Spencer, 1994). It is thus surprising that interictal spikes or electrographic seizures were not observed in HCN1−/− mice in vivo (Fig 1 and see Nolan et al. (2004)). However, the amplified IPSC frequency, even though this was to a much lower extent than excitatory synaptic transmission (Fig 7), together with increased IPSC summation caused by loss of Ih (Chen et al., 2001) might serve to offset the heightened neural network activity and thereby, prevent the occurrence of inherent interictal spikes or seizures. Hence, though the reduction of HCN1 subunit clearly favours neural network excitability (Supp Fig 1, Fig 7), the imbalance between excitation and inhibition may be insufficient to render HCN1−/− mice spontaneously epileptic.
The HCN1−/− mice, though, were clearly more susceptible to seizures and developed chronic TLE at a much faster rate than wildtype littermates (Fig 1). Multiple studies have demonstrated that HCN1 subunit expression is reduced following status epilepticus (Brewster et al., 2002; Shah et al., 2004; Dugladze et al., 2007; Jung et al., 2007; Powell et al., 2008; Shin et al., 2008; Marcelin et al., 2009). Hence, the latent period duration might be expected to be comparable between wildtypes and HCN1−/− mice following TLE induction. However, it should be noted that HCN1 expression in wildtypes is not ablated in the hippocampus or cortex following TLE (Brewster et al., 2002; Shah et al., 2004; Dugladze et al., 2007; Jung et al., 2007; Powell et al., 2008; Shin et al., 2008; Marcelin et al., 2009). Indeed, in hippocampal CA1 dendrites, depending on the time point of measurement and model employed, the decrease in Ih following status epilepticus in wildtypes can vary between 30-50% (Jung et al., 2007; Shin et al., 2008; Marcelin et al., 2009). Further, somatic HCN1 subunit expression may also be transiently enhanced in CA1 pyramidal cells following TLE in wildtypes (Shin et al., 2008). Moreover, it is not known if status epilepticus results in altered HCN1 protein levels in all wildtype neurons expressing HCN1 subunits. In contrast, in HCN1−/− mice, Ih is persistently reduced by approx. 70% in CA1 neurons (Nolan et al., 2004). Thus, variations in HCN1 levels may explain the difference in latent period duration following termination of status epilepticus in wildtype and HCN1−/− mice. Nonetheless, our results show that the decline in HCN subunit expression following TLE induction is likely to contribute to the condition.
This work was supported an MRC New Investigator Award and Epilepsy Research Foundation UK and Royal Society project grants to MMS. We would like to express our gratitude to Mr. S. Martin (UCL Sequencing and Genotyping Facility, UK) for genotyping the HCN transgenic mice. We also wish to thank Prof. D. Johnston (UT Austin, USA), Prof. D. A. Brown (UCL, UK) and Dr. M. F. Nolan (Edinburgh University, UK) for useful discussions and critically reading our manuscript.