h channel-dependent deficit of theta oscillation resonance and phase shift in temporal lobe epilepsy

I_h tunes hippocampal CA1 pyramidal cell dendrites to optimally respond to theta inputs (4–12 Hz), and provides a negative time delay to theta inputs. Decreased I_h activity, as seen in experimental temporal lobe epilepsy (TLE), could significantly alter the response of dendrites to theta inputs. Here we report a progressive erosion of theta resonance and phase lead in pyramidal cell dendrites during epileptogenesis in a rat model of TLE. These alterations were due to decreased I_h availability, via a decline in HCN1/HCN2 subunit expression resulting in decreased h currents, and altered kinetics of the residual channels. This acquired HCN channelopathy thus compromises temporal coding and tuning to theta inputs in pyramidal cell dendrites. Decreased theta resonance in vitro also correlated with a reduction in theta frequency and power in vivo. We suggest that the neuronal/circuitry changes associated with TLE, including altered I_h-dependent inductive mechanisms, can disrupt hippocampal theta function.     

低镁诱发急性海马切片高兴奋性的多电极记录

目的 多电极记录技术在脑片研究的应用已经远远超过了10年,然而该技术却没有广泛地用于癫痫领域的研究。用经典的致痫剂低镁人工脑脊液灌流大鼠急性海马切片,诱导产生癫痫样电活动并用多电极记录技术对其放电特征及内部传导方式进行分析。 方法 用多电极阵列持续记录灌流低镁人工脑脊液后海马各区域的放电情况,并比较切断CA3与CA1区域间的Schaffer氏纤维后各区的放电情况。 结果 在急性海马切片上诱导出自发、同步、癫痫样电活动;CA3区神经元簇发放电持续时间及簇发放电内动作电位的个数与CA1及DG区相比有显著的统计学差异;剪断CA3与CA1间的Schaffer氏纤维后,CA1区的电活动消失,CA3区仍有同步放电,且其自发同步放电的频率与对照组相比无显着改变,但其簇发放电持续时间及簇发放电内动作电位的个数明显降低（P<0.05）。 结论 成功地在多电极上记录到急性海马切片自发、同步、癫痫样电活动;其中CA3区神经元兴奋性最高;在低镁灌流下自发癫痫样电活动起源于CA3区,在剪断Schaffer氏侧支后CA3区神经元群体同步放电的频率的频率没有显着变化。     

低镁诱发急性海马切片高兴奋性的多电极记录

目的 多电极记录技术在脑片研究的应用已经远远超过了10年,然而该技术却没有广泛地用于癫痫领域的研究。用经典的致痫剂低镁人工脑脊液灌流大鼠急性海马切片,诱导产生癫痫样电活动并用多电极记录技术对其放电特征及内部传导方式进行分析。 方法 用多电极阵列持续记录灌流低镁人工脑脊液后海马各区域的放电情况,并比较切断CA3与CA1区域间的Schaffer氏纤维后各区的放电情况。 结果 在急性海马切片上诱导出自发、同步、癫痫样电活动;CA3区神经元簇发放电持续时间及簇发放电内动作电位的个数与CA1及DG区相比有显著的统计学差异;剪断CA3与CA1间的Schaffer氏纤维后,CA1区的电活动消失,CA3区仍有同步放电,且其自发同步放电的频率与对照组相比无显着改变,但其簇发放电持续时间及簇发放电内动作电位的个数明显降低（P<0.05）。 结论 成功地在多电极上记录到急性海马切片自发、同步、癫痫样电活动;其中CA3区神经元兴奋性最高;在低镁灌流下自发癫痫样电活动起源于CA3区,在剪断Schaffer氏侧支后CA3区神经元群体同步放电的频率的频率没有显着变化。     

Transient cerebral ischemia increases CA1 pyramidal neuron excitability

In human and experimental animals, the hippocampal CA1 region is one of the most vulnerable areas of the brain to ischemia. Pyramidal neurons in this region die 2–3 days after transient cerebral ischemia whereas other neurons in the same region remain intact. The mechanisms underlying the selective and delayed neuronal death are unclear. We tested the hypothesis that there is an increase in post-synaptic intrinsic excitability of CA1 pyramidal neurons after ischemia that exacerbates glutamatergic excitotoxicity. We performed whole-cell patch-clamp recordings in brain slices obtained 24 h after in vivo transient cerebral ischemia. We found that the input resistance and membrane time constant of the CA1 pyramidal neurons were significantly increased after ischemia, indicating an increase in neuronal excitability. This increase was associated with a decrease in voltage sag, suggesting a reduction of the hyperpolarization-activated non-selective cationic current (I_h). Moreover, after blocking I_h with ZD7288, the input resistance of the control neurons increased to that of the post-ischemia neurons, suggesting that a decrease in I_h contributes to increased excitability after ischemia. Finally, when lamotrigine, an enhancer of dendritic I_h, was applied immediately after ischemia, there was a significant attenuation of CA1 cell loss. These data suggest that an increase in CA1 pyramidal neuron excitability after ischemia may exacerbate cell loss. Moreover, this dendritic channelopathy may be amenable to treatment.     

In vivo assessment of HCN channel current (Ih) in human motor axons

The “Trond” protocol of nerve excitability tests has been used widely to assess axonal function in peripheral nerve. In this study, the routine Trond protocol was expanded to refine assessment of cAMP‐dependent, hyperpolarization‐activated current (I_h) activity. I_h activity is generated by hyperpolarization‐activated, cyclic nucleotide–modulated (HCN) channels in response to hyperpolarization. It limits activity‐dependent hyperpolarization, contributes to neuronal automaticity, and is implicated in chronic pain states. Published data regarding I_h activity in motor nerve are scant. We used additional strong, prolonged hyperpolarizing conditioning stimuli in the threshold electrotonus component of the Trond protocol to demonstrate the time‐course of activation of I_h in motor axons. Fifteen healthy volunteers were tested on four occasions during 1 week. I_h action was revealed in the threshold electrotonus by the limiting and often reversal, after about 100 ms, of the threshold increase caused by strong hyperpolarizing currents. Statistical analysis by repeated‐measures analysis of variance enabled confidence limits to be established for variation between subjects and within subjects. The results demonstrate that, of all the excitability parameters, those dependent on I_h were the most characteristic of an individual, because variance between subjects was more than four times the variance within subjects. This study demonstrates a reliable method for in vivo assessment of I_h, and also serves to document the normal variability in nerve excitability properties within subjects. Muscle Nerve, 2010     

Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (I_h). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and I_h properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that I_h was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of I_h found in WAG/Rij rats compensate each other in a way that leaves I_h availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of I_h on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in I_h in older animals.     

Long‐term high‐intensity sound stimulation inhibits h current (Ih) in CA1 pyramidal neurons

Afferent neurotransmission to hippocampal pyramidal cells can lead to long‐term changes to their intrinsic membrane properties and affect many ion currents. One of the most plastic neuronal currents is the hyperpolarization‐activated cationic current (I_h), which changes in CA1 pyramidal cells in response to many types of physiological and pathological processes, including auditory stimulation. Recently, we demonstrated that long‐term potentiation (LTP) in rat hippocampal Schaffer‐CA1 synapses is depressed by high‐intensity sound stimulation. Here, we investigated whether a long‐term high‐intensity sound stimulation could affect intrinsic membrane properties of rat CA1 pyramidal neurons. Our results showed that I_h is depressed by long‐term high‐intensity sound exposure (1 min of 110 dB sound, applied two times per day for 10 days). This resulted in a decreased resting membrane potential, increased membrane input resistance and time constant, and decreased action potential threshold. In addition, CA1 pyramidal neurons from sound‐exposed animals fired more action potentials than neurons from control animals; however, this effect was not caused by a decreased I_h. On the other hand, a single episode (1 min) of 110 dB sound stimulation which also inhibits hippocampal LTP did not affect I_h and firing in pyramidal neurons, suggesting that effects on I_h are long‐term responses to high‐intensity sound exposure. Our results show that prolonged exposure to high‐intensity sound affects intrinsic membrane properties of hippocampal pyramidal neurons, mainly by decreasing the amplitude of I_h.     

Cocaine Sensitization Increases I h Current Channel Subunit 2 (HCN2) Protein Expression in Structures of the Mesocorticolimbic System

Alteration of the biological activity among neuronal components of the mesocorticolimbic (MCL) system has been implicated in the pathophysiology of drug abuse. Changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction. The hyperpolarization-activated cyclic nucleotide current, I _h, is a prominent mixed cation current present in neurons. The biophysical properties of the I _h and its potential modulatory role in cell excitability depend on the expression profile of the hyperpolarization-activated cyclic nucleotide gated channel (HCN) subunits. We investigated whether cocaine-induced behavioral sensitization, an animal model of drug addiction, elicits region-specific changes in the expression of the HCN₂ channel’s subunit in the MCL system. Tissue samples from the ventral tegmental area, prefrontal cortex, nucleus accumbens, and hippocampus were analyzed using Western blot. Our findings demonstrate that cocaine treatment induced a significant increase in the expression profile of the HCN₂ subunit in both its glycosylated and non-glycosylated protein isoforms in all areas tested. The increase in the glycosylated isoform was only observed in the ventral tegmental area. Together, these data suggest that the observed changes in MCL excitability during cocaine addiction might be associated with alterations in the subunit composition of their HCN channels.     

Intracellular correlates of morphine excitation in the hippocampal slice preparation

The influences of morphine and opioid peptides on hippocampal CA1 pyramidal cells were investigated using intracellular recordings from the in vitro slice preparation. Morphine applied to somal and basal dendritic areas of CA1 cells via a pressure ejection system confirmed a number of excitatory actions of opiates and opioid peptides in this brain region. These included an increase in the amplitude and duration of orthodromically (radiatum) elicited EPSPs and a decrease in amplitude of the following IPSP. The increase in EPSP amplitude was accompanied by a reduction in stimulus intensity necessary for eliciting the action potential. Morphine delivered to the slice in this manner induced synaptically elicited and spontaneous multiple spike burst discharges. In slice maintained in 10⁻⁴ M pentobarbital¹, morphine reversed the presumably GABA mediated long-duration depolarization following orthodromic stimulation. Finally, depending on the specific site of application (apical or basal dendritic region) of the opiate, morphine produced two different effects on the resting membrane potential and input resistance of CA1 pyramidal cells. These findings are discussed as to whether opiates act directly excitatory influences in the hippocampus, or via blockade of GABA mediated inhibitory mechanisms.     

The influence of electric fields on the epileptiform bursts induced by high potassium in CA3 region of rat hippocampal slice

Abstract

The effects of pulsed direct current (de) electric fields on the frequency of spontaneous bursting in a model epileptic focus were studied. The high potassium hippocampal slice model was used to generate spontaneous burst firing activity similar to interictal spikes in the pyramidal cell layer of CA3. Electric fields were generated from platinum subdural electrodes placed in the perfusion bath. Three hundred and seventy-eight experimental trials were performed on 10 hippocampal slices from 10 rats and the effects of field polarity, field strength and duration of stimuli on firing frequency was examined. Hippocampal slices were oriented horizontally with the CA3 layer towards the positive electrodel the average interburst interval did not correlate significantly with polarity of the delivering pulses (one-way ANOVA, p = 0.96). Average interburst interval showed a significant correlation with pulse duration of200 and 400msec (p = 0.030 and p = 0.004, respectively). As a function of field strength, there were significant average interval changes for fields of 33, 46, and 73 mV/mm (p = 0.024, P = 0.001 and p = 0.001, respectively). In conclusion, CA3 burst firing activity in high potassium concentration can therefore be altered by electric fields. [Neural Res 1998; 20: 542-548]     

Long-term maintenance of mature hippocampal slices in vitro

Cultures of primary neurons or thin brain slices are typically prepared from immature animals. We introduce a method to prepare hippocampal slice cultures from mature rats aged 20-30 days. Mature slice cultures retain hippocampal cytoarchitecture and synaptic connections up to 3 months in vitro. Spontaneous epileptiform activity is rarely observed suggesting long-term retention of normal neuronal excitability and of excitatory and inhibitory synaptic networks. Picrotoxin, a GABAergic Cl(-) channel antagonist, induced characteristic interictal-like bursts that originated in the CA3 region, but not in the CA1 region. These data suggest that mature slice cultures displayed long-term retention of GABAergic inhibitory synapses that effectively suppressed synchronized burst activity via recurrent excitatory synapses of CA3 pyramidal cells. Mature slice cultures lack the reactive synaptogenesis, spontaneous epileptiform activity, and short life span that limit the use of slice cultures isolated from immature rats. Mature slice cultures are anticipated to be a useful addition for the in vitro study of normal and pathological hippocampal function.     

Absence epilepsy in apathetic, a spontaneous mutant mouse lacking the h channel subunit, HCN2

Analysis of naturally occurring mutations that cause seizures in rodents has advanced understanding of the molecular mechanisms underlying epilepsy. Abnormalities of I_h and h channel expression have been found in many animal models of absence epilepsy. We characterized a novel spontaneous mutant mouse, apathetic (ap/ap), and identified the ap mutation as a 4 base pair insertion within the coding region of Hcn2, the gene encoding the h channel subunit 2 (HCN2). We demonstrated that Hcn2^ap mRNA is reduced by 90% compared to wild type, and the predicted truncated HCN2^ap protein is absent from the brain tissue of mice carrying the ap allele. ap/ap mice exhibited ataxia, generalized spike–wave absence seizures, and rare generalized tonic–clonic seizures. ap/+ mice had a normal gait, occasional absence seizures and an increased severity of chemoconvulsant-induced seizures. These findings help elucidate basic mechanisms of absence epilepsy and suggest HCN2 may be a target for therapeutic intervention.     

Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (I_Na) amplitude, causing a hyperpolarizing shift in the inactivation curve of the I_Na, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (I_NaP). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner.     

Activation of extrasynaptic GABAA receptors inhibits cyclothiazide-induced epileptiform activity in hippocampal CA1 neurons

Extrasynaptic GABA_A receptors (GABA_ARs)-mediated tonic inhibition is reported to involve in the pathogenesis of epilepsy. In this study, we used cyclothiazide (CTZ)-induced in vitro brain slice seizure model to explore the effect of selective activation of extrasynaptic GABA_ARs by 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-ol (THIP) on the CTZ-induced epileptiform activity in hippocampal neurons. Perfusion with CTZ dose-dependently induced multiple epileptiform peaks of evoked population spikes (PSs) in CA1 pyramidal neurons, and treatment with THIP (5 μmol/L) significantly reduced the multiple PS peaks induced by CTZ stimulation. Western blot showed that the δ-subunit of the GABA_AR, an extrasynaptic specific GABA_AR subunit, was also significantly down-regulated in the cell membrane 2 h after CTZ treatment. Our results suggest that the CTZ-induced epileptiform activity in hippocampal CA1 neurons is suppressed by the activation of extrasynaptic GABA_ARs, and further support the hypothesis that tonic inhibition mediated by extrasynaptic GABA_ARs plays a prominent role in seizure generation.     

Variations in electrophysiological properties of hippocampal neurons in different subfields

Intracellular recording were made from hippocampal pyramidal cells (HPCs) in subregions CAla, b, c, CA2 and CA3a, b of the guinea pig hippocampal slice. There were significant differences in the mode of spike discharge at various sites. Most neurons in CA1b and CA3b fired single spikes spontaneously, or during intracellular depolarizing current pulses. HPCs in the CA1a and c, as well as CA2 and CA3a subregions usually had a burst mode of discharge under the same conditions. Basic differences in neuronal properties presumably underlie these varieties of behavior between or within various regions. Specification of the site or subregion of recording is important especially in those experiments where the mode of spike discharge or membrane events in HPCs are important variables.     

Differential developmental refinement of the intrinsic electrophysiological properties of CA1 pyramidal neurons from the rat dorsal and ventral hippocampus

The dorsal and ventral regions of the rat longitudinal hippocampal axis are functionally distinct. That is, each region is associated with different behavioral tasks and disease susceptibilities due to underlying anatomical, and physiological differences. These differences are especially pronounced in area CA1, where significant differences in morphology, synaptic physiology, intrinsic excitability, and gene expression have been reported between CA1 pyramidal neurons from the dorsal (DHC) and ventral hippocampus (VHC). However, despite a significant amount of recent attention, a cogent picture of the intrinsic electrophysiological profile of DHC and VHC neurons has remained elusive, due, in part, to experiments performed on rats at different developmental time points. Moreover, the resulting intrinsic electrophysiological profiles are sufficiently different as to warrant a thorough investigation of the spatial and temporal changes in the intrinsic excitability of CA1 pyramidal neurons across developmental time. Accordingly, in this study, I have characterized the intrinsic electrophysiological properties of CA1 pyramidal neurons from acute hippocampal slices prepared from the DHC and VHC throughout an approximately 3‐week developmental period (P14–P37). DHC and VHC neurons exhibited distinct intra‐region changes (DHC or VHC) and inter‐region differences (DHC versus VHC) in their intrinsic electrophysiological properties, which yielded two developmental timelines: (a) a common developmental timeline describing changes observed in both DHC and VHC neurons, and (b) a differential developmental timeline highlighting unique features observed in DHC neurons. Specifically, DHC neurons exhibited significant inter‐region differences in RMP, input resistance, threshold, and spike frequency adaptation relative to VHC neurons, as well as an intra‐region change in the rebound slope (a proxy for I_h). These observations both integrate and reconcile previous work performed with rats at different developmental stages and suggest a distinct developmental trajectory for DHC neurons that might shed light on the normal physiological functions and disease susceptibility of the DHC.     

Effects of kainate on the excitability of rat hippocampal neurones

Intracellular recordings from CA1 pyramidal neurones of the rat hippocampal slice preparation were used to study changes in neuronal excitability induced by the excitatory amino acid analogues kainate (KA) and N-methyl-D-aspartate (NMDA). Low concentrations of bath-applied KA (50-200 nM) or NMDA (1-3 microM) elicited a relatively small membrane depolarization and increased the number of spikes fired by a constant current pulse. The spike after-hyperpolarization (AHP) was depressed by KA but enhanced by NMDA. After blockade of the voltage-sensitive Na+ conductances with tetrodotoxin, intracellularly applied current pulses elicited Ca2+ spikes. Whereas NMDA always increased the duration (and number) of Ca2+ spikes and of their AHP, KA conversely reduced these spikes and (in almost half of the cells tested) the late phase of their AHP. When Ba2+ was used to replace extracellular Ca2+, prolonged plateau potentials developed and were also blocked by KA. NMDA had no effect on Ba2(+)-dependent responses. These results suggest that low concentrations of KA profoundly modified the electroresponsiveness of CA1 neurones perhaps by depressing a Ca2(+)-dependent K+ conductance mechanism responsible for dampening the excitability of these cells.