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KEY POINTS Stimulation of postsynaptic muscarinic receptors was shown to excite principal hippocampal neurons by modulating several membrane ion conductances. We show here that activation of postsynaptic muscarinic receptors also causes neuronal excitation by inhibiting Na+ /K+ -ATPase activity. Muscarinic Na+ /K+ -ATPase inhibition is mediated by two separate signalling pathways that lead downstream to enhanced Na+ /K+ -ATPase phosphorylation by activating protein kinase C and protein kinase G. Muscarinic excitation through Na+ /K+ -ATPase inhibition is probably involved in cholinergic modulation of hippocampal activity and may turn out to be a widespread mechanism of neuronal excitation in the brain. ABSTRACT Stimulation of muscarinic cholinergic receptors on principal hippocampal neurons enhances intrinsic neuronal excitability by modulating several membrane ion conductances. The electrogenic Na+ /K+ -ATPase (NKA; the 'Na+ pump') is a ubiquitous regulator of intrinsic neuronal excitability, generating a hyperpolarizing current to thwart excessive neuronal firing. Using electrophysiological and pharmacological methodologies in rat hippocampal slices, we show that neuronal NKA pumping activity is also subjected to cholinergic regulation. Stimulation of postsynaptic muscarinic, but not nicotinic, cholinergic receptors activates membrane-bound phospholipase C and hydrolysis of membrane-integral phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3 ). Along one signalling pathway, DAG activates protein kinase C (PKC). Along a second signalling pathway, IP3 causes Ca2+ release from the endoplasmic reticulum, facilitating nitric oxide (NO) production. The rise in NO levels stimulates cGMP synthesis by guanylate-cyclase, activating protein kinase G (PKG). The two pathways converge to cause partial NKA inhibition through enzyme phosphorylation by PKC and PKG, leading to a marked increase in intrinsic neuronal excitability. This novel mechanism of neuronal NKA regulation probably contributes to the cholinergic modulation of hippocampal activity in spatial navigation, learning and memory.

Brain insults, such as trauma, stroke, anoxia, and status epilepticus (SE), cause multiple changes in synaptic function and intrinsic properties of surviving neurons that may lead to the development of epilepsy. Experimentally, a single SE episode, induced by the convulsant pilocarpine, initiates the development of an epileptic condition resembling human temporal lobe epilepsy (TLE). Principal hippocampal neurons from such epileptic animals display enhanced spike output in response to excitatory stimuli compared with neurons from nonepileptic animals. This enhanced firing is negatively related to the size of the slow afterhyperpolarization (sAHP), which is reduced in the epileptic neurons. The sAHP is an intrinsic neuronal negative feedback mechanism consisting normally of two partially overlapping components produced by disparate mechanisms. One component is generated by activation of Ca2+-gated K+(KCa) channels, likely KCa3.1, consequent to spike Ca2+influx (the KCa-sAHP component). The second component is generated by enhancement of the electrogenic Na+/K+ATPase (NKA) by spike Na+influx (NKA-sAHP component). Here we show that the KCa-sAHP component is markedly reduced in male rat epileptic neurons, whereas the NKA-sAHP component is not altered. The KCa-sAHP reduction is due to the downregulation of KCa3.1 channels, mediated by cAMP-dependent protein kinase A (PKA). This sustained effect can be acutely reversed by applying PKA inhibitors, leading also to normalization of the spike output of epileptic neurons. We propose that the novel “acquired channelopathy” described here, namely, PKA-mediated downregulation of KCa3.1 activity, provides an innovative target for developing new treatments for TLE, hopefully overcoming the pharmacoresistance to traditional drugs.SIGNIFICANCE STATEMENTEpilepsy, a common neurological disorder, often develops following a brain insult. Identifying key molecular and cellular mechanisms underlying acquired epilepsy is critical for developing effective antiepileptic therapies. In an experimental model of acquired epilepsy, we show that principal hippocampal neurons become intrinsically hyperexcitable. This alteration is due predominantly to the downregulation of a ubiquitous class of potassium ion channels, KCa3.1, whose main function is to dampen neuronal excitability. KCa3.1 downregulation is mediated by the cAMP-dependent protein kinase A (PKA) signaling pathway. Most importantly, it can be acutely reversed by PKA inhibitors, leading to recovery of KCa3.1 function and normalization of neuronal excitability. The discovery of this novel epileptogenic mechanism hopefully will facilitate the development of more efficient pharmacotherapy for acquired epilepsy.

Stimulation of postsynaptic muscarinic receptors was shown to excite principal hippocampal neurons by modulating several membrane ion conductances. We show here that activation of postsynaptic muscarinic receptors also causes neuronal excitation by inhibiting NaStimulation of muscarinic cholinergic receptors on principal hippocampal neurons enhances intrinsic neuronal excitability by modulating several membrane ion conductances. The electrogenic Na

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K+ current (I M), Ca2+‐gated K+ currents (I Ca(K)'s) and Na+/K+‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I M contribution to the sAHP was found in these neurons. Both I Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α1‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α1‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.

The Na(+)/K(+)-ATPase (NKA) is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na(+) and K(+) electrochemical gradients across the plasmalemma of living cells. Numerous studies in non-neuronal tissues have shown that this transport mechanism is reversibly regulated by phosphorylation/dephosphorylation of the catalytic α subunit and/or associated proteins. In neurons, Na(+)/K(+) transport by NKA is essential for almost all neuronal operations, consuming up to two-thirds of the neuron's energy expenditure. However, little is known about its cellular regulatory mechanisms. Here we have used an electrophysiological approach to monitor NKA transport activity in male rat hippocampal neurons in situ. We report that this activity is regulated by a balance between serine/threonine phosphorylation and dephosphorylation. Phosphorylation by the protein kinases PKG and PKC inhibits NKA activity, whereas dephosphorylation by the protein phosphatases PP-1 and PP-2B (calcineurin) reverses this effect. Given that these kinases and phosphatases serve as downstream effectors in key neuronal signaling pathways, they may mediate the coupling of primary messengers, such as neurotransmitters, hormones, and growth factors, to the NKAs, through which multiple brain functions can be regulated or dysregulated. SIGNIFICANCE STATEMENT The Na(+)/K(+)-ATPase (NKA), known as the “Na(+) pump,” is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na(+) and K(+) electrochemical gradients across the plasma membrane of living cells. In neurons, as in most types of cells, the NKA generates the negative resting membrane potential, which is the basis for almost all aspects of cellular function. Here we used an electrophysiological approach to monitor physiological NKA transport activity in single hippocampal pyramidal cells in situ. We have found that neuronal NKA activity is oppositely regulated by phosphorylation and dephosphorylation, and we have identified the main protein kinases and phosphatases mediating this regulation. This fundamental form of NKA regulation likely plays a role in multiple brain functions.

Small-conductance Ca(2+)-activated K(+) (SK or K(Ca)2) channels are widely expressed in the CNS. In several types of neurons, these channels were shown to become activated during repetitive firing, causing early spike frequency adaptation. In CA1 pyramidal cells, SK channels in dendritic spines were shown to regulate synaptic transmission. However, the presence of functional SK channels in the somata and their role in controlling the intrinsic firing of these neurons has been controversial. Using whole-cell voltage-clamp and current-clamp recordings in acute hippocampal slices and focal applications of irreversible and reversible SK channel blockers, we provide evidence that functional SK channels are expressed in the somata and proximal dendrites of adult rat CA1 pyramidal cells. Although these channels can generate a medium duration afterhyperpolarizing current, they play only an auxiliary role in controlling the intrinsic excitability of these neurons, secondary to the low voltage-activating, noninactivating K(V)7/M channels. As long as K(V)7/M channels are operative, activation of SK channels during repetitive firing does not notably affect the spike output of CA1 pyramidal cells. However, when K(V)7/M channel activity is compromised, SK channel activation significantly and uniquely reduces spike output of these neurons. Therefore, proximal SK channels provide a "second line of defense" against intrinsic hyperexcitability, which may play a role in multiple conditions in which K(V)7/M channels activity is compromised, such as hyposmolarity.

Extracellular zinc can induce numerous acute and persistent physiological and toxic effects in neurons by acting at their plasma membrane or intracellularly following permeation or uptake into them. Zinc acutely and reversibly blocks T-type voltage-gated calcium current (I(CaT)), but the long-term effect of zinc on this current has not been studied. Because chemically induced status epilepticus (SE) results in the release of zinc into the extracellular space, as well as in a long-lasting increase in I(CaT) in CA1 pyramidal cells, we hypothesized that zinc may play a causative role in I(CaT) upregulation. We tested this hypothesis by monitoring for 18 days the effects of zinc and ibotenic acid (a neurotoxic agent serving as control for zinc), injected into the right lateral ventricle, on I(CaT) in rat CA1 pyramidal cells. Both zinc and ibotenic acid caused marked hippocampal lesions on the side of injection, but only minor damage to contralateral hippocampi. Zinc, but not ibotenic acid, caused upregulation of a nickel-sensitive I(CaT) in a subset of contralateral CA1 pyramidal cells, appearing 2 days after injection and lasting for about 2 weeks thereafter. In contrast, acute application of zinc to CA1 pyramidal cells promptly blocked I(CaT). These data indicate that extracellular zinc has a dual effect on I(CaT), blocking it acutely while causing its long-term upregulation. Through the latter effect, zinc may regulate the intrinsic excitability of principal neurons, particularly in pathological conditions associated with enhanced release of zinc, such as SE.

The pore-forming Ca(2+) channel subunit Ca(V)3.2 mediates a low voltage-activated (T-type) Ca(2+) current (I(CaT)) that contributes pivotally to neuronal and cardiac pacemaker activity. Despite the importance of tightly regulated Ca(V)3.2 levels, the mechanisms regulating its transcriptional dynamics are not well understood. Here, we have identified two key factors that up- and down-regulate the expression of the gene encoding Ca(V)3.2 (Cacna1h). First, we determined the promoter region and observed several stimulatory and inhibitory clusters. Furthermore, we found binding sites for the transcription factor early growth response 1 (Egr1/Zif268/Krox-24) to be highly overrepresented within the Ca(V)3.2 promoter region. mRNA expression analyses and dual-luciferase promoter assays revealed that the Ca(V)3.2 promoter was strongly activated by Egr1 overexpression in vitro and in vivo. Subsequent chromatin immunoprecipitation assays in NG108-15 cells and mouse hippocampi confirmed specific Egr1 binding to the Ca(V)3.2 promoter. Congruently, whole-cell I(CaT) values were significantly larger after Egr1 overexpression. Intriguingly, Egr1-induced activation of the Ca(V)3.2 promoter was effectively counteracted by the repressor element 1-silencing transcription factor (REST). Thus, Egr1 and REST can bi-directionally regulate Ca(V)3.2 promoter activity and mRNA expression and, hence, the size of I(CaT). This mechanism has critical implications for the regulation of neuronal and cardiac Ca(2+) homeostasis under physiological conditions and in episodic disorders such as arrhythmias and epilepsy.

Modest decreases in extracellular osmolarity induce brain hyperexcitability that may culminate in epileptic seizures. At the cellular level, moderate hyposmolarity markedly potentiates the intrinsic neuronal excitability of principal cortical neurons without significantly affecting their volume. The most conspicuous cellular effect of hyposmolarity is converting regular firing neurons to burst-firing mode. This effect is underlain by hyposmotic facilitation of the spike afterdepolarization (ADP), but its ionic mechanism is unknown. Because blockers of KV7 (KCNQ) channels underlying neuronal M-type K+currents (KV7/M channels) also cause spike ADP facilitation and bursting, we hypothesized that lowering osmolarity inhibits these channels. Using current- and voltage-clamp recordings in CA1 pyramidal cellsin situ, we have confirmed this hypothesis. Furthermore, we show that hyposmotic inhibition of KV7/M channels is mediated by an increase in intracellular Ca2+concentration via release from internal stores but not via influx of extracellular Ca2+. Finally, we show that interfering with internal Ca2+-mediated inhibition of KV7/M channels entirely protects against hyposmotic ADP facilitation and bursting, indicating the exclusivity of this novel mechanism in producing intrinsic neuronal hyperexcitability in hyposmotic conditions.

Early in development, network activity in the hippocampus is characterized by recurrent synchronous bursts, whose cellular correlates are giant depolarizing potentials (GDPs). The propensity for generating GDPs is attributed to GABAergic synaptic transmission being depolarizing and excitatory in neonatal neurons. However, developmental regulation of intrinsic conductances may also influence GDPs generation. A likely candidate is the non-inactivating, low-threshold, muscarinic-sensitive K+ current (M current; Im), which down-regulates intrinsic bursting activity in adult hippocampal pyramidal neurons. Western blot analysis of homogenates of the CA3 hippocampal region showed that expression of the Kv7.2 subunit, one of the constituents of neuronal M channels, is weak in neonatal neurons, and markedly increases after the first postnatal week. Likewise, the density of Im was very low in neonatal CA3 pyramidal cells and increased later on. Spontaneously occurring intrinsic bursts in neonatal neurons were longer and more robust, and recurred more regularly, than in juvenile neurons. The Im blocker linopirdine only mildly affected intrinsic bursting in neonatal neurons, but strongly facilitated and regularized it in juvenile neurons. We conclude that the low expression of Kv7/M channels and the depolarizing action of GABA early after birth enhance intrinsic bursting and neuronal synchronization leading to generation of GDPs within the hippocampal network.

The intrinsic properties of a neuron profoundly influence neuronal function and are governed by the ion channels found in different neuronal compartments. Here, Beck and Yaari discuss the mechanisms underlying the plasticity of these properties in various CNS disorders. The input–output relationship of neuronal networks depends both on their synaptic connectivity and on the intrinsic properties of their neuronal elements. In addition to altered synaptic properties, profound changes in intrinsic neuronal properties are observed in many CNS disorders. These changes reflect alterations in the functional properties of dendritic and somatic voltage- and Ca2+-gated ion channels. The molecular mechanisms underlying this intrinsic plasticity comprise the highly specific transcriptional or post-transcriptional regulation of ion-channel expression, trafficking and function. The studies reviewed here show that intrinsic plasticity, in conjunction with synaptic plasticity, can fundamentally alter the input–output properties of neuronal networks in CNS disorders.

In principal brain neurons, activation of Ca2+ channels during an action potential, or spike, causes Ca2+ entry into the cytosol within a millisecond. This in turn causes rapid activation of large conductance Ca2+-gated channels, which enhances repolarization and abbreviates the spike. Here we describe another remarkable consequence of spike Ca2+ entry: enhancement of the spike afterdepolarization. This action is also mediated by intracellular modulation of a particular class of K+ channels, namely by inhibition of KV7 (KCNQ) channels. These channels generate the subthreshold, non-inactivating M-type K+ current, whose activation curtails the spike afterdepolarization. Inhibition of KV7/M by spike Ca2+ entry allows the spike afterdepolarization to grow and can convert solitary spikes into high-frequency bursts of action potentials. Through this novel intracellular modulatory action, Ca2+ spike entry regulates the discharge mode and the signalling capacity of principal brain neurons.

A single episode of status epilepticus (se) induced in rodents by the convulsant pilocarpine, produces, after a latent period of ≥ 2 weeks, a chronic epileptic condition. During the latent period of epileptogenesis, most CA1 pyramidal cells that normally fire in a regular pattern, acquire low-threshold bursting behaviour, generating high-frequency clusters of 3–5 spikes as their minimal response to depolarizing stimuli. Recruitment of a Ni2+- and amiloride-sensitive T-type Ca2+ current (ICaT), shown to be up-regulated after se, plays a critical role in burst generation in most cases. Several lines of evidence suggest that ICaT driving bursting is located in the apical dendrites. Thus, bursting was suppressed by focally applying Ni2+ to the apical dendrites, but not to the soma. It was also suppressed by applying either tetrodotoxin or the KV7/M-type K+ channel agonist retigabine to the apical dendrites. Severing the distal apical dendrites ∼150 μm from the pyramidal layer also abolished this activity. Intradendritic recordings indicated that evoked bursts are associated with local Ni2+-sensitive slow spikes. Blocking persistent Na+ current did not modify bursting in most cases. We conclude that se-induced increase in ICaT density in the apical dendrites facilitates their depolarization by the backpropagating somatic spike. The ICaT-driven dendritic depolarization, in turn, spreads towards the soma, initiating another backpropagating spike, and so forth, thereby creating a spike burst. The early appearance and predominance of ICaT-driven low-threshold bursting in CA1 pyramidal cells that experienced se most probably contribute to the emergence of abnormal network discharges and may also play a role in the circuitry reorganization associated with epileptogenesis.

The M-type K+ current (M-current), encoded by Kv7.2/3 (KCNQ2/3) K+ channels, plays a critical role in regulating neuronal excitability because it counteracts subthreshold depolarizations. Here we have characterized the functions of pre- and postsynaptic M-channels using a novel Kv7.2/3 channel opener, NH6, which we synthesized as a new derivative of N-phenylanthranilic acid. NH6 exhibits a good selectivity as it does not affect Kv7.1 and IKS K+ currents as well as NR1/NR2B, AMPA, and GABAA receptor-mediated currents. Superfusion of NH6 increased recombinant Kv7.2/3 current amplitude (EC50 = 18 μM) by causing a hyperpolarizing shift of the voltage activation curve and by markedly slowing the deactivation kinetics. Activation of native M-currents by NH6 robustly reduced the number of evoked and spontaneous action potentials in cultured cortical, hippocampal and dorsal root ganglion neurons. In hippocampal slices, NH6 decreased somatically evoked spike afterdepolarization of CA1 pyramidal neurons and induced regular firing in bursting neurons. Activation of M-channels by NH6, potently reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents. Activation of M-channels also decreased the frequency of miniature excitatory (mEPSC) and inhibitory (mIPSC) postsynaptic currents without affecting their amplitude and waveform, thus suggesting that M-channels presynaptically inhibit glutamate and GABA release. Our results suggest a role of presynaptic M-channels in the release of glutamate and GABA. They also indicate that M-channels act pre- and postsynaptically to dampen neuronal excitability.

Temporal lobe epilepsy (TLE) is the most common focal seizure disorder in adults. In many patients, transient brain insults, including status epilepticus (SE), are followed by a latent period of epileptogenesis, preceding the emergence of clinical seizures. In experimental animals, transcriptional upregulation of CaV3.2 T-type Ca2+-channels, resulting in an increased propensity for burst discharges of hippocampal neurons, is an important trigger for epileptogenesis. Here we provide evidence that the metal-regulatory transcription factor 1 (MTF1) mediates the increase of CaV3.2 mRNA and intrinsic excitability consequent to a rise in intracellular Zn2+ that is associated with SE. Adeno-associated viral (rAAV) transfer of MTF1 into murine hippocampi leads to increased CaV3.2 mRNA. Conversely, rAAV-mediated expression of a dominant-negative MTF1 abolishes SE-induced CaV3.2 mRNA upregulation and attenuates epileptogenesis. Finally, data from resected human hippocampi surgically treated for pharmacoresistant TLE support the Zn2+-MTF1-CaV3.2 cascade, thus providing new vistas for preventing and treating TLE., Temporal lobe epilepsy can cause ionic imbalance in the brain and alter transcriptional activities. Here, van Loo et al. show that the increase in neuronal zinc following status epilepticus can induce transcriptional change via metal-regulatory transcription factor 1, and alter voltage-gated calcium channel CaV3.2 and intrinsic neuronal excitability.

Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na+current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendriticINaPis due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendriticINaPcauses augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy.SIGNIFICANCE STATEMENTIn this paper, we describe a novel mechanism that causes increased dendritic persistent Na+current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na+currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.

Kv7/KCNQ/M channel subunits are widely expressed in peripheral and central neurons, where they give rise to a muscarinic-sensitive, subthreshold, and noninactivating K+ current (M current). Immunohistochemical data suggest that Kv7/M channels are expressed in both axons, somata and dendrites, but their distinctive roles in these compartments are not known. Here we used intracellular microelectrode recordings to monitor the effects of selective Kv7/M channel modulators focally applied to the axo-somatic region and to the apical dendrites of adult rat CA1 pyramidal cells. We show that both compartments express functional Kv7/M channels that synergistically control intrinsic neuronal excitability, albeit in different ways. Axo-somatic Kv7/M channels activate during the spike afterdepolarization (ADP) and counteract the depolarizing drive furnished by conjointly activated persistent Na+ channels. Thereby they limit the size and duration of the spike ADP and prevent its escalation into a somatic spike burst. Apical dendritic Kv7/M channels do not ordinarily regulate the somatic spike ADP and spike output. In hyperexcitable conditions that promote Ca2+ electrogenesis in these dendrites, they elevate the threshold for initiating Ca2+ spikes and associated downstream spike bursts. Thus the concerted activity of Kv7/M channels in both compartments serves to reduce the propensity to generate self-sustained burst responses and fosters a regular, stimulus-graded spike output of the neuron. Given that the activity of Kv7/M channels is regulated by multiple neurotransmitters, they may provide a substrate for neuromodulation of neuronal input/output relations at both the axo-somatic and apical dendritic regions.

In many principal brain neurons, the fast, all-or-none Na+spike initiated at the proximal axon is followed by a slow, graded afterdepolarization (ADP). The spike ADP is critically important in determining the firing mode of many neurons; large ADPs cause neurons to fire bursts of spikes rather than solitary spikes. Nonetheless, not much is known about how and where spike ADPs are initiated. We addressed these questions in adult CA1 pyramidal cells, which manifest conspicuous somatic spike ADPs and an associated propensity for bursting, using sharp and patch microelectrode recordings in acutely isolated hippocampal slices and single neurons. Voltage-clamp commands mimicking spike waveforms evoked transient Na+spike currents that declined quickly after the spike but were followed by substantial sustained Na+spike aftercurrents. Drugs that blocked the persistent Na+current (INaP), markedly suppressed the sustained Na+spike aftercurrents, as well as spike ADPs and associated bursting. Ca2+spike aftercurrents were much smaller, and reducing them had no noticeable effect on the spike ADPs. Truncating the apical dendrites affected neither spike ADPs nor the firing modes of these neurons. Application ofINaPblockers to truncated neurons, or their focal application to the somatic region of intact neurons, suppressed spike ADPs and associated bursting, whereas their focal application to distal dendrites did not. We conclude that the somatic spike ADPs are generated predominantly by persistent Na+channels located at or near the soma. Through this action, proximalINaPcritically determines the firing mode and spike output of adult CA1 pyramidal cells.

During postnatal development neurones display discharge behaviours that are not present in the adult, yet they are essential for the normal maturation of the nervous system. Neonatal CA1 pyramidal cells, like their adult counterparts, fire regularly, but excitatory GABAergic transmission drives them to generate spontaneous high-frequency bursts until postnatal day (P) 15. Using intracellular recordings in hippocampal slices from rats at P8 to P25, we show herein that as the network-driven burst activity fades out, most CA1 pyramidal cells become intrinsically bursting neurones. The incidence of intrinsic bursters begins to rise at P11 and attains a peak of 74% by P18–P19, after which it decreases over the course of a week, disappearing almost entirely at P25. Analysis of the effects of different voltage-gated Ca2+ and Na+ channel antagonists, applied focally to proximal and distal parts of developing neurones, revealed a complex burst mechanism. Intrinsic bursting in developing neurones results from ‘ping-pong’ interplay between a back-propagating spike that activates T/R- and L-type voltage-gated Ca2+ channels in the distal apical dendrites and persistent voltage-gated Na+ channels in the somatic region. Thus, developing pyramidal neurones transitionally express not only distinctive synaptic properties, but also unique intrinsic firing patterns, that may contribute to the ongoing formation and refinement of synaptic connections.

A single episode of status epilepticus (SE) induces neuropathological changes in the brain that may lead to the development of a permanent epileptic condition. Most studies of this plasticity have focused on the hippocampus, where both synaptic function and intrinsic neuronal excitability have been shown to be persistently modified by SE. However, many other brain structures are activated during SE and may also be involved in the subsequent epileptogenic process. Here we have investigated whether SE, induced in rats with pilocarpine and terminated after 40 min with diazepam, persistently modifies the intrinsic excitability of pyramidal neurons in the subiculum. Subicular slices were prepared from control and SE-experienced rats (2‐5 weeks after SE). In the control group, only 4% of the neurons fired bursts in response to intrasomatic, threshold-straddling depolarizing current pulses (low-threshold bursters). The remaining neurons either fired bursts in response to strong (33 threshold) depolarizations (35%; high-threshold bursters) or fired in a completely regular mode (61%; nonbursters). In the SE-experienced group, the fractions of low- and high-threshold bursters markedly increased to 29% and 53%, respectively. This change in firing behaviour was associated with a marked increase in the size of the spike afterdepolarization, particularly in low-threshold bursters. Experimental suppression of Ca 2+ currents selectively blocked low-threshold bursting but did not affect high-threshold bursting, suggesting that a dual Ca 2+ - dependent and Ca 2+ - independent mechanism controls bursting in these neurons. The persistent upregulation of intrinsic bursting in the subiculum, in concert with similar changes in the hippocampus, undoubtedly contributes to epileptogenesis following pilocarpine-induced SE.

Poisoning with organophosphates (OPs) may induce status epilepticus (SE), leading to severe brain damage. Our objectives were to investigate whether OP-induced SE leads to the emergence of spontaneous recurrent seizures (SRSs), the hallmark of chronic epilepsy, and if so, to assess the efficacy of benzodiazepine therapy following SE onset in preventing the epileptogenesis. We also explored early changes in hippocampal pyramidal cells excitability in this model. Adult rats were poisoned with the paraoxon (450μg/kg) and immediately treated with atropine (3mg/kg) and obidoxime (20mg/kg) to reduce acute mortality due to peripheral acetylcholinesterase inhibition. Electrical brain activity was assessed for two weeks during weeks 4-6 after poisoning using telemetric electrocorticographic intracranial recordings. All OP-poisoned animals developed SE, which could be suppressed by midazolam. Most (88%) rats which were not treated with midazolam developed SRSs, indicating that they have become chronically epileptic. Application of midazolam 1min following SE onset had a significant antiepileptogenic effect (only 11% of the rats became epileptic; p=0.001 compared to non-midazolam-treated rats). Applying midazolam 30min after SE onset did not significantly prevent chronic epilepsy. The electrophysiological properties of CA1 pyramidal cells, assessed electrophysiologically in hippocampal slices, were not altered by OP-induced SE. Thus we show for the first time that a single episode of OP-induced SE in rats leads to the acquisition of chronic epilepsy, and that this epileptogenic outcome can be largely prevented by immediate, but not delayed, administration of midazolam. Extrapolating these results to humans would suggest that midazolam should be provided together with atropine and an oxime in the immediate pharmacological treatment of OP poisoning.

A simple system for cutting dendrites of single neurons without damaging the viability of the cell is described. The system utilizes a rapidly vibrating (100 Hz) micropipette (

1. Whole-cell patch-clamp recordings of iontophoretically induced N-methyl-D-aspartate (NMDA) receptor-mediated currents (INMDA) in CA1 pyramidal cells in hippocampal slices from 1- to 40-day-old rats were used to characterize developmental changes in the Mg2+ sensitivity of NMDA receptors. 2. The dose-response relations for extracellular Mg2+ blockade of INMDA indicated a high affinity binding of Mg2+ to NMDA receptors at membrane potentials more negative than -60 mV, independent of postnatal age. 3. Depolarizing the cells unblocked NMDA receptors by decreasing their affinity for Mg2+. The efficacy of depolarization in unblocking NMDA receptors markedly increased after postnatal day 4 (P4), endowing the receptors with a greater voltage dependence. 4. The NR2B subunit-specific NMDA antagonist ifenprodil reduced INMDA in pyramidal cells of all ages. The sensitivity of INMDA to ifenprodil was greatest during the first postnatal week and decreased thereafter, indicating an enhanced contribution of NR2B subunit-containing NMDA receptors to INMDA in the first week after birth. 5. In the first postnatal week, the ifenprodil-insensitive INMDA component had a lower voltage dependence than the total INMDA. In older pyramidal cells, the voltage dependence of the ifenprodil-insensitive component and the total INMDA were similar. 6. In another set of CA1 pyramidal cells, single-cell reverse transcription and polymerase chain reaction (RT-PCR) were used to characterize concomitant developmental changes in NMDA subunit mRNA expression. The mRNA for the NR2D subunit was detected during the first postnatal week in 50 % of the cells and disappeared thereafter. The proportion of cells expressing the NR2A and NR2B subunits remained relatively constant throughout the first five postnatal weeks. 7. We conclude that NMDA receptors in hippocampal CA1 pyramidal cells are effectively blocked by Mg2+ at all ages. After 4 days they become much less sensitive to Mg2+ at depolarized membrane potentials. This postnatal switch in voltage control of Mg2+ binding to NMDA receptors may be due to the downregulation of NR2D subunit expression in developing CA1 pyramidal cells.

Acetylcholine released from basal forebrain cholinergic fibres suppresses intrinsic bursting in cortical pyramidal cells through activation of muscarinic receptors. The signal transduction pathway mediating this action is not known. We used intracellular recordings from CA1 pyramidal cells in hippocampal slices to investigate the involvement of protein kinase C (PKC) in this cholinergic function. Bath-applied carbachol (CCh; 5 μM) consistently suppressed intrinsic bursting in an atropine-sensitive (1 μM) manner. Intrinsic bursting was suppressed by 4β-phorbol 12,13-dibutyrate (PDBu; 5-10 μM), a potent PKC activator, but not by the inactive phorbol ester 4α-phorbol 12,13-didecanoate (PDC; 50 μM). Prior application of the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7; 10 μM) extracellularly or intracellularly prevented the PDBu effect. Pretreatment with H7, but not with the broad-spectrum kinase inhibitor N-(2-guanidino-ethyl)-5-isoquinoline-sulfonyl hydrochloride (HA1004; 10 μM), prevented the CCh-induced suppression of bursting. The active component of the spike after-depolarization (ADP) was reduced by CCh in an atropine-sensitive manner. This effect was mimicked by PDBu, but not by PDC. It was prevented by pretreatment with H7, but not with HA1004. Blocking most K+ currents with Ca2+-free, TEA-containing saline induced large TTX-sensitive plateau potentials lasting > 150 ms, driven by a persistent Na+ current. These potentials were suppressed by PDBu, but not by PDC. Pretreatment with H7 prevented the PDBu-induced suppression of the plateau potentials. We conclude that cholinergic suppression of intrinsic bursting in hippocampal CA1 pyramidal cells is mediated by muscarinic activation of PKC, which down-regulates the persistent Na+ current underlying slow depolarizing potentials in these neurons. Neurons in the mammalian brain vary with respect to their intrinsic firing patterns. In cortical (Agmon & Connors, 1989; Chagnac Amitai et al. 1990; Jensen et al. 1994) and subcortical structures (McCormick & Prince, 1988; McCormick & Feeser, 1990; Silva et al. 1991), a subgroup of neurons exhibits intrinsic bursting. An intrinsic burst in these neurons consists of three to seven closely spaced action potentials, capping a distinct slow depolarizing envelope. The spontaneous generation of intrinsic bursts is a probable driving force for physiological and pathophysiological brain rhythms (Wong & Prince, 1979; Jensen & Yaari, 1997). We have previously shown that muscarinic receptor activation by endogenously released acetylcholine or exogenously applied carbachol (CCh) converts intrinsically bursting hippocampal CA1 pyramidal cells into regularly firing (i.e. non-bursting) neurons (Azouz et al. 1994). A similar muscarinic suppression of intrinsic bursting was seen in neocortical pyramidal neurons (Metherate et al. 1992). This effect was independent of an associated muscarinic depolarization, suggesting that muscarinic receptors directly modulate the membrane currents that generate the burst (Azouz et al. 1994). However, the nature of the signal transduction pathway that mediates this muscarinic effect is not known. Central muscarinic receptors are a heterogeneous group of metabotropic receptors that interact with various membrane-associated guanine nucleotide-binding proteins (G-proteins). These, in turn, are negatively coupled to adenylyl cyclase or positively coupled to guanylyl cyclase or to phospholipase C (for review see Brown et al. 1997). Activation of the m1, m3 and m5 muscarinic receptor subtypes induces the breakdown of inositol phospholipids, which leads to the production of inositol trisphosphate and diacylglycerol. Diacylglycerol, in turn, activates protein kinase C (PKC; Nishizuka, 1984b). In the hippocampus, CCh acting on muscarinic receptors has been shown to elicit the breakdown of inositol phospholipids (Downes, 1982), suggesting that this pathway may mediate muscarinic action in this region. Indeed, several muscarinic effects in CA1 pyramidal cells were shown to involve PKC activation, including block of the slow after-hyperpolarization (slow AHP) and the associated spike frequency accommodation (Malenka et al. 1986), enhancement of NMDA receptor-mediated response (e.g. Segal, 1992), and induction of cholinergic long-term potentiation (Auerbach & Segal, 1996). In hippocampal CA1 (Azouz et al. 1996) and neocortical pyramidal cells (Franceschetti et al. 1995) alike, intrinsic bursting is generated by a persistent Na+ current (INa,P; French et al. 1990; Alzheimer et al. 1993). It was recently shown that muscarinic activation of PKC reduces INa,P (Cantrell et al. 1996; Mittmann & Alzheimer, 1998). This mechanism may constitute the link between muscarinic receptor activation and block of intrinsic bursting. We tested this hypothesis in rat hippocampal slices using pharmacological manipulations of PKC activity. Our results suggest that muscarinic receptor activation blocks intrinsic bursting by a PKC-dependent action, most probably by suppression of INa,P. A preliminary report of these findings has appeared in a recent abstract (Alroy & Yaari, 1997).

Whole-cell patch-clamp recordings in adult mouse hippocampal slices were used to test the mechanism by which the volatile anesthetic halothane inhibits glutamate receptor-mediated synaptic transmission. Non-N-methyl-D-aspartate (nonNMDA) and NMDA receptor-mediated currents in CA1 pyramidal cells were pharmacologically isolated by bath application of D,L-2-amino-5-phosphonovaleric acid (APV; 100 μM) or 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 5 μM), respectively. Halothane blocked both nonNMDA and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) to a similar extent (IC50 values of 0.66 and 0.57 mM, respectively). Partial blockade of the EPSCs by lowering the extracellular concentration of calcium ([Ca2+]o), but not by application of CNQX (1 μM), was accompanied by an increase in paired-pulse facilitation (PPF). Halothane-induced blockade of the EPSCs also was associated with an increase in PPF. The effects of halothane on α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA receptor-mediated currents induced by agonist iontophoresis, were compared. AMPA-induced currents were blocked with an IC50 of 1.7 mM. NMDA-induced currents were significantly less sensitive to halothane (IC50 of 5.9 mM). The effect of halothane on iontophoretic AMPA dose-response curves was tested. Halothane suppressed the maximal response to AMPA without affecting its EC50, suggesting a noncompetitive mechanism of inhibition. All effects of halothane were reversible upon termination of the exposure to the drug. These data suggest that halothane blocks central glutamatergic synaptic transmission by presynaptically inhibiting glutamate release and postsynaptically blocking the AMPA subtype of glutamate receptors. British Journal of Pharmacology (1998) 124, 1607–1614; doi:10.1038/sj.bjp.0701996

1. The properties of ryanodine-sensitive Ca2+ stores in CA1 pyramidal cells were investigated in rat hippocampal slices by using whole-cell patch-clamp recordings combined with fura-2-based fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2. Brief pressure applications of caffeine onto the somata of pyramidal cells caused large transient increases in [Ca2+]i (Ca2+ transients) of 50-600 nM above baseline. 3. The Ca2+ transients evoked by caffeine at -60 mV were not associated with an inward current, persisted after blocking voltage-activated Ca2+ currents and were completely blocked by bath-applied ryanodine. Similar transients were also evoked at +60 mV. Thus, these transients reflect Ca2+ release from intracellular ryanodine-sensitive Ca2+ stores. 4. The Ca2+ transients evoked by closely spaced caffeine pulses rapidly decreased in amplitude, indicating progressive depletion of the Ca2+ stores. The amplitude of the Ca2+ transients recovered spontaneously with an exponential time constant of 59 s. Recovery was accelerated by depolarization-induced elevations in [Ca2+]i and blocked by cyclopiazonic acid (CPA) and thapsigargin, indicating that store refilling is mediated by endoplasmic reticulum Ca(2+)-ATPases. 5. Even without prior store depletion the caffeine-induced Ca2+ transients disappeared after 6 min exposure to CPA, suggesting that ryanodine-sensitive Ca2+ stores are maintained at rest by continuous Ca2+ sequestration. 6. Caffeine-depleted Ca2+ stores did not refill in Ca(2+)-free saline, suggesting that the refilling of the stores depends upon Ca2+ influx through a 'capacitative-like' transmembrane influx pathway operating at resting membrane potential. The refilling of the stores was also blocked by Ni2+ and gallopamil (D600). 7. Elevations of basal [Ca2+]i produced by bath-applied KCl markedly potentiated (up to 6-fold) the caffeine-induced Ca2+ transients. The degree of potentiation was positively related to the increase in basal [Ca2+]i. The Ca2+ transients remained potentiated up to 9 min after reversing the KCl-induced [Ca2+]i increase. Thus, the ryanodine-sensitive Ca2+ stores can 'overcharge' when challenged with an increase in [Ca2+]i and slowly discharge excess Ca2+ after basal [Ca2+]i returns to its resting level. 8. Pressure applications of caffeine onto pyramidal cell dendrites evoked local Ca2+ transients similar to those separately evoked in the respective somata. Thus, dendritic ryanodine-sensitive Ca2+ stores are also loaded at rest and can function as independent compartments. 9. In conclusion, the ryanodine-sensitive Ca2+ stores in hippocampal pyramidal neurones contain a releasable pool of Ca2+ that is maintained by a Ca2+ entry pathway active at subthreshold membrane potentials. Ca2+ entry through voltage-gated Ca2+ channels transiently overcharges the stores. Thus, by acting as powerful buffers at rest and as regulated sources during activity, Ca2+ stores may control the waveform of physiological Ca2+ signals in CA1 hippocampal pyramidal neurones.

Background The effects of halothane on excitatory synaptic transmission in the central nervous system of mammals have been studied in vivo and in vitro in several investigations with partially contradicting results. Direct measurements of the effects of halothane on isolated glutamate receptor-mediated (glutamatergic) excitatory postsynaptic currents (EPSCs), however, have not been reported to date. Methods The effects of halothane on glutamatergic EPSCs were studied in vitro by using tight-seal, whole-cell recordings from CA1 pyramidal cells in thin slices from the adult mouse hippocampus. The EPSCs were pharmacologically isolated into their non-N-methyl-D-aspartate (non-NMDA) and NMDA receptor-mediated components by using selective antagonists. The effects of halothane on EPSC amplitude and kinetics were analyzed at various membrane potentials and were compared with its effects on currents evoked by exogenously applied glutamatergic agonists. Results Halothane (0.2-5.1%; 0.37-2.78 mM) reversibly blocked non-NMDA and NMDA EPSCs. This effect was voltage independent; concentrations producing 50% inhibition were 0.87% (0.66 mM) and 0.69% (0.57 mM), respectively. Currents induced by bath-applied glutamatergic agonists were not affected even by the high concentrations of halothane. Conclusions Halothane depresses glutamatergic EPSCs irrespective of receptor subtype, most likely by inhibition of glutamate release.

Extracellular zinc can induce numerous acute and persistent physiological and toxic effects in neurons by acting at their plasma membrane or intracellularly following permeation or uptake into them. Zinc acutely and reversibly blocks T-type voltage-gated calcium current (I(CaT)), but the long-term effect of zinc on this current has not been studied. Because chemically induced status epilepticus (SE) results in the release of zinc into the extracellular space, as well as in a long-lasting increase in I(CaT) in CA1 pyramidal cells, we hypothesized that zinc may play a causative role in I(CaT) upregulation. We tested this hypothesis by monitoring for 18 days the effects of zinc and ibotenic acid (a neurotoxic agent serving as control for zinc), injected into the right lateral ventricle, on I(CaT) in rat CA1 pyramidal cells. Both zinc and ibotenic acid caused marked hippocampal lesions on the side of injection, but only minor damage to contralateral hippocampi. Zinc, but not ibotenic acid, caused upregulation of a nickel-sensitive I(CaT) in a subset of contralateral CA1 pyramidal cells, appearing 2 days after injection and lasting for about 2 weeks thereafter. In contrast, acute application of zinc to CA1 pyramidal cells promptly blocked I(CaT). These data indicate that extracellular zinc has a dual effect on I(CaT), blocking it acutely while causing its long-term upregulation. Through the latter effect, zinc may regulate the intrinsic excitability of principal neurons, particularly in pathological conditions associated with enhanced release of zinc, such as SE.

α-synuclein (α-Syn) is a neuronal protein that accumulates progressively in Parkinson’s disease and related synucleinopathies. Attempting to identify cellular factors that affect α-Syn neuropathology, we previously reported that polyunsaturated fatty acids (PUFAs) promote α-Syn oligomerization and aggregation in cultured cells. We now report that docosahexaenoic acid (DHA) a 22:6 PUFA affects α-Syn oligomerization by activating retinoic X receptor (RXR) and peroxisome proliferator-activated receptor γ2 (PPARγ2). In addition, we show that dietary changes in brain DHA levels affect α-Syn cytopathology in mice transgenic for the Parkinson’s disease-causing A53T mutation in human α-Syn. A diet enriched in docosahexaenoic acid, an activating ligand of RXR, increased the accumulation of soluble and insoluble neuronal α-Syn, neuritic injury and astrocytosis. Conversely, abnormal accumulations of α-Syn and its deleterious effects were significantly attenuated by low dietary docosahexaenoic acid levels. Our results suggest a role for activated RXR/PPARγ 2, obtained by elevated brain polyunsaturated fatty acids levels, in α-Syn neuropathology.

Dendrites of pyramidal neurons integrate multiple synaptic inputs and transform them into axonal action potential output. This fundamental process is controlled by a variety of dendritic channels. The properties of dendritic ion channels are not static but can be modified by neuronal activity. Activity-dependent changes in the density, localization, or biophysical properties of dendritic voltage-gated channels can persistently alter the integration of synaptic inputs. Furthermore, dendritic intrinsic plasticity can induce neuronal output mode transitions (e.g. from regular spiking to burst firing). Recent advances in the field reviewed here represent an important step toward uncovering the principles of neuronal input/output transformations in response to various patterns of brain activity.

CA1 pyramidal cells (PCs) provide a major output of the hippocampus proper. They integrate information arriving directly from the entorhinal cortex via the temporoammonic pathway, and indirectly via the polysynaptic dentate gyrus–CA3–CA1 loop. Both pathways terminate in a spatially segregated manner onto CA1 PC apical dendrites. The temporoammonic pathway targets the distal dendrites in the stratum lacunosum moleculare, while CA3 PC axons terminate within the stratum radiatum. These inputs are integrated in the somatodendritic compartment and converted to an output consisting of action potentials propagating along the CA1 axons, a process controlled by the intrinsic membrane properties of CA1 neurons. We will first consider the mechanisms determining input–output properties of normal CA1 PCs. We will then address how the properties of CA1 PCs change in acute and chronic epilepsy models, and how they might contribute to increased excitability.

In both humans and animals, an insult to the brain can lead, after a variable latent period, to the appearance of spontaneous epileptic seizures that persist for life. The underlying processes, collectively referred to as epileptogenesis, include multiple structural and functional neuronal alterations. We have identified the T-type Ca2+channel Cav3.2 as a central player in epileptogenesis. We show that a transient and selective upregulation of Cav3.2 subunits on the mRNA and protein levels after status epilepticus causes an increase in cellular T-type Ca2+currents and a transitional increase in intrinsic burst firing. These functional changes are absent in mice lacking Cav3.2 subunits. Intriguingly, the development of neuropathological hallmarks of chronic epilepsy, such as subfield-specific neuron loss in the hippocampal formation and mossy fiber sprouting, was virtually completely absent in Cav3.2−/−mice. In addition, the appearance of spontaneous seizures was dramatically reduced in these mice. Together, these data establish transcriptional induction of Cav3.2 as a critical step in epileptogenesis and neuronal vulnerability.

Early in development, network activity in the hippocampus is characterized by recurrent synchronous bursts, whose cellular correlates are giant depolarizing potentials (GDPs). The propensity for generating GDPs is attributed to GABAergic synaptic transmission being depolarizing and excitatory in neonatal neurons. However, developmental regulation of intrinsic conductances may also influence GDPs generation. A likely candidate is the non-inactivating, low-threshold, muscarinic-sensitive K(+) current (M current; I(m)), which down-regulates intrinsic bursting activity in adult hippocampal pyramidal neurons. Western blot analysis of homogenates of the CA3 hippocampal region showed that expression of the Kv7.2 subunit, one of the constituents of neuronal M channels, is weak in neonatal neurons, and markedly increases after the first postnatal week. Likewise, the density of I(m) was very low in neonatal CA3 pyramidal cells and increased later on. Spontaneously occurring intrinsic bursts in neonatal neurons were longer and more robust, and recurred more regularly, than in juvenile neurons. The I(m) blocker linopirdine only mildly affected intrinsic bursting in neonatal neurons, but strongly facilitated and regularized it in juvenile neurons. We conclude that the low expression of Kv7/M channels and the depolarizing action of GABA early after birth enhance intrinsic bursting and neuronal synchronization leading to generation of GDPs within the hippocampal network.

In principal brain neurons, activation of Ca(2+) channels during an action potential, or spike, causes Ca(2+) entry into the cytosol within a millisecond. This in turn causes rapid activation of large conductance Ca(2+)-gated channels, which enhances repolarization and abbreviates the spike. Here we describe another remarkable consequence of spike Ca(2+) entry: enhancement of the spike afterdepolarization. This action is also mediated by intracellular modulation of a particular class of K(+) channels, namely by inhibition of K(V)7 (KCNQ) channels. These channels generate the subthreshold, non-inactivating M-type K(+) current, whose activation curtails the spike afterdepolarization. Inhibition of K(V)7/M by spike Ca(2+) entry allows the spike afterdepolarization to grow and can convert solitary spikes into high-frequency bursts of action potentials. Through this novel intracellular modulatory action, Ca(2+) spike entry regulates the discharge mode and the signalling capacity of principal brain neurons.

A single episode of status epilepticus (SE) induced in rodents by the convulsant pilocarpine, produces, after a latent period ofor = 2 weeks, a chronic epileptic condition. During the latent period of epileptogenesis, most CA1 pyramidal cells that normally fire in a regular pattern, acquire low-threshold bursting behaviour, generating high-frequency clusters of 3-5 spikes as their minimal response to depolarizing stimuli. Recruitment of a Ni(2+)- and amiloride-sensitive T-type Ca(2+) current (I(CaT)), shown to be up-regulated after SE, plays a critical role in burst generation in most cases. Several lines of evidence suggest that I(CaT) driving bursting is located in the apical dendrites. Thus, bursting was suppressed by focally applying Ni(2+) to the apical dendrites, but not to the soma. It was also suppressed by applying either tetrodotoxin or the K(V)7/M-type K(+) channel agonist retigabine to the apical dendrites. Severing the distal apical dendrites approximately 150 microm from the pyramidal layer also abolished this activity. Intradendritic recordings indicated that evoked bursts are associated with local Ni(2+)-sensitive slow spikes. Blocking persistent Na(+) current did not modify bursting in most cases. We conclude that SE-induced increase in I(CaT) density in the apical dendrites facilitates their depolarization by the backpropagating somatic spike. The I(CaT)-driven dendritic depolarization, in turn, spreads towards the soma, initiating another backpropagating spike, and so forth, thereby creating a spike burst. The early appearance and predominance of I(CaT)-driven low-threshold bursting in CA1 pyramidal cells that experienced SE most probably contribute to the emergence of abnormal network discharges and may also play a role in the circuitry reorganization associated with epileptogenesis.

The intrinsic firing modes of adult CA1 pyramidal cells vary along a continuum of “burstiness” from regular firing to rhythmic bursting, depending on the ionic composition of the extracellular milieu. Burstiness is low in neurons exposed to a normal extracellular Ca2+ concentration ([Ca2+]o), but is markedly enhanced by lowering [Ca2+]o, although not by blocking Ca2+ and Ca2+-activated K+ currents. We show, using intracellular recordings, that burstiness in low [Ca2+]o persists even after truncating the apical dendrites, suggesting that bursts are generated by an interplay of membrane currents at or near the soma. To study the mechanisms of bursting, we have constructed a conductance-based, one-compartment model of CA1 pyramidal neurons. In this neuron model, reduced [Ca2+]o is simulated by negatively shifting the activation curve of the persistent Na+ current ( INaP) as indicated by recent experimental results. The neuron model accounts, with different parameter sets, for the diversity of firing patterns observed experimentally in both zero and normal [Ca2+]o. Increasing INaP in the neuron model induces bursting and increases the number of spikes within a burst but is neither necessary nor sufficient for bursting. We show, using fast-slow analysis and bifurcation theory, that the M-type K+ current ( IM) allows bursting by shifting neuronal behavior between a silent and a tonically active state provided the kinetics of the spike generating currents are sufficiently, although not extremely, fast. We suggest that bursting in CA1 pyramidal cells can be explained by a single compartment “square bursting” mechanism with one slow variable, the activation of IM.

KCNQ channel subunits are widely expressed in peripheral and central neurons, where they give rise to a muscarinic-sensitive, subthreshold, and noninactivating K+current (M-current). It is generally agreed that activation of KCNQ/M channels contributes to spike frequency adaptation during sustained depolarizations but is too slow to influence the repolarization of solitary spikes. This concept, however, is based mainly on experiments with muscarinic agonists, the multiple effects on membrane conductances of which may overshadow the distinctive effects of KCNQ/M channel block. Here, we have used selective modulators of KCNQ/M channels to investigate their role in spike electrogenesis in CA1 pyramidal cells. Solitary spikes were evoked by brief depolarizing current pulses injected into the neurons. The KCNQ/M channel blockers linopirdine and XE991 markedly enhanced the spike afterdepolarization (ADP) and, in most neurons, converted solitary (“simple”) spikes to high-frequency bursts of three to seven spikes (“complex” spikes). Conversely, the KCNQ/M channel opener retigabine reduced the spike ADP and induced regular firing in bursting neurons. Selective block of BK or SK channels had no effect on the spike ADP or firing mode in these neurons. We conclude that KCNQ/M channels activate during the spike ADP and limit its duration, thereby precluding its escalation to a burst. Consequently, down-modulation of KCNQ/M channels converts the neuronal firing pattern from simple to complex spiking, whereas up-modulation of these channels exerts the opposite effect.

A single episode of status epilepticus (SE) induces neuropathological changes in the brain that may lead to the development of a permanent epileptic condition. Most studies of this plasticity have focused on the hippocampus, where both synaptic function and intrinsic neuronal excitability have been shown to be persistently modified by SE. However, many other brain structures are activated during SE and may also be involved in the subsequent epileptogenic process. Here we have investigated whether SE, induced in rats with pilocarpine and terminated after 40 min with diazepam, persistently modifies the intrinsic excitability of pyramidal neurons in the subiculum. Subicular slices were prepared from control and SE-experienced rats (2-5 weeks after SE). In the control group, only 4% of the neurons fired bursts in response to intrasomatic, threshold-straddling depolarizing current pulses (low-threshold bursters). The remaining neurons either fired bursts in response to strong (3x threshold) depolarizations (35%; high-threshold bursters) or fired in a completely regular mode (61%; nonbursters). In the SE-experienced group, the fractions of low- and high-threshold bursters markedly increased to 29% and 53%, respectively. This change in firing behaviour was associated with a marked increase in the size of the spike after depolarization, particularly in low-threshold bursters. Experimental suppression of Ca2+ currents selectively blocked low-threshold bursting but did not affect high-threshold bursting, suggesting that a dual Ca2+- dependent and Ca2+- independent mechanism controls bursting in these neurons. The persistent up-regulation of intrinsic bursting in the subiculum, in concert with similar changes in the hippocampus, undoubtedly contributes to epileptogenesis following pilocarpine-induced SE.

The generation of high-frequency spike bursts (“complex spikes”), either spontaneously or in response to depolarizing stimuli applied to the soma, is a notable feature in intracellular recordings from hippocampal CA1 pyramidal cells (PCs)in vivo. There is compelling evidence that the bursts are intrinsically generated by summation of large spike afterdepolarizations (ADPs). Using intracellular recordings in adult rat hippocampal slices, we show that intrinsic burst-firing in CA1 PCs is strongly dependent on the extracellular concentration of Ca2+([Ca2+]o). Thus, lowering [Ca2+]o(by equimolar substitution with Mn2+or Mg2+) induced intrinsic bursting in nonbursters, whereas raising [Ca2+]osuppressed intrinsic bursting in native bursters. The induction of intrinsic bursting by low [Ca2+]owas associated with enlargement of the spike ADP. Low [Ca2+]o-induced intrinsic bursts and their underlying ADPs were suppressed by drugs that reduce the persistent Na+current (INaP), indicating that this current mediates the slow burst depolarization. Blocking Ca2+-activated K+currents with extracellular Ni2+or intracellular chelation of Ca2+did not induce intrinsic bursting. This and other evidence suggest that lowering [Ca2+]omay induce intrinsic bursting by augmentingINaP. Because repetitive neuronal activity in the hippocampus is associated with marked decreases in [Ca2+]o, the regulation of intrinsic bursting by extracellular Ca2+may provide a mechanism for preferential recruitment of this firing mode during certain forms of hippocampal activation.

Chronically epileptic rats, produced by prior injection of pilocarpine, were used to investigate whether changes in intrinsic neuronal excitability may contribute to the epileptogenicity of the hippocampus in experimental temporal lobe epilepsy (TLE). Paired extra-/intracellular electrophysiological recordings were made in the CA1 pyramidal layer in acute hippocampal slices prepared from control and epileptic rats and perfused with artificial cerebrospinal fluid (ACSF). Whereas orthodromic activation of CA1 neurons evoked only a single, stimulus-graded population spike in control slices, it produced an all-or-none burst of population spikes in epileptic slices. The intrinsic firing patterns of CA1 pyramidal cells were determined by intrasomatic positive current injection. In control slices, the vast majority (97%) of the neurons were regular firing cells. In epileptic slices, only 53% the pyramidal cells fired in a regular mode. The remaining 47% of the pyramidal cells were intrinsic bursters. These neurons generated high-frequency bursts of three to six spikes in response to threshold depolarizations. A subgroup of these neurons (10.1% of all cells) also burst fired spontaneously even after suppression of synaptic activity. In epileptic slices, burst firing in most cases (ca 70%) was completely blocked by adding the Ca2+ channel blocker Ni2+ (1 mM) to, or removing Ca2+ from, the ACSF, but not by intracellular application of the Ca2+ chelater 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N '-tetra-acetic acid (BAPTA), suggesting it was driven by a Ca2+ current. Spontaneously recurring population bursts were observed in a subset of epileptic slices. They were abolished by adding 2 M 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) to the ACSF, indicating that synaptic excitation is critical for the generation of these events. All sampled pyramidal cells fired repetitively during each population burst. The firing of spontaneously active bursters anteceded the population discharge, whereas most other pyramidal cells began to fire conjointly with the first population spike. Thus, spontaneous bursters are likely to be the initiators of spontaneous population bursts in epileptic slices. The dramatic up-regulation of intrinsic bursting in CA1 pyramidal cells, particularly the de novo appearance of Ca2+-dependent bursting, may contribute to the epileptogenicity of the hippocampus in the pilocarpine model of TLE. These findings have important implications for the pharmacological treatment of medically refractory human TLE.

In the hippocampus, fast excitatory synaptic transmission of principal projection neurons is mediated by non-NMDA glutamate receptors, whereas NMDA glutamate receptors serve a slower modulatory role. We used the whole-cell patch-clamp technique in adult hippocampal slices to assess the role of NMDA receptors in synaptic excitation of a recently discovered excitatory projection neuron, the CA1 radiatum giant cell (RGC). Glutamatergic synaptic activation, even after blocking non-NMDA receptors, fired an NMDA receptor-dependent burst of action potentials in RGCs. In contrast, the contribution of NMDA receptors to synaptic activation of pyramidal cells (PCs) was minimal. Stimulation of the same synaptic inputs evoked greater than threefold larger EPSCs in RGCs than in PCs. Isolated NMDA receptor-mediated EPSCs were significantly less sensitive to blockade by extracellular Mg2+and had slower decay kinetics in RGCs than in PCs. Thus, unique properties of synaptic NMDA receptors underlie enhanced synaptic excitability in a newly discovered excitatory hippocampal projection neuron.

(1) We have investigated the effect of halothane on glutamate receptor-mediated excitation in pyramidal cells and interneurons of the hippocampal CA1 area. (2) Halothane similarly inhibited non-NMDA and NMDA receptor-mediated excitatory postsynaptic currents in both cell types at low concentrations but preferentially blocked responses to exogenously applied AMPA at higher concentrations. (3) Synaptically but not directly evoked action potentials in interneurons were inhibited by halothane.

1. Intracellular recordings in adult rat hippocampal slices were used to investigate the modulation of endogenous neuronal firing patterns by moderate changes (+/-13%) in the extracellular osmotic pressure (pi o). The responses of CA1 pyramidal cells to graded depolarizing current pulses were used to differentiate between regular and burst-firing patterns and to characterize the stimulus requirements for evoking endogenous burst discharge. 2. Decreasing or increasing pi o had no significant effects on resting membrane potential and input resistance, spike threshold and amplitude, and the amplitudes of the fast, medium and slow spike after-hyperpolarizations (AHPs). The apparent membrane time constant (tau m) increased in low pi o and decreased in high pi o. 3. Reducing pi o converted non-bursting neurones (non-bursters) to bursting neurones (bursters) and decreased the stimulus requirements for evoking burst firing in native bursters. Increasing pi o suppressed endogenous burst firing. 4. Lowering pi o increased the size of the 'active' (i.e. re-depolarizing) component of the spike after-depolarization (ADP). Conversely, increasing pi o suppressed the active ADP component. 5. The sensitivity of spike ADPs and firing patterns of pyramidal cells to the changes in pi o persisted also in Ca(2+)-free saline, indicating that the osmotic effects are not imparted by modulation of Ca2+ and/or Ca(2+)-activated K+ currents. 6. Blocking most K+ currents with Ca(2+)-free, TEA-containing saline induced large and prolonged (up to 1 s), TTX-sensitive plateau potentials following the primary fast spikes. These potentials were augmented by low pi o and abated by high pi o. 7. When injected with subthreshold depolarizing current pulses in Ca(2+)-free saline, pyramidal cells displayed a distinct TTX-sensitive inward rectification. This rectification was augmented by low pi o and reduced by high pi o. 8. The various effects of low-pi o and high-pi o saline solutions were reversible upon washing with normosmotic saline. 9. We conclude that pi o is a critical determinant of the endogenous firing patterns of CA1 pyramidal cells. The data suggest that the osmotic effects are most likely to be mediated by changes in the persistent Na+ current, which underlies the active spike ADP and the burst potential in CA1 pyramidal neurones. The possible contribution of these effects to changes in brain excitability in various abnormal osmotic states in discussed.

Jensen, Morten S. and Yoel Yaari. Role of intrinsic burst firing, potassium accumulation, and electrical coupling in the elevated potassium model of hippocampal epilepsy. J. Neurophysiol. 77: 1224–1233, 1997. Perfusing rat hippocampal slices with high-K+ (7.5 mM) saline induced brief population bursts originating in CA3 at 0.5–1 Hz and spreading synaptically into CA1. In 42% of the slices the brief bursts evoked in CA1 gave way every 0.5–2 s to sustained ictal (or seizure) episodes with tonic and clonic components. Paired intra- and extracellular recordings in the CA1 pyramidal layer were used to characterize the synaptic and nonsynaptic mechanisms generating the brief and sustained epileptiform events. The interictal, tonic, or clonic primary burst response in CA1 comprised a spindle-shaped, tight cluster (170–180 Hz) of five to seven population spikes. Bursts evoked between sequential seizures (interictal bursts) were initially small and progressively increased in size. Concurrently, basal extracellular K+ concentration ([K+]o) increased from 6.5 to 7.5 mM. The tonic event emanated from a large primary burst and comprised prolonged (>1 s), self-sustained afterdischarge, associated with a rise in [K+]o to 12 mM. Bursts generated during the subsequent [K+]o decline (clonic bursts) also were large and followed by some afterdischarge. They became small during [K+]o undershoot to 6.5 mM. Intrinsically burst firing pyramidal cells (PCs) were recruited before or at the very onset of the primary population burst and fired repetitively during its course. Nonbursters were recruited ≥10 ms after the beginning of the primary burst and fired, on average, only one spike. The PCs depolarized during the primary burst and subsequent afterdischarge. The primary depolarizing shift was larger in bursters than in nonbursters. Both bursters and nonbursters fired repetitively, albeit intermittently, during tonic and clonic afterdischarge. Throughout the interictal-ictal cycle intracellular spikes were time-locked to population spikes, indicating that PCs fire in tight synchrony. Differential recording of transmembrane potentials unmasked rapid (4–7 ms) transmembrane depolarizing potentials of up to 10 mV, coincident with population spikes. We conclude that in the high-K+ model of hippocampal epilepsy, the local generation of population bursts in CA1 is led by intrinsic bursters, which recruit and synchronize other PCs by synaptic, electrical, and K+-mediated excitatory interactions. The cycling between interictal, tonic, and clonic events appears to result from feedback interactions between neuronal discharge and [K+]o.

1. The distribution of distinctive firing modes within the population of CA1 pyramidal cells and their modulation by the extracellular concentration of potassium ([K+]o) were investigated with intracellular recordings in rat hippocampal slices. 2. Pyramidal cells were injected with long (> 250 ms) and brief (3-5 ms) positive current pulses of increasing intensity. In normal [K+]o (3.5 mM), most cells (38 of 46 cells; 83%) were regular spiking neurons (generating accommodating trains of independent action potentials during long depolarizations and a single spike in response to brief stimuli). The remaining pyramidal cells (8 of 46; 17%) displayed differential tendencies to generate stereotyped clusters of action potentials, or bursts, according to which they were grouped into three subsets of endogenous bursters: grade I, bursting only when stimulated with long depolarizing current pulses (6 of 46; 13%); grade II, bursting also in response to brief stimulation (1 of 46; 2%); grade III, bursting also spontaneously even in absence of synaptic transmission (1 of 46; 2%). 3. Raising [K+]o from 3.5 to 7.5 mM (high [K+]o) significantly reduced resting membrane potential and input impedance but did not change the threshold potential for eliciting an action potential. 4. Raising [K+]o to 7.5 mM reversibly converted many regular spiking cells to bursters. Likewise, the burst tendency of normally bursting pyramidal cells increased to a higher grade in high [K+]o. Consequently, the fraction of bursters in high [K+]o (17 of 41 cells; 42%) was approximately 2.5-fold higher than in normal [K+]o and their differential distribution was shifted toward higher grades of bursting.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Whole-cell tight-seal recordings were obtained from visually identified pyramidal cells (PCs) and interneurones (INs) in the CA1 field of thin hippocampal slices from 13- to 23-day-old rats. The INs sampled were classified according to their location either in the molecular layer (M-INs) or in the oriens layer and alveus (OA-INs). PCs and INs differed in their mode of firing when depolarized by a prolonged current pulse. Whereas PCs fired a single action potential, most INs responded with non-accommodating high frequency spike firing. 2. In the presence of 1 microM tetrodotoxin (TTX), bath application of either 50 microM L-glutamate with 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 2.5 microM N-methyl-D-aspartate (NMDA), induced a similar conductance increase in PCs and INs that was completely blocked by 200 microM DL-2-amino-5-phosphonovaleric acid (APV). The NMDA receptor-mediated currents reversed around 4 mV and exhibited an area of negative slope conductance at potentials more negative than -20 to -30 mV in the presence of 1-2 mM Mg2+. 3. Dual-component excitatory postsynaptic currents (EPSCs) were evoked in PCs and INs by stimulating afferent fibres close to the neurone. The NMDA receptor-mediated component of the EPSCs (NMDA EPSC) was isolated by adding 10 microM CNQX to block non-NMDA receptors. The NMDA EPSCs in all cell types reversed around 1.5 mV and were abolished by 50 microM APV. 4. In saline containing 1 mM Mg2+, the peak current-voltage (I-V) relationship of NMDA EPSCs in PCs and INs showed an area of negative slope conductance at voltages more negative than -20 to -30 mV. In nominally Mg(2+)-free saline, the peak I-V relation was linear over a much wider voltage range in both cell types. 5. The 10-90% rise times of NMDA EPSCs at -60 mV ranged from 4.5 to 16 ms in PCs (mean 8.7 ms; n = 25) and in M-INs (mean 9.1 ms; n = 10). Their decay could be best fitted with the sum of two exponentials. The decay of NMDA EPSCs in PCs was significantly slower than that recorded in INs. The average fast (tau f) and slow (tau s) time constants of decay were, respectively, 66.5 and 353.9 ms in PCs, and 34.4 and 212.5 ms in M-INs.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The effects of raising the concentration of extracellular potassium ([K+]o) on gamma aminobutyric acid (GABA)-mediated inhibitory postsynaptic potentials (IPSPs) were investigated in adult rat hippocampal slices using intracellular recording techniques. IPSPs were evoked in CA1 pyramidal neurons by direct activation of inhibitory interneurons in slices treated with glutamatergic antagonists to block excitatory synaptic transmission. The fast (Cl(-)-dependent, GABAA receptor-mediated) IPSPs (fIPSPs) were isolated from the slow (K(+)-dependent; GABAB receptor-mediated) IPSPs (sIPSPs) by intracellular injection of QX-314, which also suppressed fast (Na(+)-dependent) action potentials. 2. In normal (3.5 mM) and in high (7.5 mM) [K+]o, the peak fIPSP amplitude changed nonlinearly with membrane potential (VM) in a way consistent with outward rectification of the underlying conductance. The input conductance of the fIPSP (GfIPSP) measured around resting VM (about -67 mV) increased 1.7-fold on changing from normal to high-K+ saline, whereas resting VM depolarized 6.8 mV. Repolarizing VM reversed the increase in GfIPSP, suggesting that it was due to outward rectification. The resting input conductance of the neurons increased 1.4-fold in high K+. 3. The time course of fIPSPs was prolonged by high K+. The half time of fIPSP decay increased 1.4-fold, and in half of the neurons the decay became conspicuously multipeaked, suggesting that neurally evoked GABA release from inhibitory interneurons was prolonged. 4. In normal K+, the reversal potentials of fIPSPs (EfIPSP; -76.5 mV) was 9.7 mV more negative than resting VM. Polarizing VM 10–20 mV for 15 min with current injection did not change EfIPSP appreciably.(ABSTRACT TRUNCATED AT 250 WORDS)