Yoel Yaari

KCNQ/M Channels Control Spike Afterdepolarization and Burst Generation in Hippocampal Neurons

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.

Ionic basis of spike after‐depolarization and burst generation in adult rat hippocampal CA1 pyramidal cells.

1. Intracellular recordings in adult rat hippocampal slices were used to identify the ionic conductances underlying active spike after‐depolarization (ADP) and intrinsic burst firing in the somata of CA1 pyramidal cells (PCs). To test the ‘Ca2+ hypothesis’, Ca2+ currents were suppressed by replacing the Ca2+ in the saline with either Mn2+ or Mg2+. Alternatively, the inorganic Ca2+ channel blockers Cd2+ (0.5 mM) or Ni2+ (2 mM) were added to the saline. To test the ‘Na+ hypothesis’, Na+ currents were blocked with tetrodotoxin (TTX; 0.5 microM). 2. The suppression of Ca2+ currents blocked the fast after‐hyperpolarization (AHP) generated by the fast Ca(2+)‐gated K+ current Ic, while enhancing the amplitude and duration of active spike ADPS. 3. Evoked and spontaneous burst firing was preserved undiminished following Ca2+ current suppression, while the propensity to fire bursts increased in many cases. The postburst medium AHP (generated primarily by the muscarine‐sensitive voltage‐gated K+ current, IM) was not affected by this treatment, which blocked the slow AHP (generated by the slow Ca(2+)‐gated K+ current, IAHP). 4. TTX strongly suppressed active ADPs and intrinsic bursts before substantially reducing the threshold, rate of rise and amplitude of solitary spikes. 5. In Ca(2+)‐free saline, caesium‐filled PCs generated large, plateau ADPs following an initial burst of fast spikes. Application of TTX suppressed these ADPs before solitary fast spikes appeared to be reduced. 6. Injection of brief, just subthreshold depolarizing current pulses into bursters evoked slow depolarizing potentials lasting up to 50 ms. These persisted after suppression of Ca2+ currents and were entirely blocked by TTX. 7. We conclude that active spike ADPs and intrinsic bursts in the somata of adult CA1 PCs are generated by a low voltage‐gated, persistent Na+ current. Burst termination is mediated by voltage‐gated K+ currents activated during the burst (most likely IM), rather than by the Ca(2+)‐gated K+ currents Ic and IAHP. The latter currents downregulate the innate tendency of CA1 PCs to burst (Ic) and limit the rate of spontaneous burst firing (IAHP).

Release and sequestration of calcium by ryanodine‐sensitive stores in rat hippocampal neurones

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 Ca2+‐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 Ca2+‐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.

Initiation of network bursts by Ca2+‐dependent intrinsic bursting in the rat pilocarpine model of temporal lobe epilepsy

1 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). 2 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. 3 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. 4 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. 5 Spontaneously recurring population bursts were observed in a subset of epileptic slices. They were abolished by adding 2 μm 6‐cyano‐7‐nitro‐quinoxaline‐2,3‐dione (CNQX) to the ACSF, indicating that synaptic excitation is critical for the generation of these events. 6 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. 7 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.

Proximal Persistent Na+ Channels Drive Spike Afterdepolarizations and Associated Bursting in Adult CA1 Pyramidal Cells

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 of INaP blockers 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, proximal INaP critically determines the firing mode and spike output of adult CA1 pyramidal cells.

Plasticity of intrinsic neuronal properties in 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.

SYNAPTIC NMDA RECEPTORS IN DEVELOPING MOUSE HIPPOCAMPAL NEURONES : FUNCTIONAL PROPERTIES AND SENSITIVITY TO IFENPRODIL

1. Whole‐cell patch‐clamp techniques were used to record pharmacologically isolated NMDA receptor‐mediated EPSCs (NMDA EPSCs) from CA1 pyramidal cells (PCs) in hippocampal slices from 4‐day‐old to 36‐week‐old mice, in order to characterize developmental changes in functional properties and subunit composition of synaptic NMDA receptors. 2. During the first postnatal weeks the dendritic tree of CA1 PCs stained with biocytin increased both in size and in complexity. This was associated with an increase in amplitude of the focally evoked NMDA EPSCs recorded either in nominally Mg(2+)‐free or Mg(2+)‐containing saline. In adult PCs (> 5 weeks old) EPSC amplitude was 4‐fold larger than in very young (up to 2 weeks old) neurones. 3. The sensitivity of NMDA EPSCs to blockade by Mg2+ did not change with age. In very young, intermediate and adult PCs the EPSC‐voltage relation displayed an area of negative slope conductance at membrane potentials more negative than ‐30 mV. The apparent Kd values of the NMDA receptors for Mg2+ at 0 mV were 7.8 +/‐ 6.4, 10.4 +/‐ 14.1 and 6.5 +/‐ 4.7 mM in very young, intermediate and adult neurones, respectively. 4. The decay of the NMDA EPSC in both young and adult neurones could be described by the sum of a fast and a slow exponential function. Both EPSC rise time and fast and slow decay time constants measured at ‐60 mV, decreased with age. 5. The decay of NMDA EPSCs in young versus adult PCs was differentially modulated by membrane voltage. In young PCs depolarization slowed both the fast and the slow EPSC components. In adult PCs depolarization slightly accelerated the initial EPSC decay, though the overall duration of the EPSC did not change. The rise time of the EPSCs was not affected by voltage at any age. 6. The subunit‐selective NMDA receptor antagonist ifenprodil similarly blocked iontophoretic NMDA‐induced currents and NMDA EPSCs. In both young and adult PCs, the concentration‐response curves for this effect disclosed distinct low and high affinity binding sites for ifenprodil. 7. In young PCs, low and high affinity binding sites for ifenprodil were about equally expressed (57 versus 43%, respectively), whereas in adult PCs, synaptic NMDA receptors expressed a majority (78%) of low affinity binding sites for ifenprodil. 8. The long duration of NMDA EPSCs (and by implication, of Ca2+ transfer through NMDA receptor channels) and its further prolongation by depolarization in young PCs are consistent with heightened NMDA‐dependent neuronal plasticity early in development. The age‐related changes in these properties may result from a developmental change in NMDA receptor subunit composition.

Transcriptional Upregulation of Cav3.2 Mediates Epileptogenesis in the Pilocarpine Model of Epilepsy

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.

Role of axonal NaV1.6 sodium channels in action potential initiation of CA1 pyramidal neurons.

In many neuron types, the axon initial segment (AIS) has the lowest threshold for action potential generation. Its active properties are determined by the targeted expression of specific voltage-gated channel subunits. We show that the Na+ channel NaV1.6 displays a striking aggregation at the AIS of cortical neurons. To assess the functional role of this subunit, we used Scn8amed mice that are deficient for NaV1.6 subunits but still display prominent Na+ channel aggregation at the AIS. In CA1 pyramidal cells from Scn8amed mice, we found a depolarizing shift in the voltage dependence of activation of the transient Na+ current (INaT), indicating that NaV1.6 subunits activate at more negative voltages than other NaV subunits. Additionally, persistent and resurgent Na+ currents were significantly reduced. Current-clamp recordings revealed a significant elevation of spike threshold in Scn8amed mice as well as a shortening of the estimated delay between spike initiation at the AIS and its arrival at the soma. In combination with simulations using a realistic computer model of a CA1 pyramidal cell, our results imply that a hyperpolarized voltage dependence of activation of AIS NaV1.6 channels is important both in determining spike threshold and localizing spike initiation to the AIS. In addition to altered spike initiation, Scn8amed mice also showed a strongly reduced spike gain as expected with combined changes in persistent and resurgent currents and spike threshold. These results suggest that NaV1.6 subunits at the AIS contribute significantly to its role as spike trigger zone and shape repetitive discharge properties of CA1 neurons.

Effects of halothane on glutamate receptor-mediated excitatory postsynaptic currents. A patch-clamp study in adult mouse hippocampal slices.

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.