Stefan Remy

A novel mechanism underlying drug resistance in chronic epilepsy.

The development of resistance to pharmacological treatment is common to many human diseases. In chronic epilepsy, many patients develop resistance to anticonvulsant drug treatment during the course of their disease, with the underlying mechanisms remaining unclear. We have studied cellular mechanisms underlying drug resistance in resected hippocampal tissue from patients with temporal lobe epilepsy by comparing two groups of patients, the first displaying a clinical response to the anticonvulsant carbamazepine and a second group with therapy‐resistant seizures. Using patch‐clamp recordings, we show that the mechanism of action of carbamazepine, use‐dependent block of voltage‐dependent Na+ channels, is completely lost in carbamazepine‐resistant patients. Likewise, seizure activity elicited in human hippocampal slices is insensitive to carbamazepine. In marked contrast, carbamazepine‐induced use‐dependent block of Na+ channels and blocked seizure activity in vitro in patients clinically responsive to this drug. Consistent with these results in human patients, we also show that use‐dependent block of Na+ channels by carbamazepine is absent in chronic experimental epilepsy. Taken together, these data suggest that a loss of Na+ channel drug sensitivity may constitute a novel mechanism underlying the development of drug‐resistant epilepsy. Ann Neurol 2003

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.

Molecular and functional changes in voltage-dependent na+ channels following pilocarpine-induced status epilepticus in rat dentate granule cells

Status epilepticus (S.E.) is known to lead to a large number of changes in the expression of voltage-dependent ion channels and neurotransmitter receptors. In the present study, we examined whether an episode of S.E. induced by pilocarpine in vivo alters functional properties and expression of voltage-gated Na(+) channels in dentate granule cells (DGCs) of the rat hippocampus. Using patch-clamp recordings in isolated DGCs, we show that the voltage-dependent inactivation curve is significantly shifted toward depolarizing potentials following S.E. (half-maximal inactivation at -43.2+/-0.6 mV) when compared with control rats (-48.2+/-0.8 mV, P<0.0001). The voltage-dependent activation curve is significantly shifted to more negative potentials following S.E., with half-maximal activation at -28.6+/-0.8 mV compared with -25.8+/-0.9 mV in control animals (P<0.05). The changes in voltage dependence resulted in an augmented window current due to increased overlap between the activation and inactivation curve. In contrast to Na(+) channel voltage-dependence, S.E. caused no changes in the kinetics of fast or slow recovery from inactivation. The functional changes were accompanied by altered expression of Na(+) channel subunits measured by real-time reverse transcription-polymerase chain reaction in dentate gyrus microslices. We investigated expression of the pore-forming alpha subunits Na(v)1.1-Na(v)1.3 and Na(v)1.5-Na(v)1.6, in addition to the accessory subunits beta(1) and beta(2). The Na(v)1.2 and Na(v)1.6 subunit as well as the beta(1) subunit were persistently down-regulated up to 30 days following S.E. The beta(2) subunit was transiently down-regulated on the first and third day following S.E. These results indicate that differential changes in Na(+) channel subunit expression occur in concert with functional changes. Because coexpression of beta subunits is known to robustly shift the voltage dependence of inactivation in a hyperpolarizing direction, we speculate that a down-regulation of beta-subunit expression may contribute to the depolarizing shift in the inactivation curve following S.E.

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.

Anticonvulsant pharmacology of voltage‐gated Na+ channels in hippocampal neurons of control and chronically epileptic rats

Voltage‐gated Na+ channels are a main target of many first‐line anticonvulsant drugs and their mechanism of action has been extensively investigated in cell lines and native neurons. Nevertheless, it is unknown whether the efficacy of these drugs might be altered following chronic epileptogenesis. We have, therefore, analysed the effects of phenytoin (100 µm), lamotrigine (100 µm) and valproate (600 µm) on Na+ currents in dissociated rat hippocampal granule neurons in the pilocarpine model of chronic epilepsy. In control animals, all three substances exhibited modest tonic blocking effects on Na+ channels in their resting state. These effects of phenytoin and lamotrigine were reduced (by 77 and 64%) in epileptic compared with control animals. Phenytoin and valproate caused a shift in the voltage dependence of fast inactivation in a hyperpolarizing direction, while all three substances shifted the voltage dependence of activation in a depolarizing direction. The anticonvulsant effects on Na+ channel voltage dependence proved to be similar in control and epileptic animals. The time course of fast recovery from inactivation was potently slowed by lamotrigine and phenytoin in control animals, while valproate had no effect. Interestingly, the effects of phenytoin on fast recovery from inactivation were significantly reduced in chronic epilepsy. Taken together, these results reveal that different anticonvulsant drugs may exert a distinct pattern of effects on native Na+ channels. Furthermore, the reduction of phenytoin and, to a less pronounced extent, lamotrigine effects in chronic epilepsy raises the possibility that reduced pharmacosensitivity of Na+ channels may contribute to the development of drug resistance.

Dendritic Integration in Hippocampal Dentate Granule Cells

Hippocampal granule cells are important relay stations that transfer information from the entorhinal cortex into the hippocampus proper. This process is critically determined by the integrative properties of granule cell dendrites. However, their small diameter has so far hampered efforts to examine their properties directly. Using a combination of dual somatodendritic patch-clamp recordings and multiphoton glutamate uncaging, we now show that the integrative properties of granule cell dendrites differ substantially from other principal neurons. Due to a very strong dendritic voltage attenuation, the impact of individual synapses on granule cell output is low. At the same time, integration is linearized by voltage-dependent boosting mechanisms, only weakly affected by input synchrony, and independent of input location. These experiments establish that dentate granule cell dendritic properties are optimized for linear integration and strong attenuation of synaptic input from the entorhinal cortex, which may contribute to the sparse activity of granule cells in vivo.

Locomotion, Theta Oscillations, and the Speed-Correlated Firing of Hippocampal Neurons Are Controlled by a Medial Septal Glutamatergic Circuit

Before the onset of locomotion, the hippocampus undergoes a transition into an activity-state specialized for the processing of spatially related input. This brain-state transition is associated with increased firing rates of CA1 pyramidal neurons and the occurrence of theta oscillations, which both correlate with locomotion velocity. However, the neural circuit by which locomotor activity is linked to hippocampal oscillations and neuronal firing rates is unresolved. Here we reveal a septo-hippocampal circuit mediated by glutamatergic (VGluT2(+)) neurons that is activated before locomotion onset and that controls the initiation and velocity of locomotion as well as the entrainment of theta oscillations. Moreover, via septo-hippocampal projections onto alveus/oriens interneurons, this circuit regulates feedforward inhibition of Schaffer collateral and perforant path input to CA1 pyramidal neurons in a locomotion-dependent manner. With higher locomotion speed, the increased activity of medial septal VGluT2 neurons is translated into increased axo-somatic depolarization and higher firing rates of CA1 pyramidal neurons. VIDEO ABSTRACT.

Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer's disease.

Dendritic structure critically determines the electrical properties of neurons and, thereby, defines the fundamental process of input-to-output conversion. The diversity of dendritic architectures enables neurons to fulfill their specialized circuit functions during cognitive processes. It is known that this dendritic integrity is impaired in patients with Alzheimers disease and in relevant mouse models. It is unknown, however, whether this structural degeneration translates into aberrant neuronal function. Here we use in vivo whole-cell patch-clamp recordings, high-resolution STED imaging, and computational modeling of CA1 pyramidal neurons in a mouse model of Alzheimers disease to show that structural degeneration and neuronal hyperexcitability are crucially linked. Our results demonstrate that a structure-dependent amplification of synaptic input to action potential output conversion might constitute a novel cellular pathomechanism underlying network dysfunction with potential relevance for other neurodegenerative diseases with abnormal changes of dendritic morphology.

Activity-Dependent Control of Neuronal Output by Local and Global Dendritic Spike Attenuation

Neurons possess elaborate dendritic arbors which receive and integrate excitatory synaptic signals. Individual dendritic subbranches exhibit local membrane potential supralinearities, termed dendritic spikes, which control transfer of local synaptic input to the soma. Here, we show that dendritic spikes in CA1 pyramidal cells are strongly regulated by specific types of prior input. While input in the linear range is without effect, supralinear input inhibits subsequent spikes, causing them to attenuate and ultimately fail due to dendritic Na(+) channel inactivation. This mechanism acts locally within the boundaries of the input branch. If an input is sufficiently strong to trigger axonal action potentials, their back-propagation into the dendritic tree causes a widespread global reduction in dendritic excitability which is prominent after firing patterns occurring in vivo. Together, these mechanisms control the capability of individual dendritic branches to trigger somatic action potential output. They are invoked at frequencies encountered during learning, and impose limits on the storage and retrieval rates of information encoded as branch excitability.

An Increase in Persistent Sodium Current Contributes to Intrinsic Neuronal Bursting After Status Epilepticus

Brain damage causes multiple changes in synaptic function and intrinsic properties of surviving neurons, leading to the development of chronic epilepsy. In the widely used pilocarpine-status epilepticus (SE) rat model of temporal lobe epilepsy (TLE), a major alteration is the marked increase in the fraction of intrinsically bursting CA1 pyramidal cells. Here we have differentiated between two types of bursting phenotypes: 1) bursting in response to threshold-straddling excitatory current pulses (low-threshold bursting) and 2) bursting only in response to suprathreshold stimuli (high-threshold bursting). Low-threshold bursting prevailed in 46.5% of SE-experienced neurons sampled 1-4 wk after pilocarpine-SE, but was rarely seen in control neurons (1.9%). As previously shown, it appeared to be driven predominantly by a T-type Ca(2+) current (I(CaT)) in the apical dendrites. After blocking low-threshold bursting with Ni(2+), the same neurons still manifested a high-threshold bursting phenotype. Another 40.1% of SE-experienced neurons displayed only a high-threshold bursting phenotype and the remaining 13.4% of these neurons were nonbursters. Altogether, high-threshold bursting prevailed in 86.6% of SE-experienced neurons, but only in 33.0% of control neurons. Several lines of evidence indicated that high-threshold bursting is driven by persistent Na(+) current (I(NaP)) at or near the soma. Congruently, I(NaP) was 1.5-fold larger in SE-experienced versus control neurons. We conclude that an increase in I(NaP), conjointly with an increase in I(CaT), strongly contributes to the predominance of bursting phenotypes in CA1 pyramidal cells early after pilocarpine-SE and thus likely plays a role in the development of a chronic epileptic condition in this TLE model.