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Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding and deficits in spatial memory. Two-photon glutamate uncaging experiments revealed a marked increase in the fraction of hippocampal first-order CA1 pyramidal cell dendrites capable of generating dendritic spikes in the kainate model of chronic epilepsy. Moreover, in epileptic mice dendritic spikes were generated with lower input synchrony, and with a lower threshold. The Nav1.3/1.1 selective Na+ channel blocker ICA-121431 reversed dendritic hyperexcitability in epileptic mice, while the Nav1.2/1.6 preferring anticonvulsant S-Lic did not. We used in vivo two-photon imaging to determine if aberrant dendritic excitability is associated with altered place-related firing of CA1 neurons. We show that ICA-121431 improves degraded hippocampal spatial representations in epileptic mice. Finally, behavioural experiments show that reversing aberrant dendritic excitability with ICA-121431 reverses hippocampal memory deficits. Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition.

Objective Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood. Methods In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats. Results We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 μM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca2+ -permeable AMPA receptors completely prevents upmodulation of feedback inhibition. Significance These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.

Background: Despite the key role of optimized fasting in modern perioperative patient management, little current data exist on perioperative fasting intervals in routine clinical practice. Methods: In this multicenter prospective study, the length of pre- and postoperative fasting intervals was assessed with the use of a specifically developed questionnaire. Between 15 January 2021 and 31 May 2022, 924 gynecology patients were included, from 13 German gynecology departments. Results: On average, patients remained fasting for about three times as long as recommended for solid foods (17:02 ± 06:54 h) and about five times as long as recommended for clear fluids (9:21 ± 5:48 h). The average perioperative fasting interval exceeded one day (28:23 ± 14:02 h). Longer fasting intervals were observed before and after oncological or extensive procedures, while shorter preoperative fasting intervals were reported in the participating university hospitals. Smoking, treatment in a non-university hospital, an increased Charlson Comorbidity Index and extensive surgery were significant predictors of longer preoperative fasting from solid foods. In general, prolonged preoperative fasting was tolerated well and quality of patient information was perceived as good. Conclusion: Perioperative fasting intervals were drastically prolonged in this cohort of 924 gynecology patients. Our data indicate the need for better patient education about perioperative fasting.

Maintaining an appropriate balance between excitation and inhibition is critical for neuronal information processing. Cortical neurons can cell-autonomously adjust the inhibition they receive to individual levels of excitatory input, but the underlying mechanisms are unclear. We describe that Ste20-like kinase (SLK) mediates cell-autonomous regulation of excitation-inhibition balance in the thalamocortical feedforward circuit, but not in the feedback circuit. This effect is due to regulation of inhibition originating from parvalbumin-expressing interneurons, while inhibition via somatostatin-expressing interneurons is unaffected. Computational modeling shows that this mechanism promotes stable excitatory-inhibitory ratios across pyramidal cells and ensures robust and sparse coding. Patch-clamp RNA sequencing yields genes differentially regulated by SLK knockdown, as well as genes associated with excitation-inhibition balance participating in transsynaptic communication and cytoskeletal dynamics. These data identify a mechanism for cell-autonomous regulation of a specific inhibitory circuit that is critical to ensure that a majority of cortical pyramidal cells participate in information coding.

Dendrites of hippocampal CA1 pyramidal cells amplify clustered glutamatergic input by activation of voltage-gated sodium channels and N-methyl-D-aspartate receptors (NMDARs). NMDAR activity depends on the presence of NMDAR co-agonists such as D-serine, but how co-agonists influence dendritic integration is not well understood. Using combinations of whole-cell patch clamp, iontophoretic glutamate application, two-photon excitation fluorescence microscopy and glutamate uncaging we found that exogenous D-serine reduces the threshold of dendritic spikes and increases their amplitude. Triggering an astrocytic mechanism controlling endogenous D-serine supply via endocannabinoid receptors (CBRs) also increased dendritic spiking. Unexpectedly, this pathway was activated by pyramidal cell activity primarily in the theta range, which required HCN channels and astrocytic CB1Rs. Therefore, astrocytes close a positive and frequency-dependent feedback loop between pyramidal cell activity and their integration of dendritic input. Its disruption led to an impairment of spatial memory, which demonstrates its behavioral relevance.

Ischemic stroke (IS) is still among the leading causes of death and disability worldwide. The pathogenic mechanisms beyond its development are several and are complex and this is the main reason why a functional therapy is still missed. The beneficial effects of natural compounds against cardiovascular diseases and IS have been investigated for a long time. In this article, we reviewed the association between the most studied polyphenols and stroke protection in terms of prevention, effect on acute phase, and rehabilitation. We described experimental and epidemiological studies reporting the role of flavonols, phenolic acid, and stilbens on ischemic mechanisms leading to stroke. We analyzed the principal animal models used to evaluate the impact of these micronutrients to cerebral blood flow and to molecular pathways involved in oxidative stress and inflammation modulation, such as sirtuins. We reported the most significant clinical trials demonstrated as the persistent use of polyphenols is clinically relevant in terms of the reduction of vascular risk factors for IS, such as Atrial Fibrillation. Interestingly, different kinds of polyphenols provide brain protection by activating different pathways and mechanisms, like inducing antithrombotic effect, such as Honokiol. For this reason, we discussed an appropriate integrative use of them as a possible therapeutic alternative against stroke.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. GABAergic silencing using Clozapine-N-Oxide (CNO) demonstrated reliable induction of local epileptiform events in the electroencephalogram signal of awake freely moving mice. Anesthetized mice experiments showed consistent induction of focal epileptiform-events in both the barrel cortex (BC) and the medial prefrontal cortex (mPFC), accompanied by high-frequency oscillations, a known characteristic of human seizures. Epileptiform-events showed propagation indication with favored propagation pathways: from the BC on 1 hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, sensory whisker-pad stimulation evoked BC epileptiform events post-CNO, highlighting the potential use of this model in studying sensory-evoked seizures. Combined, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile, and reliable model of focal cortical epileptic activity suitable for systematically studying cortical ictogenesis in different cortical areas.

The hippocampal dentate gyrus is an important relay conveying sensory information from the entorhinal cortex to the hippocampus proper. During exploration, the dentate gyrus has been proposed to act as a pattern separator. However, the dentate gyrus also shows structured activity during immobility and sleep. The properties of these activity patterns at cellular resolution, and their role in hippocampal-dependent memory processes have remained unclear. Using dual-color in-vivo two-photon Ca2+ imaging, we show that in immobile mice dentate granule cells generate sparse, synchronized activity patterns associated with entorhinal cortex activation. These population events are structured and modified by changes in the environment; and they incorporate place- and speed cells. Importantly, they are more similar than expected by chance to population patterns evoked during self-motion. Using optogenetic inhibition, we show that granule cell activity is not only required during exploration, but also during immobility in order to form dentate gyrus-dependent spatial memories.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance. SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

SummaryThe size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ 3rd, order dendrites. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, while excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and of inhibitory synapse maintenance.

Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding, and deficits in spatial memory.Two-photon glutamate uncaging experiments revealed that the mechanisms constraining the generation of Na+ spikes in hippocampal 1st order pyramidal cell dendrites are profoundly degraded in experimental epilepsy. A selective Nav1.3 sodium channel blocker reversed this effect, and Nav1.3 channels were up-regulated selectively in principal neurons at the mRNA level. Finally, in-vivo two-photon imaging revealed that the Nav1.3 channel blocker improves degraded hippocampal spatial representations, and reverses hippocampal memory deficits.Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition.One Sentence SummaryImpaired input computations via aberrant dendritic spikes in chronic epilepsy degrade neuronal place codes and spatial memory

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in-vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. Following focal silencing of GABAergic neurons by administration of Clozapine-N-Oxide (CNO), we demonstrated reliable induction of local epileptiform events in the electroencephalogram (EEG) signal of awake freely moving mice. Experiments in anesthetized mice showed consistent induction of focal seizures in two different brain regions – the barrel cortex (BC) and at the medial prefrontal cortex (mPFC). Seizures were accompanied by high frequency oscillations, a known characteristic of human focal seizures. Seizures propagated, but an analysis of seizure propagation revealed favored propagation pathways. CNO-induced epileptiform events propagated from the BC on one hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, post-CNO epileptiform events in the BC could be triggered by sensory whisker-pad stimulation, indicating that this model, applied to sensory cortices, may be useful to study sensory-evoked seizures. Taken together, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile and reliable model of focal cortical epilepsy suitable to systematically study cortical ictogenesis in different cortical areas.Significance StatementFocal cortical epilepsies are often hard to alleviate using current anticonvulsant therapies while further drug discovery is impeded by the limited variety of suitable animal models. In this study, we established a novel model of focal cortical seizures induced by spatially-restricted chemogenetic silencing of cortical inhibitory neurons. We have shown this method to be effective at various cortical regions and reliably induce seizures that share key characteristics with known human epilepsy traits, including sensory triggering and seizure propagation. This model may thus be used to advance the discovery of new remedies for focal cortical epilepsies, as well as to improve our understanding of seizure spread along different cortical pathways.

The hippocampal dentate gyrus is an important relay conveying sensory information from the entorhinal cortex to the hippocampus proper. During exploration, the dentate gyrus has been proposed to act as a pattern separator. However, the dentate gyrus also shows structured activity during immobility and sleep. The properties of these activity patterns at cellular resolution, and their role in hippocampal-dependent memory processes have remained unclear. Using dual-color in-vivo two-photon Ca2+ imaging, we show that in immobile mice dentate granule cells generate sparse, synchronized activity patterns associated with entorhinal cortex activation. These population events are structured and modified by changes in the environment; and they incorporate place- and speed cells. Importantly, they recapitulate population patterns evoked during self-motion. Using optogenetic inhibition during immobility, we show that granule cell activity during immobility is required to form dentate gyrus-dependent spatial memories. These data suggest that memory formation is supported by dentate gyrus replay of population codes of the current environment.

Objective Pharmacoresistance is a problem affecting ∼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy. Methods We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation. Results S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 μmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 μmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Nav 1.2 and Nav 1.6 subunits, but not in Nav 1.1 and Nav 1.3 subunits. Significance From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Nav 1.2 and Nav 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.

Polyamine Modulation of Anticonvulsant Drug Response: A Potential Mechanism Contributing to Pharmacoresistance in Chronic Epilepsy Beckonert NM, Opitz T, Pitsch J, Soares da Silva P, Beck H. J Neurosci. 2018;38(24):5596-5605. https://doi.org/10.1523/JNEUROSCI.0640-18.2018.Despite the development of numerous novel anticonvulsant drugs, ∼30% of patients with epilepsy remain refractory to antiepileptic drugs (AEDs). Many established and novel AEDs reduce hyperexcitability via voltage- and use-dependent inhibition of voltage-gated Na+ channels. For the widely used anticonvulsant carbamazepine (CBZ), use-dependent block of Na+ channels is significantly reduced both in experimental and human epilepsies. However, the molecular underpinnings of this potential cellular mechanism for pharmacoresistance have remained enigmatic. Here, we describe the mechanism that leads to the emergence of CBZ-resistant Na+ channels. We focused on the endogenous polyamine system, which powerfully modulates Na+ channels in a use-dependent manner. We had shown previously that the intracellular polyamine spermine is reduced in chronic epilepsy, resulting in increased persistent Na+ currents. Because both spermine and CBZ bind use-dependently in spatial proximity within the Na+ channel pore, we hypothesized that spermine loss might also be related to diminished CBZ response. Using the pilocarpine model of refractory epilepsy in male rats and whole-cell patch-clamp recordings, we first replicated the reduction of use-dependent block by CBZ in chronically epileptic animals. We then substituted intracellular spermine via the patch pipette in different concentrations. Under these conditions, we found that exogenous spermine significantly rescues use-dependent block of Na+ channels by CBZ. These findings indicate that an unexpected modulatory mechanism, depletion of intracellular polyamines, leads both to increased persistent Na+ currents and to diminished CBZ sensitivity of Na+ channels. These findings could lead to novel strategies for overcoming pharmacoresistant epilepsy that target the polyamine system. Significance Statement: Pharmacoresistant epilepsy affects ∼18 million people worldwide, and intense efforts have therefore been undertaken to uncover the underlying molecular and cellular mechanisms. One of the key known candidate mechanisms of pharmacoresistance has been a loss of use-dependent Na+ channel block by the anticonvulsant CBZ, both in human and experimental epilepsies. Despite intense scrutiny, the molecular mechanisms underlying this phenomenon have not been elucidated. We now show that a loss of intracellular spermine in chronic epilepsy is a major causative factor leading to the development of CBZ-resistant Na+ currents. This finding can be exploited both for the screening of anticonvulsants in expression systems and for novel strategies to overcome pharmacoresistance that target the polyamine system.

Focal cortical dysplasias (FCDs) represent common cortical malformations that are frequently associated with epilepsy. They have so far not been well understood in terms of their molecular pathogenesis, and with respect to mechanisms of seizure emergence.Several recent studies have succeeded in making significant advances in understanding the molecular genetics, in particular FCD type II. A second major advance has been the development of novel rodent models of FCDs that replicate a somatic mutation seen in humans, lead to a focal lesion, and recapitulate many phenotypic features of human FCDs. We will discuss these recent advances.These advances promise significant advances in understanding the heterogeneity of FCDs at the molecular genetic level. They also promise a much better understanding of cell-intrinsic and network mechanisms underlying increased seizure susceptibility and altered cognition. Systematic studies utilizing the approaches summarized here promise to lead to specific strategies regarding when and how to treat specific subgroups of FCDs.

Modern neuroscientific techniques that specifically manipulate and measure neuronal activity in behaving animals now allow bridging of the gap from the cellular to the behavioral level. However, in doing so, they also pose new challenges. Research using incompletely defined manipulations in a high-dimensional space without clear hypotheses is likely to suffer from multiple well-known conceptual and statistical problems. In this context it is essential to develop hypotheses with testable implications across levels. Here we propose that a focus on circuit motifs can help achieve this goal. Viewing neural structures as an assembly of circuit motif building blocks is not new. However, recent tool advances have made it possible to extensively map, specifically manipulate, and quantitatively investigate circuit motifs and thereby reexamine their relevance to brain function.

The firing of neurons throughout the brain is determined by the precise relations between excitatory and inhibitory inputs, and disruption of their balance underlies many psychiatric diseases. Whether or not these inputs covary over time or between repeated stimuli remains unclear due to the lack of experimental methods for measuring both inputs simultaneously. We developed a new analytical framework for instantaneous and simultaneous measurements of both the excitatory and inhibitory neuronal inputs during a single trial under current clamp recording. This can be achieved by injecting a current composed of two high frequency sinusoidal components followed by analytical extraction of the conductances. We demonstrate the ability of this method to measure both inputs in a single trial under realistic recording constraints and from morphologically realistic CA1 pyramidal model cells. Future experimental implementation of our new method will facilitate the understanding of fundamental questions about the health and disease of the nervous system., Author summary Most neurons in the brain receive synaptic inputs from both excitatory and inhibitory neurons. Together, these inputs determine neuronal activity: excitatory synapses shift the electrical potential across the membrane towards the threshold for generation of action potentials, whereas inhibitory synapses lower this potential away from the threshold. Action potentials are the rapid electrochemical signals that transmit information to other neurons and they are critical for the information processing abilities of the brain. Although there are many ways to measure either excitatory or inhibitory inputs, these methods have been unable to measure both at the same time. Measuring both inputs together is essential towards understanding how neurons integrate information. We developed a new analytical method to measure excitatory and inhibitory inputs at the same time from the voltage response to injection of an alternating current into a neuron. We describe the foundation of this new method and find that it works in biologically realistic simulations of neurons. By using this technique in real neurons, scientists could investigate basic principles of information processing in the healthy and diseased brain.

1AbstractTransgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. Particularly, the investigation of interneurons has benefitted from the ability to target genetic constructs to defined cell types. However, the cell type specificity of some mouse lines has been called into questions. Here we show for the first time the functional consequences of unspecific expression in a somatostatin-Cre (SST-Cre) mouse line. We find large optogenetically evoked excitatory currents originating from unspecifically targeted CA3 pyramidal cells. We also used public Allen Brain Institute data to estimate expression specificity in other Cre lines. Another SST-Cre mouse lines shows comparable unspecificity, whereas a Parvalbumin-Cre mouse line shows much less unspecific expression. Finally, we make suggestions to ensure that the results from in-vivo use of Cre mouse lines are interpretable.

Inhibitory interneurons, organized into canonical feedforward and feedback motifs, play a key role in controlling normal and pathological neuronal activity. We demonstrate prominent quantitative changes in the dynamics of feedback inhibition in a rat model of chronic epilepsy (male Wistar rats). Systematic interneuron recordings revealed a large decrease in intrinsic excitability of basket cells and oriens-lacunosum moleculare interneurons in epileptic animals. Additionally, the temporal dynamics of interneuron recruitment by recurrent feedback excitation were strongly altered, resulting in a profound loss of initial feedback inhibition during synchronous CA1 pyramidal activity. Biophysically constrained models of the complete feedback circuit motifs of normal and epileptic animals revealed that, as a consequence of altered feedback inhibition, burst activity arising in CA3 is more strongly converted to a CA1 output. This suggests that altered dynamics of feedback inhibition promote the transmission of epileptiform bursts to hippocampal projection areas.SIGNIFICANCE STATEMENTWe quantitatively characterized changes of the CA1 feedback inhibitory circuit in a model of chronic temporal lobe epilepsy. This study shows, for the first time, that dynamic recruitment of inhibition in feedback circuits is altered and establishes the cellular mechanisms for this change. Computational modeling revealed that the observed changes are likely to systematically alter CA1 input-output properties leading to (1) increased seizure propagation through CA1 and (2) altered computation of synchronous CA3 input.

SummaryObjective In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. Methods We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na+ currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. Results We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Significance Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance.

Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this ‘PAC-K’ silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.

The medial septum/diagonal band of Broca complex (MSDB) is a key structure that modulates hippocampal rhythmogenesis. Cholinergic neurons of the MSDB play a central role in generating and pacing theta-band oscillations in the hippocampal formation during exploration, novelty detection, and memory encoding. How precisely cholinergic neurons affect hippocampal network dynamicsin vivo, however, has remained elusive. In this study, we show that stimulation of cholinergic MSDB neurons in urethane-anesthetized mice acts on hippocampal networks via two distinct pathways. A direct septo-hippocampal cholinergic projection causes increased firing of hippocampal inhibitory interneurons with concomitantly decreased firing of principal cells. In addition, cholinergic neurons recruit noncholinergic neurons within the MSDB. This indirect pathway is required for hippocampal theta synchronization. Activation of both pathways causes a reduction in pyramidal neuron firing and a more precise coupling to the theta oscillatory phase. These two anatomically and functionally distinct pathways are likely relevant for cholinergic control of encoding versus retrieval modes in the hippocampus.

Cellular behavior is orchestrated by the complex interactions of a myriad of intracellular signal transduction pathways. To understand and investigate the role of individual components in such signaling networks, the availability of specific inhibitors is of paramount importance. We report the generation and validation of a novel variant of an RNA aptamer that selectively inhibits the mitogen‐activated kinase pathway in neurons. We demonstrate that the aptamer retains function under intracellular conditions and that application of the aptamer through the patch‐clamp pipette efficiently inhibits mitogen‐activated kinase‐dependent synaptic plasticity. This approach introduces synthetic aptamers as generic tools, readily applicable to inhibit different components of intraneuronal signaling networks with utmost specificity.

Guillain-Barre syndrome (GBS) is an acute, immune-mediated polyradiculoneuropathy characterized by rapidly progressive paresis and sensory disturbances. Moderate to severe and often intractable neuropathic pain is a common symptom of GBS, but its underlying mechanisms are unknown. Pathology of GBS is classically attributed to demyelination of large, myelinated peripheral fibers. However, there is increasing evidence that neuropathic pain in GBS is associated with impaired function of small, unmyelinated, nociceptive fibers. We therefore examined the functional properties of small DRG neurons, the somata of nociceptive fibers, in a rat model of GBS (experimental autoimmune neuritis=EAN). EAN rats developed behavioral signs of neuropathic pain. This was accompanied by a significant shortening of action potentials due to a more rapid repolarization and an increase in repetitive firing in a subgroup of capsaicin-responsive DRG neurons. Na+ current measurements revealed a significant increase of the fast TTX-sensitive current and a reduction of the persistent TTX-sensitive current component. These changes of Na+ currents may account for the significant decrease in AP duration leading to an overall increase in excitability and are therefore possibly directly linked to pathological pain behavior. Thus, like in other animal models of neuropathic and inflammatory pain, Na+ channels seem to be crucially involved in the pathology of GBS and may constitute promising targets for pain modulating pharmaceuticals.

SummaryObjective The maturation of adult-born granule cells and their functional integration into the network is thought to play a key role in the proper functioning of the dentate gyrus. In temporal lobe epilepsy, adult-born granule cells in the dentate gyrus develop abnormally and possess a hilar basal dendrite (HBD). Although morphological studies have shown that these HBDs have synapses, little is known about the functional properties of these HBDs or the intrinsic and network properties of the granule cells that possess these aberrant dendrites. Methods We performed patch-clamp recordings of granule cells within the granule cell layer “normotopic” from sham-control and status epilepticus (SE) animals. Normotopic granule cells from SE animals possessed an HBD (SE+HBD+ cells) or not (SE+HBD− cells). Apical and basal dendrites were stimulated using multiphoton uncaging of glutamate. Two-photon Ca2+ imaging was used to measure Ca2+ transients associated with back-propagating action potentials (bAPs). Results Near-synchronous synaptic input integrated linearly in apical dendrites from sham-control animals and was not significantly different in apical dendrites of SE+HBD− cells. The majority of HBDs integrated input linearly, similar to apical dendrites. However, 2 of 11 HBDs were capable of supralinear integration mediated by a dendritic spike. Furthermore, the bAP-evoked Ca2+ transients were relatively well maintained along HBDs, compared with apical dendrites. This further suggests an enhanced electrogenesis in HBDs. In addition, the output of granule cells from epileptic tissue was enhanced, with both SE+HBD− and SE+HBD+ cells displaying increased high-frequency (>100 Hz) burst-firing. Finally, both SE+HBD− and SE+HBD+ cells received recurrent excitatory input that was capable of generating APs, especially in the absence of feedback inhibition. Significance Taken together, these data suggest that the enhanced excitability of HBDs combined with the altered intrinsic and network properties of granule cells collude to promote excitability and synchrony in the epileptic dentate gyrus.

SummaryNeuronal processing is classically conceptualized as dendritic input, somatic integration, and axonal output. The axon initial segment, the proposed site of action potential generation, usually emanates directly from the soma. However, we found that axons of hippocampal pyramidal cells frequently derive from a basal dendrite rather than from the soma. This morphology is particularly enriched in central CA1, the principal hippocampal output area. Multiphoton glutamate uncaging revealed that input onto the axon-carrying dendrites (AcDs) was more efficient in eliciting action potential output than input onto regular basal dendrites. First, synaptic input onto AcDs generates action potentials with lower activation thresholds compared with regular dendrites. Second, AcDs are intrinsically more excitable, generating dendritic spikes with higher probability and greater strength. Thus, axon-carrying dendrites constitute a privileged channel for excitatory synaptic input in a subset of cortical pyramidal cells.Video Abstract

Summary Objective SCN1A encodes the alpha subunit of the voltage-gated sodium channel and plays a crucial role in several epilepsy syndromes. The common SCN1A splice-site polymorphism rs3812718 (IVS5N+5 G>A) might contribute to the pathophysiology underlying genetic generalized epilepsies and is associated with electrophysiologic properties of the channel and the effect of sodium-channel blocking antiepileptic drugs. We assessed the effects of the rs3812718 genotype on cortical excitability at baseline and after administration of carbamazepine in order to investigate the mechanism of this association. Methods Paired-pulse transcranial magnetic stimulation (TMS) was applied in 92 healthy volunteers with the homozygous genotypes AA or GG of rs3812718 at baseline and after application of 400 mg of carbamazepine or placebo in a double-blind, randomized, crossover design. Resting motor threshold (RMT), short interval intracortical inhibition (SICI), intracortical facilitation (ICF), and cortical silent period (CSP) were determined. Results At baseline there was no significant difference in any TMS parameter. Genotype GG was associated with a higher carbamazepine-induced increase in CSP duration as compared to AA (multivariate analysis of covariance [MANCOVA], p = 0.013). An expected significant increase in RMT was genotype independent. Significance We found that the rs3812718 genotype modifies the effect of carbamazepine on CSP duration (mainly reflecting modulation of γ-aminobutyric acid (GABA)ergic inhibition), but not on RMT (mainly reflecting modulation of voltage-gated sodium channels). This provides evidence that rs3812718 affects the pharmacoresponse to carbamazepine via an effect on GABAergic cortical interneurons. Our results also confirm that TMS is useful to investigate the effect of genetic variants on cortical excitability and pharmacoresponse.

Summary Eslicarbazepine is the major active metabolite of eslicarbazepine acetate, a once-daily antiepileptic drug approved in Europe as adjunctive therapy for refractory partial-onset seizures in adults. This study was aimed to determine the effects of eslicarbazepine on persistent Na + currents ( I NaP ) and the role of β subunits in modulating these effects. To study the role of β subunits of the Na + channel we used a mouse line genetically lacking either the β 1 or β 2 subunit, encoded by the SCN1B or SCN2B gene, respectively. Whole cell patch-clamp recordings were performed on CA1 neurons in hippocampal slices under control conditions and application of 300μM eslicarbazepine. We examined I NaP in acutely isolated CA1 neurons and repetitive firing in hippocampal slices of mice lacking β subunits and corresponding wild-type littermates. We found that eslicarbazepine caused a significant reduction of maximal I NaP conductance and an efficient reduction of the firing rate in wild-type mice. We have shown previously a paradoxical increase of conductance of I NaP caused by carbamazepine in mice lacking β 1 subunits in the subthreshold range, leading to a failure in affecting neuronal firing (Uebachs et al., 2010). In contrast, eslicarbazepine did not cause this paradoxical effect on I NaP in SCN1B null mice. Consequently, the effects of eslicarbazepine on repetitive firing were maintained in these animals. These results indicate that eslicarbazepine exerts effects on I NaP similar to those known for carbamazepine. However, in animals lacking the β 1 Na + channel subunit these effects are maintained. Therefore, eslicarbazepine potentially overcomes a previously described putative mechanism of resistance to established Na + channel acting antiepileptic drugs.

Background Smad-interacting protein 1 (also named Zeb2 and Zfhx1b) is a transcription factor that plays an important role in neuronal development and, when mutated, causes Mowat–Wilson syndrome (MWS). A corresponding mouse model carrying a heterozygous Zeb2 deletion was comprehensively analysed in the German Mouse Clinic. The most prominent phenotype was the reduced pain sensitivity. In this study, we investigated the role of Zeb2 in inflammatory and neuropathic pain. Methods For this, we tested mutant Zeb2 animals in different models of inflammatory pain like abdominal constriction, formalin and carrageenan test. Furthermore, we studied the pain reactivity of the mice after peripheral nerve ligation. To examine the nociceptive transmission of primary sensory dorsal root ganglia (DRG) neurons, we determined the neuronal activity in the spinal dorsal horn after the formalin test using staining of c-Fos. Next, we characterized the neuronal cell population in the DRGs and in the sciatic nerve to study the effect of the Zeb2 mutation on peripheral nerve morphology. Results The present data show that Zeb2 is involved in the development of primary sensory DRG neurons, especially of C- and Aδ fibres. These alterations contribute to a hypoalgesic phenotype in inflammatory but not in neuropathic pain in these Zeb2+/− mice. Conclusion Our data suggest that the under-reaction to pain observed in MWS patients results from a reduced responsivity to nociceptive stimulation rather than an inability to communicate discomfort.

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/β and RIM2α/β, have been shown to be centrally involved in mediating presynaptic active zone function. The RIM protein family contains two additional small isoforms, RIM3γ and RIM4γ, which are composed only of the RIM-specific C-terminal C2B domain and varying N-terminal sequences and whose function remains to be elucidated. Here, we report that both, RIM3γ and RIM4γ, play an essential role for the development of neuronal arborization and of dendritic spines independent of synaptic function. γ-RIM knock-down in rat primary neuronal cultures andin vivoresulted in a drastic reduction in the complexity of neuronal arborization, affecting both axonal and dendritic outgrowth, independent of the time point of γ-RIM downregulation during dendrite development. Rescue experiments revealed that the phenotype is caused by a function common to both γ-RIMs. These findings indicate that γ-RIMs are involved in cell biological functions distinct from the regulation of synaptic vesicle exocytosis and play a role in the molecular mechanisms controlling the establishment of dendritic complexity and axonal outgrowth.

Genetic mutations causing dysfunction of both voltage- and ligand-gated ion channels make a major contribution to the cause of many different types of familial epilepsy. Key mechanisms comprise defective Na(+) channels of inhibitory neurons, or GABA(A) receptors affecting pre- or postsynaptic GABAergic inhibition, or a dysfunction of different types of channels at axon initial segments. Many of these ion channel mutations have been modelled in mice, which has largely contributed to the understanding of where and how the ion channel defects lead to neuronal hyperexcitability. Animal models of febrile seizures or mesial temporal epilepsy have shown that dendritic K(+) channels, hyperpolarization-activated cation channels and T-type Ca(2+) channels play important roles in the generation of seizures. For the latter, it has been shown that suppression of their function by pharmacological mechanisms or in knock-out mice can antagonize epileptogenesis. Defects of ion channel function are also associated with forms of acquired epilepsy. Autoantibodies directed against ion channels or associated proteins, such as K(+) channels, LGI1 or NMDA receptors, have been identified in epileptic disorders that can largely be included under the term limbic encephalitis which includes limbic seizures, status epilepticus and psychiatric symptoms. We conclude that ion channels and associated proteins are important players in different types of genetic and acquired epilepsies. Nevertheless, the molecular bases for most common forms of epilepsy are not yet clear, and evidence to be discussed indicates just how much more we need to understand about the complex mechanisms that underlie epileptogenesis.