Neurochemical Profile of BRAFV600E/AktT308D/S473D Mouse Gangliogliomas Reveals Impaired GABAergic System Inhibition.

Gangliogliomas (GGs), composed of dysmorphic neurons and neoplastic astroglia, represent the most frequent tumor entity associated with chronic recurrent epileptic seizures. So far, a systematic analysis of potential differences in neurochemical profiles of dysmorphic tumoral neurons as well as neurons of the peritumoral microenvironment (PTME) was hampered by the inability to unequivocally differentiate between the distinct neuronal components in human GG biopsies. Here, we have applied a novel GG mouse model that allows to clearly resolve the neurochemical profiles of GG-intrinsic versus PTME neurons. For this purpose, glioneuronal tumors in mice were induced by intraventricular in utero electroporation (IUE) of piggyBac-based plasmids for BRAFV600E and activated Akt (AktT308D/S473D, further referred to as AktDD) and analyzed neurochemically by immunocytochemistry against specific marker proteins. IUE of BRAFV600E/AktDD in mice resulted in tumors with the morphological features of human GGs. Our immunocytochemical analysis revealed a strong reduction of GABAARα1 immunoreactivity in the tumor compared to the PTME. In contrast, the extent of NMDAR1 immunoreactivity in the tumor appeared comparable to the PTME. Interestingly, tumor cells maintained the potential to express both receptors. Fittingly, the abundance of the presynaptic vesicular neurotransmitter transporters VGLUT1 and VGAT was also decreased in the tumor. Additionally, the fraction of parvalbumin and somatostatin nonneoplastic interneurons was reduced. In conclusion, changes in the levels of key proteins in neurotransmitter signaling suggest a loss of synapses and may thereby lead to neuronal network alterations in mouse GGs.

Ste20-like Kinase Is Critical for Inhibitory Synapse Maintenance and Its Deficiency Confers a Developmental Dendritopathy.

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.

CD8+ T-Lymphocyte-Driven Limbic Encephalitis Results in Temporal Lobe Epilepsy.

OBJECTIVE: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved. METHODS: Here, we scrutinized pathogenic consequences emerging from CD8+ T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1-/- mice (termed "OVA-CD8+ LE model"). RESULTS: Viral-mediated antigen transfer caused dense CD8+ T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8+ T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8+ LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8+ T cells specifically targeted OVA-expressing, SIINFEKL-H-2Kb -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8+ T cells escorted by NK cells. INTERPRETATION: These data indicate that a CD8+ T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8+ T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.

Complex effects of eslicarbazepine on inhibitory micro networks in chronic experimental epilepsy.

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.

Polyamine Modulation of Anticonvulsant Drug Response: A Potential Mechanism Contributing to Pharmacoresistance in Chronic Epilepsy.

Despite the development of numerous novel anticonvulsant drugs, ∼30% of epilepsy patients 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 epilepsy. 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 spermine and CBZ both 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 carbamazepine (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.

Effects of eslicarbazepine on slow inactivation processes of sodium channels in dentate gyrus granule cells.

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.

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

OBJECTIVE: 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.

Downregulation of Spermine Augments Dendritic Persistent Sodium Currents and Synaptic Integration after Status Epilepticus.

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 dendritic INaP 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 INaP causes 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 STATEMENT: In 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.

RIM3γ and RIM4γ are key regulators of neuronal arborization.

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/ß and RIM2α/ß, 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 and in vivo resulted 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.

The presynaptic active zone protein RIM1α controls epileptogenesis following status epilepticus.

To ensure operation of synaptic transmission within an appropriate dynamic range, neurons have evolved mechanisms of activity-dependent plasticity, including changes in presynaptic efficacy. The multidomain protein RIM1α is an integral component of the cytomatrix at the presynaptic active zone and has emerged as key mediator of presynaptically expressed forms of synaptic plasticity. We have therefore addressed the role of RIM1α in aberrant cellular plasticity and structural reorganization after an episode of synchronous neuronal activity pharmacologically induced in vivo [status epilepticus (SE)]. Post-SE, all animals developed spontaneous seizure events, but their frequency was dramatically increased in RIM1α-deficient mice (RIM1α(-/-)). We found that in wild-type mice (RIM1α(+/+)) SE caused an increase in paired-pulse facilitation in the CA1 region of the hippocampus to the level observed in RIM1α(-/-) mice before SE. In contrast, this form of short-term plasticity was not further enhanced in RIM1α-deficient mice after SE. Intriguingly, RIM1α(-/-) mice showed a unique pattern of selective hilar cell loss (i.e., endfolium sclerosis), which so far has not been observed in a genetic epilepsy animal model, as well as less severe astrogliosis and attenuated mossy fiber sprouting. These findings indicate that the decrease in release probability and altered short- and long-term plasticity as present in RIM1α(-/-) mice result in the formation of a hyperexcitable network but act in part neuroprotectively with regard to neuropathological alterations associated with epileptogenesis. In summary, our results suggest that presynaptic plasticity and proper function of RIM1α play an important part in a neuron's adaptive response to aberrant electrical activity.

Emergence of nociceptive functionality and opioid signaling in human induced pluripotent stem cell-derived sensory neurons.

ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

Loss of ß1 accessory Na+ channel subunits causes failure of carbamazepine, but not of lacosamide, in blocking high-frequency firing via differential effects on persistent Na+ currents.

PURPOSE: In chronic epilepsy, a substantial proportion of up to 30% of patients remain refractory to antiepileptic drugs (AEDs). An understanding of the mechanisms of pharmacoresistance requires precise knowledge of how AEDs interact with their targets. Many commonly used AEDs act on the transient and/or the persistent components of the voltage-gated Na(+) current (I(NaT) and I(NaP) , respectively). Lacosamide (LCM) is a novel AED with a unique mode of action in that it selectively enhances slow inactivation of fast transient Na(+) channels. Given that functional loss of accessory Na(+) channel subunits is a feature of a number of neurologic disorders, including epilepsy, we examined the effects of LCM versus carbamazepine (CBZ) on the persistent Na(+) current (I(NaP) ), in the presence and absence of accessory subunits within the channel complex. METHODS: Using patch-clamp recordings in intact hippocampal CA1 neurons of Scn1b null mice, I(NaP) was recorded using slow voltage ramps. Application of 100 µm CBZ or 300 µm LCM reduced the maximal I(NaP) conductance in both wild-type and control mice. KEY FINDINGS: As shown previously by our group in Scn1b null mice, CBZ induced a paradoxical increase of I(NaP) conductance in the subthreshold voltage range, resulting in an ineffective block of repetitive firing in Scn1b null neurons. In contrast, LCM did not exhibit such a paradoxical increase, and accordingly maintained efficacy in blocking repetitive firing in Scn1b null mice. SIGNIFICANCE: These results suggest that the novel anticonvulsant LCM maintains activity in the presence of impaired Na(+) channel ß(1) subunit expression and thus may offer an improved efficacy profile compared with CBZ in diseases associated with an impaired expression of ß sub-units as observed in epilepsy.

A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration.

An intriguing question in human embryonic stem cell (hESC) biology is whether these pluripotent cells can give rise to stably expandable somatic stem cells, which are still amenable to extrinsic fate instruction. Here, we present a pure population of long-term self-renewing rosette-type hESC-derived neural stem cells (lt-hESNSCs), which exhibit extensive self-renewal, clonogenicity, and stable neurogenesis. Although lt-hESNSCs show a restricted expression of regional transcription factors, they retain responsiveness to instructive cues promoting the induction of distinct subpopulations, such as ventral midbrain and spinal cord fates. Using lt-hESNSCs as a donor source for neural transplantation, we provide direct evidence that hESC-derived neurons can establish synaptic connectivity with the mammalian nervous system. Combining long-term stability, maintenance of rosette-properties and phenotypic plasticity, lt-hESNSCs may serve as useful tool to study mechanisms of human NSC self-renewal, lineage segregation, and functional in vivo integration.

Characterisation of NLRP3 pathway-related neuroinflammation in temporal lobe epilepsy.

OBJECTIVE: Inflammation of brain structures, in particular the hippocampal formation, can induce neuronal degeneration and be associated with increased excitability manifesting as propensity for repetitive seizures. An increase in the abundance of individual proinflammatory molecules including interleukin 1 beta has been observed in brain tissue samples of patients with pharmacoresistant temporal lobe epilepsy (TLE) and corresponding animal models. The NLRP3-inflammasome, a cytosolic protein complex, acts as a key regulator in proinflammatory innate immune signalling. Upon activation, it leads to the release of interleukin 1 beta and inflammation-mediated neurodegeneration. Transient brain insults, like status epilepticus (SE), can render hippocampi chronically hyperexcitable and induce segmental neurodegeneration. The underlying mechanisms are referred to as epileptogenesis. Here, we have tested the hypothesis that distinct NLRP3-dependent transcript and protein signalling dynamics are induced by SE and whether they differ between two classical SE models. We further correlated the association of NLRP3-related transcript abundance with convulsive activity in human TLE hippocampi of patients with and without associated neurodegenerative damage. METHODS: Hippocampal mRNA- and protein-expression of NLRP3 and associated signalling molecules were analysed longitudinally in pilocarpine- and kainic acid-induced SE TLE mouse models. Complementarily, we studied NLRP3 inflammasome-associated transcript patterns in epileptogenic hippocampi with different damage patterns of pharmacoresistant TLE patients that had undergone epilepsy surgery for seizure relief. RESULTS: Pilocarpine- and kainic acid-induced SE elicit distinct hippocampal Nlrp3-associated molecular signalling. Transcriptional activation of NLRP3 pathway elements is associated with seizure activity but independent of the particular neuronal damage phenotype in KA-induced and in human TLE hippocampi. SIGNIFICANCE: These data suggest highly dynamic inflammasome signalling in SE-induced TLE and highlight a vicious cycle associated with seizure activity. Our results provide promising perspectives for the inflammasome signalling pathway as a target for anti-epileptogenic and -convulsive therapeutic strategies. The latter may even applicable to a particularly broad spectrum of TLE patients with currently pharmacoresistant disease.

An astrocytic signaling loop for frequency-dependent control of dendritic integration and spatial learning.

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 in acute rat and mouse brain slices we found that exogenous D-serine reduced the threshold of dendritic spikes and increased 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 in mice led to an impairment of spatial memory, which demonstrated its behavioral relevance.

Circuit-selective cell-autonomous regulation of inhibition in pyramidal neurons by Ste20-like kinase.

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.

Efficacy loss of the anticonvulsant carbamazepine in mice lacking sodium channel beta subunits via paradoxical effects on persistent sodium currents.

Neuronal excitability is critically determined by the properties of voltage-gated Na(+) currents. Fast transient Na(+) currents (I(NaT)) mediate the fast upstroke of action potentials, whereas low-voltage-activated persistent Na(+) currents (I(NaP)) contribute to subthreshold excitation. Na(+) channels are composed of a pore-forming alpha subunit and beta subunits, which modify the biophysical properties of alpha subunits. We have examined the idea that the presence of beta subunits also modifies the pharmacological properties of the Na(+) channel complex using mice lacking either the beta(1) (Scn1b) or beta(2) (Scn2b) subunit. Classical effects of the anticonvulsant carbamazepine (CBZ), such as the use-dependent reduction of I(NaT) and effects on I(NaT) voltage dependence of inactivation, were unaltered in mice lacking beta subunits. Surprisingly, CBZ induced a small but significant shift of the voltage dependence of activation of I(NaT) and I(NaP) to more hyperpolarized potentials. This novel CBZ effect on I(NaP) was strongly enhanced in Scn1b null mice, leading to a pronounced increase of I(NaP) within the subthreshold potential range, in particular at low CBZ concentrations of 10-30 microm. A combination of current-clamp and computational modeling studies revealed that this effect causes a complete loss of CBZ efficacy in reducing repetitive firing. Thus, beta subunits modify not only the biophysical but also the pharmacological properties of Na(+) channels, in particular with respect to I(NaP). Consequently, altered expression of beta subunits in other neurological disorders may cause altered neuronal sensitivity to drugs targeting Na(+) channels.

Stable mossy fiber long-term potentiation requires calcium influx at the granule cell soma, protein synthesis, and microtubule-dependent axonal transport.

The synapses formed by the mossy fiber (MF) axons of hippocampal dentate gyrus granule neurons onto CA3 pyramidal neurons exhibit an intriguing form of experience-dependent synaptic plasticity that is induced and expressed presynaptically. In contrast to most other CNS synapses, long-term potentiation (LTP) at the MF-CA3 synapse is readily induced even during blockade of postsynaptic glutamate receptors. Furthermore, blocking voltage-gated Ca(2+) channels prevents MF-LTP, supporting an involvement of presynaptic Ca(2+) signaling via voltage-gated Ca(2+) channels in MF-LTP induction. We examined the contribution of activity in both dentate granule cell somata and MF terminals to MF-LTP. We found that the induction of stable MF-LTP requires tetanization-induced action potentials not only at MF boutons, but also at dentate granule cell somata. Similarly, blocking Ca(2+) influx via voltage-gated Ca(2+) channels only at the granule cell soma was sufficient to disrupt MF-LTP. Finally, blocking protein synthesis or blocking fast axonal transport mechanisms via disruption of axonal tubulin filaments resulted in decremental MF-LTP. Collectively, these data suggest that-in addition to Ca(2+) influx at the MF terminals-induction of MF synaptic plasticity requires action potential-dependent Ca(2+) signaling at granule cell somata, protein synthesis, and fast axonal transport along MFs. A parsimonious interpretation of these results is that somatic activity triggers protein synthesis at the soma; newly synthesized proteins are then transported to MF terminals, where they contribute to the stabilization of MF-LTP. Finally, the present data imply that synaptic plasticity at the MF-CA3 synapse can be affected by local modulation of somatic and presynaptic Ca(2+) channel activity.

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.

Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo.

The controlled in vitro differentiation of human embryonic stem cells (hESCs) and other pluripotent stem cells provides interesting prospects for generating large numbers of human neurons for a variety of biomedical applications. A major bottleneck associated with this approach is the long time required for hESC-derived neural cells to give rise to mature neuronal progeny. In the developing vertebrate nervous system, Notch signaling represents a key regulator of neural stem cell (NSC) maintenance. Here, we set out to explore whether this signaling pathway can be exploited to modulate the differentiation of hESC-derived NSCs (hESNSCs). We assessed the expression of Notch pathway components in hESNSCs and demonstrate that Notch signaling is active under self-renewing culture conditions. Inhibition of Notch activity by the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) in hESNSCs affects the expression of human homologues of known targets of Notch and of several cell cycle regulators. Furthermore, DAPT-mediated Notch inhibition delays G1/S-phase transition and commits hESNSCs to neurogenesis. Combined with growth factor withdrawal, inhibition of Notch signaling results in a marked acceleration of differentiation, thereby shortening the time required for the generation of electrophysiologically active hESNSC-derived neurons. This effect can be exploited for neural cell transplantation, where transient Notch inhibition before grafting suffices to promote the onset of neuronal differentiation of hESNSCs in the host tissue. Thus, interference with Notch signaling provides a tool for controlling human NSC differentiation both in vitro and in vivo.