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Computation within cortical microcircuits is determined by functional properties of the neurons and their synaptic interactions. While heterogeneity of inhibitory interneurons is well established, the anatomical, physiological, and molecular differentiation of excitatory pyramidal neurons is not fully resolved. To identify functional subtypes within the pyramidal neuron population, we focused on human layer 2-3 cortex which greatly expanded during evolution. We performed multi-neuron patch-clamp recordings in brain slices from the temporal cortex of 22 epilepsy patients. We characterized the electrophysiological properties of up to 80 pyramidal neurons per patient, enabling us to assess inter- and intra-individual functional variability. Hierarchical clustering of the high-dimensional parameter space yielded functionally distinct clusters of pyramidal neurons which were present across individuals. This may represent a generic organizational principle converging with previously described transcriptomic heterogeneity. We further observed substantial heterogeneity in physiological parameters with intra-individual variability being severalfold larger than inter-individual variability. The phenotypic variability within and across pyramidal neuron subtypes has important implications for the computational capacity of the cortical microcircuit.

Background and purpose The KCNQ2 gene encodes for the Kv 7.2 subunit of non-inactivating potassium channels. KCNQ2-related diseases range from autosomal dominant neonatal self-limited epilepsy, often caused by KCNQ2 haploinsufficiency, to severe encephalopathies caused by KCNQ2 missense variants. In vivo and in vitro effects of the sodium channel blocker eslicarbazepine acetate (ESL) and eslicarbazepine metabolite (S-Lic) in a mouse model of self-limited neonatal epilepsy as a first attempt to assess the utility of ESL in the KCNQ2 disease spectrum was investigated. Experimental approach Effects of S-Lic on in vitro physiological and pathological hippocampal neuronal activity in slices from mice carrying a heterozygous deletion of Kcnq2 (Kcnq2+/- ) and Kcnq2+/+ mice were investigated. ESL in vivo efficacy was investigated in the 6-Hz psychomotor seizure model in both Kcnq2+/- and Kcnq2+/+ mice. Key results S-Lic increased the amplitude and decreased the incidence of physiological sharp wave-ripples in a concentration-dependent manner and slightly decreased gamma oscillations frequency. 4-Aminopyridine-evoked seizure-like events were blocked at high S-Lic concentrations and substantially reduced in incidence at lower concentrations. These results were not different in Kcnq2+/+ and Kcnq2+/- mice, although the EC50 estimation implicated higher efficacy in Kcnq2+/- animals. In vivo, Kcnq2+/- mice had a lower seizure threshold than Kcnq2+/+ mice. In both genotypes, ESL dose-dependently displayed protection against seizures. Conclusions and implications S-Lic slightly modulates hippocampal oscillations and blocks epileptic activity in vitro and in vivo. Our results suggest that the increased excitability in Kcnq2+/- mice is effectively targeted by S-Lic high concentrations, presumably by blocking diverse sodium channel subtypes.

Despite its decades' long therapeutic use in psychiatry, the biological mechanisms underlying lithium's mood-stabilizing effects have remained largely elusive. Here, we investigated the effect of lithium on tryptophan breakdown via the kynurenine pathway using immortalized human microglia cells, primary human microglia isolated from surgical specimens, and microglia-like cells differentiated from human induced pluripotent stem cells. Interferon (IFN)-gamma, but not lipopolysaccharide, was able to activate immortalized human microglia, inducing a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3 beta(S9) phosphorylation and reducing STAT1(S727) and STAT3(Y705) phosphorylation levels in IFN-gamma treated cells. Studies in primary human microglia and hiPSC-derived microglia confirmed the anti-inflammatory effects of lithium, highlighting that IDO activity is reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1(S727) and P-STAT3(Y705). Primary human microglia differed from immortalized human microglia and hiPSC derived microglia-like cells in their strong sensitivity to LPS, resulting in robust upregulation of IDO1 and anti-inflammatory cytokine IL-10. While lithium again decreased IDO1 activity in primary cells, it further increased release of IL-10 in response to LPS. Taken together, our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.

Epileptiform activity alters gene expression in the central nervous system, a phenomenon that has been studied extensively in animal models. Here, we asked whether also in vitro models of seizures are in principle suitable to investigate changes in gene expression due to epileptiform activity and tested this hypothesis mainly in rodent and additionally in some human brain slices. We focused on three genes relevant for seizures and epilepsy: FOS proto-oncogene (c-Fos), inducible cAMP early repressor (Icer) and mammalian target of rapamycin (mTor). Seizure-like events (SLEs) were induced by 4-aminopyridine (4-AP) in rat entorhinal-hippocampal slices and by 4-AP/8 mM potassium in human temporal lobe slices obtained from surgical treatment of epilepsy. SLEs were monitored simultaneously by extracellular field potentials and intrinsic optical signals (IOS) for 1–4 h, mRNA expression was quantified by real time PCR. In rat slices, both duration of SLE exposure and SLE onset region were associated with increased expression of c-Fos and Icer while no such association was shown for mTor expression. Similar to rat slices, c-FOS induction in human tissue was increased in slices with epileptiform activity. Our results indicate that irrespective of limitations imposed by ex vivo conditions, in vitro models represent a suitable tool to investigate gene expression. Our finding is of relevance for the investigation of human tissue that can only be performed ex vivo. Specifically, it presents an important prerequisite for future studies on transcriptome-wide and cell-specific changes in human tissue with the goal to reveal novel candidates involved in the pathophysiology of epilepsy and possibly other CNS pathologies.

Synthetic receptor biology and genome editing are emerging techniques, both of which are currently beginning to be used in preclinical and clinical applications. We were interested in whether a combination of these techniques approaches would allow for the generation of a novel type of reporter cell that would recognize transient cellular events through specifically designed synthetic receptors and would permanently store information about these events via associated gene editing. Reporting cells could be used in the future to detect alterations in the cellular microenvironment, including degenerative processes or malignant transformation into cancer cells. Here, we explored synthetic Notch (synNotch) receptors expressed in human embryonic kidney cells to investigate the efficacy of antigen recognition events in a time- and dose-dependent manner. First, we evaluated the most suitable conditions for synNotch expression based on dsRed-Express fluorophore expression. Then, we used a synNotch receptor coupled to transcriptional activators to induce the expression of a Cas9 nuclease targeted to a specific genomic DNA site. Our data demonstrate that recognition of various specific antigens via synNotch receptors robustly induced Cas9 expression and resulted in an indel formation frequency of 34.5%&ndash, 45.5% at the targeted CXCR4 locus. These results provide proof of concept that reporter cells can be designed to recognize a given event and to store transient information permanently in their genomes.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The active electrical properties of dendrites shape neuronal input and output and are fundamental to brain function. However, our knowledge of active dendrites has been almost entirely acquired from studies of rodents. In this work, we investigated the dendrites of layer 2 and 3 (L2/3) pyramidal neurons of the human cerebral cortex ex vivo. In these neurons, we discovered a class of calcium-mediated dendritic action potentials (dCaAPs) whose waveform and effects on neuronal output have not been previously described. In contrast to typical all-or-none action potentials, dCaAPs were graded; their amplitudes were maximal for threshold-level stimuli but dampened for stronger stimuli. These dCaAPs enabled the dendrites of individual human neocortical pyramidal neurons to classify linearly nonseparable inputs—a computation conventionally thought to require multilayered networks., This is the author's version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science, 03 Jan 2020: Vol. 367, Issue 6473, pp. 83-87 (DOI: 10.1126/science.aax6239). We thank U. Schneider for providing the human tissue; L. Kraus, A. Ragot, O. Kruchik, and I. Wolter for assisting with human tissue processing; and S. Grosser and F. J. Barreda Tomás for assisting with confocal imaging. This work was supported by Deutsche Forschungsgemeinschaft DFG: 2112280105 (to T.A.Z.), EXC 257 (to I.V., P.P., and M.E.L.), FOR 2143 (to I.V.), EXC 2049 (to P.F.), LA 3442/3-1 (to M.E.L.), SPP1665 (to M.E.L.), and SFB1078 B2 (to M.E.L.); 7FP Health-F2-602531-2013 DESIRE (to M.H.); Hellenic Foundation for Research and Innovation HFRI and the General Secretariat for Research and 15 Technology GSRT 1357 (to A.P.); Humboldt Foundation Friedrich Wilhelm Bessel Research Award (to P.P.); H2020 European Research Council ERC STG 311435 (to P.P.); H2020 Research and Innovation Programme 720270/HBP SGA1, 785907/HBP SGA2, and 670118/ERC ActiveCortex (to M.E.L.); and EMBO ALTF 1144-2012 (to A.G.)

Das Verstandnis pathophysiologischer Grundlagen der Entstehung von epileptischen Anfallen und Epilepsien ist Voraussetzung fur die Entwicklung neuer Therapiekonzepte. Aus ethischen Grunden sind der Forschung an Menschen enge Grenzen gesetzt, daher muss haufig auf Tiermodelle zuruckgegriffen werden. Die Entstehung eines akut symptomatischen Anfalls ist – in Abhangigkeit von der Ursache – in der Regel auf eine reversible Verschiebung im Verhaltnis von Inhibition zu Exzitation zuruckzufuhren. Bei Epilepsien stehen eher die Mechanismen, die zur Epileptogenese beigetragen haben, im Vordergrund. Diese fuhren uber atiologieabhangige Wege zu einer reduzierten Anfallsschwelle, die das Auftreten von unprovozierten Anfallen erleichtert. Tiermodelle sollen zur Erforschung pathophysiologischer Mechanismen beitragen, sie dienen zudem zum Screening von neuen potenziell antiepileptisch wirksamen Substanzen. Kein Modell ist ideal, es werden je nach Fragestellung unterschiedliche Modelle eingesetzt.

Malformations of cortical development (MCD) represent a group of rare diseases with severe clinical presentation as epileptic and pharmacoresistant encephalopathies. Morphological studies in tissue from MCD patients have revealed reduced GABAergic efficacy and increased intracellular chloride concentration in neuronal cells as important pathophysiological mechanisms in MCD. Also, in various animal models, alterations of GABAergic inhibition have been postulated as a predominant factor contributing to perilesional hyperexcitability. Along with this line, the NKCC1 inhibitor bumetanide has been postulated as a potential drug for treatment of epilepsy, mediating its antiepileptic effect by reduction of the intracellular chloride and increased inhibitory efficacy of GABAergic transmission. In the present study, we focused on the focal freeze-lesion model of MCD to compare antiepileptic drugs with distinct mechanisms of action, including NKCC1 inhibition by bumetanide. For this purpose, we combined electrophysiological and optical methods in slice preparations and assessed the properties of seizure like events (SLE) induced by 4-aminopyridine. In freeze-lesioned but not control slices, SLE onset was confined to the perilesional area, confirming that this region is hyperexcitable and likely triggers pathological activity. Bumetanide selectively reduced epileptic activity in lesion-containing slices but not in slices from sham-treated control rats. Moreover, bumetanide caused a shift in the SLE onset site away from the perilesional area. In contrast, effects of other antiepileptic drugs including carbamazepine, lacosamide, acezatolamide and zonisamide occurred mostly independently of the lesion and did not result in a shift of the onset region. Our work adds evidence for the functional relevance of chloride homeostasis in the pathophysiology of microgyrus formation as represented in the focal freeze-lesion model. Further studies in different MCD models and human tissue will be required to validate the effects across different MCD subtypes and species and to assess the translational value of our findings.

The dentate gyrus (DG) as part of the hippocampal formation is believed to serve as a gatekeeper with strong inhibitory properties against uncontrolled propagation of neuronal activity from the entorhinal cortex and neocortical structures. In temporal lobe epilepsy, the DG becomes hyperexcitable and loses its gate function, enabling propagation of ictal activity into downstream structures such as CA3 and CA1 areas. Furthermore, the DG, apart from facilitating propagation, may also be able to autonomously generate ictal activity, but this point has remained open so far. To tackle this question, we used intrinsic optical imaging in combination with electrophysiological recordings in brain slice preparations from rats in which status epilepticus had been induced electrically several weeks prior to measurements. Upon omission of Mg++ from the artificial cerebrospinal fluid, in 15 out of 33 slices (45.4%) from 9 out of 13 epileptic animals (69.2%), spontaneous and autonomous ictal activity, mostly seizure-like events (SLE), was observed in the DG. This activity manifested independently from SLE generated in adjacent cortices and never occurred in slices from control animals. SLE generated in the DG differed from those with origin in the entorhinal or temporal cortex by longer latency to the first event after Mg++ omission (p

Objective Maternal autoantibodies are a risk factor for impaired brain development in offspring. Antibodies (ABs) against the NR1 (GluN1) subunit of the N-methyl-d-aspartate receptor (NMDAR) are among the most frequently diagnosed anti-neuronal surface ABs, yet little is known about effects on fetal development during pregnancy. Methods We established a murine model of in utero exposure to human recombinant NR1 and isotype-matched nonreactive control ABs. Pregnant C57BL/6J mice were intraperitoneally injected on embryonic days 13 and 17 each with 240μg of human monoclonal ABs. Offspring were investigated for acute and chronic effects on NMDAR function, brain development, and behavior. Results Transferred NR1 ABs enriched in the fetus and bound to synaptic structures in the fetal brain. Density of NMDAR was considerably reduced (up to -49.2%) and electrophysiological properties were altered, reflected by decreased amplitudes of spontaneous excitatory postsynaptic currents in young neonates (-34.4%). NR1 AB-treated animals displayed increased early postnatal mortality (+27.2%), impaired neurodevelopmental reflexes, altered blood pH, and reduced bodyweight. During adolescence and adulthood, animals showed hyperactivity (+27.8% median activity over 14 days), lower anxiety, and impaired sensorimotor gating. NR1 ABs caused long-lasting neuropathological effects also in aged mice (10 months), such as reduced volumes of cerebellum, midbrain, and brainstem. Interpretation The data collectively support a model in which asymptomatic mothers can harbor low-level pathogenic human NR1 ABs that are diaplacentally transferred, causing neurotoxic effects on neonatal development. Thus, AB-mediated network changes may represent a potentially treatable neurodevelopmental congenital brain disorder contributing to lifelong neuropsychiatric morbidity in affected children. ANN NEUROL 2019;86:656-670.

Background In patients taking antiepileptic drugs (AEDs) for epilepsy, adverse effects (AEs) often lead to unfavorable quality of life, impaired adherence, and, eventually, discontinuation of pharmacological treatment. In a true-to-life sample of subjects from our academic epilepsy outpatient clinic, we aimed to identify predictors for overall high AE burden and for specific AEs focusing on patients on monotherapy. Methods All patients ≥ 16 years of age with epilepsy for ≥ 12 months were routinely asked to complete the Liverpool Adverse Event Profile (LAEP) just before their appointment. Demographic, epilepsy, and treatment variables were derived from our comprehensive outpatient database. Results Out of 841 patients, 438 (61% female, mean age: 44.7 ± 17.1 years) on monotherapy were included in this study. Levetiracetam (n = 151), lamotrigine (n = 167), valproic acid (n = 73), or controlled-release carbamazepine (n = 47) were the most commonly used antiepileptic drugs (AEDs). Independent predictors for general high AE burden (LAEP score ≥ 45) were duration of epilepsy, lack of 12-month seizure freedom, and partial epilepsy, but none of the four individual AEDs. The most frequent LAEP-defined specific AEs were sleepiness, difficulty concentrating, tiredness, and memory problems. The three most frequent independent predictors for each of the 19 AEs were lack of 12-month seizure freedom (13/19 AEs), individual AED (7/19 AEs), and partial epilepsy (6/19 AEs). Levetiracetam was independently associated with anger/aggression, nervousness/agitation, upset stomach, depression, and sleep disturbance; lamotrigine with nervousness/agitation, upset stomach, and difficulty concentrating; and valproic acid with upset stomach and shaky hands. Conclusion Individual AEDs independently predicted some specific AEs, but not overall high AE burden. Our findings may help to characterize patients with epilepsy who are at high risk for specific AEs. Dose reduction or change to another AED may reduce LAEP score and potential nonadherence.

Human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry Böttcher C, Schlickeiser S, Sneeboer MAM, et al. Nat Neurosci. 2019;22(1):78-90.Microglia, the specialized innate immune cells of the central nervous system, play crucial roles in neural development and function. Different phenotypes and functions have been ascribed to rodent microglia, but little is known about human microglia (huMG) heterogeneity. Difficulties in procuring huMG and their susceptibility to cryopreservation damage have limited large-scale studies. Here we applied multiplexed mass cytometry for a comprehensive characterization of postmortem huMG (103-104 cells). We determined expression levels of 57 markers on huMG isolated from up to 5 different brain regions of 9 donors. We identified the phenotypic signature of huMG, which was distinct from peripheral myeloid cells but was comparable to fresh huMG. We detected microglia regional heterogeneity using a hybrid workflow combining Cytobank and R/Bioconductor for multidimensional data analysis. Together, these methodologies allowed us to perform high-dimensional, large-scale Immunophenotyping of huMG at the single-cell level, which facilitates their unambiguous profiling in health and disease.

Microglia, the specialized innate immune cells of the CNS, play crucial roles in neural development and function. Different phenotypes and functions have been ascribed to rodent microglia, but little is known about human microglia (huMG) heterogeneity. Difficulties in procuring huMG and their susceptibility to cryopreservation damage have limited large-scale studies. Here we applied multiplexed mass cytometry for a comprehensive characterization of postmortem huMG (103 – 104 cells). We determined expression levels of 57 markers on huMG isolated from up to five different brain regions of nine donors. We identified the phenotypic signature of huMG, which was distinct from peripheral myeloid cells but was comparable to fresh huMG. We detected microglia regional heterogeneity using a hybrid workflow combining Cytobank and R/Bioconductor for multidimensional data analysis. Together, these methodologies allowed us to perform high-dimensional, large-scale immunophenotyping of huMG at the single-cell level, which facilitates their unambiguous profiling in health and disease.

Background and Purpose KCNQ2-5 channels are voltage-gated potassium channels that regulate neuronal excitability and represent suitable targets for the treatment of hyperexcitability disorders. The effect of Chlor-N-(6-chlor-pyridin-3-yl)-benzamid was tested on KCNQ subtypes for its ability to alter neuronal excitability and for its anticonvulsant potential. Experimental Approach The effect of 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid was evaluated using whole-cell voltage-clamp recordings from CHO cells and Xenopus laevis oocytes expressing different types of KCNQ channels. Epileptiform afterdischarges were recorded in fully amygdala-kindled rats in vivo. Neuronal excitability was assessed using field potential and whole cell recording in rat hippocampus in vitro. Key Results 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid caused a hyperpolarizing shift of the activation curve and a pronounced slowing of deactivation in KCNQ2-mediated currents, whereas KCNQ3/5 heteromers remained unaffected. The effect was also apparent in the Retigabine-insensitive mutant KCNQ2-W236L. In fully amygdala-kindled rats, it elevated the threshold for induction of afterdischarges and reduced seizure severity and duration. In hippocampal CA1 cells, 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid strongly damped neuronal excitability caused by a membrane hyperpolarization and a decrease in membrane resistance and induced an increase of the somatic resonance frequency on the single cell level, whereas synaptic transmission was unaffected. On the network level, 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid caused a significant reduction of γ and θ oscillation peak power, with no significant change in oscillation frequency. Conclusion and Implications Our data indicate that 4-Chlor-N-(6-chlor-pyridin-3-yl)-benzamid is a potent KCNQ activator with a selectivity for KCNQ2 containing channels. It strongly reduces neuronal excitability and displays anticonvulsant activity in vivo.

The subiculum (Sub) as a part of the hippocampal formation is thought to play a functional role in learning and memory. In addition to its major input from CA1 pyramidal cells, the subiculum receives input from the entorhinal cortex (EC) via the temporoammonic pathway. Thus far, synaptic plasticity in the subiculum was mainly investigated at CA1–Sub synapses. According to their spiking pattern, pyramidal cells in the subiculum were classified as bursting cells and non-bursting cells. In the present study, we demonstrate that subicular bursting cells show input-specific forms of long-term potentiation (LTP). At CA1–Sub synapses, bursting cells have been shown to express a presynaptic NMDA receptor-dependent LTP that depends on the activation of a cAMP–PKA cascade (Wozny et al., Journal of Physiology 2008). In contrast, at EC–Sub synapses the induction of LTP in bursting cells shows a high induction-threshold and relies on the activation of postsynaptic NMDA receptors, postsynaptic depolarization and postsynaptic Ca2+ influx. Each form of LTP is input-specific and fails to induce heterosynaptic plasticity. Taken together, our data suggest that distinct, input-specific mechanisms govern high frequency-induced LTP at subicular bursting cells' synapses.

Canonical olfactory signal transduction involves the activation of cyclic AMP-activated cation channels that depolarize the cilia of receptor neurons and raise intracellular calcium. Calcium then activates Cl(-) currents that may be up to tenfold larger than cation currents and are believed to powerfully amplify the response. We identified Anoctamin2 (Ano2, also known as TMEM16B) as the ciliary Ca(2+)-activated Cl(-) channel of olfactory receptor neurons. Ano2 is expressed in the main olfactory epithelium (MOE) and in the vomeronasal organ (VNO), which also expresses the related Ano1 channel. Disruption of Ano2 in mice virtually abolished Ca(2+)-activated Cl(-) currents in the MOE and VNO. Ano2 disruption reduced fluid-phase electro-olfactogram responses by only ∼40%, did not change air-phase electro-olfactograms and did not reduce performance in olfactory behavioral tasks. In contrast with the current view, cyclic nucleotide-gated cation channels do not need a boost by Cl(-) channels to achieve near-physiological levels of olfaction.

The hippocampus plays a central role in memory formation in the mammalian brain. The subiculum is the principal target of CA1 pyramidal cells and thus serves as the major relay station for the outgoing hippocampal information. Pyramidal cells in the subiculum have been classified as burst-spiking (BS) and regular-spiking (RS) cells. In this study we demonstrate that application of the β-adrenergic agonist isoproterenol (2 μM) induces a chemical form of long-term potentiation (LTP) of responses to alvear stimulation in (BS) but not in (RS) cells. This effect is prevented by the β-adrenergic receptor antagonist propranolol (2 μM). The isoproterenol-induced LTP in (BS) cells does not depend on postsynaptic Ca(2+)-signaling, as 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) does not prevent its induction. Furthermore, paired-pulse facilitation (PPF) and coefficient of variation (CV) analysis indicate the site of the LTP expression to be presynaptic. Our findings show that activation of β-adrenergic receptors (β-ARs) at CA1-subiculum synapses induces a cell-type-specific form of chemical LTP in subicular (BS) cells that may allow a target-specific trafficking of hippocampal output.

Encoding of novel information has been proposed to rely on the time-locked release of dopamine in the hippocampal formation during novelty detection. However, the site of novelty detection in the hippocampus remains a matter of debate. According to current models, the CA1 and the subiculum act as detectors and distributors of novel sensory information. Although most CA1 pyramidal neurons exhibit regular-spiking behavior, the majority of subicular pyramidal neurons fire high-frequency bursts of action potentials. The present study investigates the efficacy of dopamine D1/D5 receptor activation to facilitate the induction of activity-dependent long-term potentiation (LTP) in rat CA1 regular-spiking and subicular burst-spiking pyramidal cells. Using a weak stimulation protocol, set at a level subthreshold for the induction of LTP, we show that activation of D1/D5 receptors for 5-10 min facilitates LTP in subicular burst-spiking neurons but not in CA1 neurons. The results demonstrate that D1/D5 receptor-facilitated LTP is NMDA receptor-dependent, and requires the activation of protein kinase A. In addition, the D1/D5 receptor-facilitated LTP is shown to be presynaptically expressed and relies on presynaptic Ca 2+ signaling. The phenomenon of dopamine-induced facilitation of presynaptic NMDA receptor-dependent LTP in subicular burst-spiking pyramidal cells is in accordance with observations of the time-locked release of dopamine during novelty detection in this brain region, and reveals an intriguing mechanism for the encoding of hippocampal output information.

Members of the CLC protein family of Cl(-) channels and transporters display the remarkable ability to function as either chloride channels or Cl(-)/H(+) antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl(-)/H(+) exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H(+) transport was abolished by mutations in either the "gating" or "proton" glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl(-)I(-) conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the "anion-coordinating" tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO(3)(-) conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl(-)/H(+) antiporter.

The subiculum (Sub) is the principal target of CA1 pyramidal cells. It serves as the final relay of hippocampal output and thus mediates hippocampal–cortical interaction. In addition, the Sub receives direct input from the entorhinal cortex via the temporoammonic pathway. In this study, we demonstrate that low-frequency stimulation of the temporoammonic pathway results in the disinhibition of excitatory synaptic transmission at CA1-Sub synapses. We provide evidence that this disinhibition is mediated by an NMDA receptor-dependent long-term depression (LTD) of GABAergic inhibition. This mechanism might bear physiological significance for the stabilization and processing of mnemonic information at hippocampal output synapses and underpins the functional role of hippocampal–entorhinal interaction in memory formation. © 2010 Wiley-Liss, Inc.

The subiculum is the principal target of CA1 pyramidal cells. It functions as a mediator of hippocampal-cortical interaction and has been proposed to play an important role in the encoding and retrieval of long-term memory. The cellular mechanisms of memory formation are thought to include long-term potentiation (LTP) and depression (LTD) of synaptic strength. This review summarizes the contemporary knowledge of LTP and LTD at CA1-subiculum synapses. The observation that the underlying mechanisms of LTP and LTD at CA1-subiculum synapses correlate with the discharge properties of subicular pyramidal cell reveals a novel and intriguing mechanism of cell-specific consolidation of hippocampal output.

Hippocampal output is mediated via the subiculum, which is the principal target of CA1 pyramidal cells, and which sends projections to a variety of cortical and subcortical regions. Pyramidal cells in the subiculum display two different firing modes and are classified as being burst-spiking or regular-spiking. In a previous study, we found that low-frequency stimulation induces an NMDA receptor-dependent long-term depression (LTD) in burst-spiking cells and a metabotropic glutamate receptor-dependent long-term potentiation (LTP) in regular-spiking cells [P. Fidzinski, O. Shor, J. Behr, Target-cell-specific bidirectional synaptic plasticity at hippocampal output synapses, Eur. J. Neurosci., 27 (2008) 1111-1118]. Here, we present evidence that this bidirectional plasticity relies upon the co-activation of muscarinic acetylcholine receptors, as scopolamine blocks synaptic plasticity in both cell types. In addition, we demonstrate that the L-type calcium channel inhibitor nifedipine converts LTD to LTP in burst-spiking cells and LTP to LTD in regular-spiking cells, indicating that the polarity of synaptic plasticity is modulated by voltage-gated calcium channels. Bidirectional synaptic plasticity in subicular cells therefore appears to be governed by a complex signaling system, involving cell-specific recruitment of ligand and voltage-gated ion channels as well as metabotropic receptors. This complex regulation might be necessary for fine-tuning of synaptic efficacy at hippocampal output synapses.

cAMP is a critical second messenger involved in synaptic transmission and synaptic plasticity. Here, we show that activation of the adenylyl cyclase by forskolin and application of the cAMP-analog Sp-5,6-DCl-cBIMPS both mimicked and occluded tetanus-induced long-term potentiation (LTP) in subicular bursting neurons, but not in subicular regular firing cells. Furthermore, LTP in bursting cells was inhibited by protein kinase A (PKA) inhibitors Rp-8-CPT-cAMP and H-89. Variations in the degree of EPSC blockade by the low-affinity competitive AMPA receptor-antagonist γ-d-glutamyl-glycine (γ-DGG), analysis of the coefficient of variance as well as changes in short-term potentiation suggest an increase of glutamate concentration in the synaptic cleft after expression of LTP. We conclude that presynaptic LTP in bursting cells requires activation of PKA by a calcium-dependent adenylyl cyclase while LTP in regular firing cells is independent of elevated cAMP levels. Our results provide evidence for a differential role of cAMP in LTP at hippocampal output synapses.

It is commonly accepted that the hippocampus is critically involved in the explicit memory formation of mammals. The subiculum is the principal target of CA1 pyramidal cells and thus serves as the major relay station for the outgoing hippocampal information. Pyramidal cells in the subiculum can be classified according to their firing properties into burst-spiking and regular-spiking cells. In the present study we demonstrate that burst-spiking and regular-spiking cells show fundamentally different forms of low frequency-induced synaptic plasticity in rats. In burst-spiking cells, low-frequency stimulation (at 0.5-5 Hz) induces frequency-dependent long-term depression (LTD) with a maximum at 1 Hz. This LTD is dependent on the activation of NMDAR and masks an mGluR-dependent long-term potentiation (LTP). In contrast, in regular-spiking cells low-frequency stimulation induces an mGluR-dependent LTP that masks an NMDAR-dependent LTD. Both processes depend on postsynaptic Ca(2+)-signaling as BAPTA prevents the induction of synaptic plasticity in both cell types. Thus, mGluR-dependent LTP and NMDAR-dependent LTD occur simultaneously at CA1-subiculum synapses and the predominant direction of synaptic plasticity relies on the cell type investigated. Our data indicate a novel mechanism for the sliding-threshold model of synaptic plasticity, in which induction of LTP and LTD seems to be driven by the relative activation state of NMDAR and mGluR. Our observation that the direction of synaptic plasticity correlates with the discharge properties of the postsynaptic cell reveals a novel and intriguing mechanism of target specificity that may serve in tuning the significance of neuronal information by trafficking hippocampal output onto either subicular burst-spiking or regular-spiking cells.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) K(+) channels dampen neuronal excitability and their functional impairment may lead to epilepsy. Less is known about KCNQ5 (Kv7.5), which also displays wide expression in the brain. Here we show an unexpected role of KCNQ5 in dampening synaptic inhibition and shaping network synchronization in the hippocampus. KCNQ5 localizes to the postsynaptic site of inhibitory synapses on pyramidal cells and in interneurons. Kcnq5(dn/dn) mice lacking functional KCNQ5 channels display increased excitability of different classes of interneurons, enhanced phasic and tonic inhibition, and decreased electrical shunting of inhibitory postsynaptic currents. In vivo, loss of KCNQ5 function leads to reduced fast (gamma and ripple) hippocampal oscillations, altered gamma-rhythmic discharge of pyramidal cells and impaired spatial representations. Our work demonstrates that KCNQ5 controls excitability and function of hippocampal networks through modulation of synaptic inhibition.

The response of living cells of the trabecular meshwork to synthetic ion channels is described. The THF-gramicidin hybrids THF-gram and THF-gram-TBDPS as well as a linked gA-TBDPS and gramicidin A were applied to cultured ocular trabecular meshwork cells. THF-gram application (minimal concentration, 10(-8) M; saturation, 10(-7) M) led to an additional conductance which displayed characteristics of weak Eisenman-I-selective cation channels, no cell destruction, an asymmetric change of the inward/outward currents, and higher current densities using Cs(+) as charge carrier compared to Na(+) and K(+). Linked-gA-TBDPS showed at 10(-12) M increases of the membrane conductance comparable to gA at 10(-7) M and a much faster response of the cells. Thus, THF-gramicidin hybrids form a basis for the use of synthetic ion channels in biological systems, which eventually may lead to new therapeutic approaches.

Encoding of novel information has been proposed to rely on the time-locked release of dopamine in the hippocampal formation during novelty detection. However, the site of novelty detection in the hippocampus remains a matter of debate. According to current models, the CA1 and the subiculum act as detectors and distributors of novel sensory information. Although most CA1 pyramidal neurons exhibit regular-spiking behavior, the majority of subicular pyramidal neurons fire high-frequency bursts of action potentials. The present study investigates the efficacy of dopamine D1/D5 receptor activation to facilitate the induction of activity-dependent long-term potentiation (LTP) in rat CA1 regular-spiking and subicular burst-spiking pyramidal cells. Using a weak stimulation protocol, set at a level subthreshold for the induction of LTP, we show that activation of D1/D5 receptors for 5-10 min facilitates LTP in subicular burst-spiking neurons but not in CA1 neurons. The results demonstrate that D1/D5 receptor-facilitated LTP is NMDA receptor-dependent, and requires the activation of protein kinase A. In addition, the D1/D5 receptor-facilitated LTP is shown to be presynaptically expressed and relies on presynaptic Ca(2+) signaling. The phenomenon of dopamine-induced facilitation of presynaptic NMDA receptor-dependent LTP in subicular burst-spiking pyramidal cells is in accordance with observations of the time-locked release of dopamine during novelty detection in this brain region, and reveals an intriguing mechanism for the encoding of hippocampal output information.

Clinical and experimental evidence suggest that the subiculum plays an important role in the maintenance of temporal lobe seizures. Using the pilocarpine-model of temporal lobe epilepsy (TLE), the present study examines the vulnerability of GABAergic subicular interneurons to recurrent seizures and determines its functional implications. In the subiculum of pilocarpine-treated animals, the density of glutamic acid decarboxylase (GAD) mRNA-positive cells was reduced in all layers. Our data indicate a substantial loss of parvalbumin-immunoreactive neurons in the pyramidal cell and molecular layer whereas calretinin-immunoreactive cells were predominantly reduced in the molecular layer. Though the subiculum of pilocarpine-treated rats showed an increased intensity of GAD65 immunoreactivity, the density of GAD65 containing synaptic terminals in the pyramidal cell layer was decreased indicating an increase in the GAD65 intensity of surviving synaptic terminals. We observed a decrease in evoked inhibitory post-synaptic currents that mediate dendritic inhibition as well as a decline in the frequency of miniature inhibitory post-synaptic currents (mIPSCs) that are restricted to the perisomatic region. The decrease in mIPSC frequency (-30%) matched with the reduced number of perisomatic GAD-positive terminals (-28%) suggesting a decrease of pre-synaptic GABAergic input onto pyramidal cells in epileptic animals. Though cell loss in the subiculum has not been considered as a pathogenic factor in human and experimental TLE, our data suggest that the vulnerability of subicular GABAergic interneurons causes an input-specific disturbance of the subicular inhibitory system.

The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na+/H+exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pHi) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1–100 nM), the rate of pHirecovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na+-dependent pHirecovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 μM) completely abolished the pHirecovery by NHE. 18α-Glycyrrhetinic acid (18α-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pHirecovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined.

The subiculum is the principal target of CA1 pyramidal cells and mediates hippocampal output to various cortical and subcortical regions of the brain. The majority of subicular pyramidal cells are burst-spiking neurons. Previous studies indicated that high frequency stimulation in subicular burst-spiking cells causes presynaptic NMDA-receptor dependent long-term potentiation (LTP) whereas low frequency stimulation induces postsynaptic NMDA-receptor-dependent long-term depression (LTD). In the present study, we investigate the effect of 5-hydroxytryptamine type 4 (5-HT4) receptor activation and blockade on both forms of synaptic plasticity in burst-spiking cells. We demonstrate that neither activation nor block of 5-HT4 receptors modulate the induction or expression of LTP. In contrast, activation of 5-HT4 receptors facilitates expression of LTD, and block of the 5-HT4 receptor prevents induction of short-term depression and LTD. As 5-HT4 receptors are positively coupled to adenylate cyclase 1 (AC1), 5-HT4 receptors might modulate PKA activity through AC1. Since LTD is blocked in the presence of 5-HT4 receptor antagonists, our data are consistent with 5-HT4 receptor activation by ambient serotonin or intrinsically active 5-HT4 receptors. Our findings provide new insight into aminergic modulation of hippocampal output.