Pawel Fidzinski

Ca2+-activated Cl- currents are dispensable for olfaction

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 Ca2+-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 Ca2+-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.

Loss of GABAergic neurons in the subiculum and its functional implications in temporal lobe epilepsy

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 Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

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 NO3− conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl−/H+ antiporter.

Synaptic plasticity in the subiculum.

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.

Activation of dopamine D1/D5 receptors facilitates the induction of presynaptic long-term potentiation at hippocampal output synapses

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 Ca2+ 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.

Specific adverse effects of antiepileptic drugs - a true-to-life monotherapy study

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 ≥16years of age with epilepsy for ≥12months 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±17.1years) 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.

Differential cAMP signaling 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.

Target‐cell‐specific bidirectional synaptic plasticity 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 Ca2+‐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.

KCNQ5 K+ channels control hippocampal synaptic inhibition and fast network oscillations

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.

Muscarinic acetylcholine receptors and voltage-gated calcium channels contribute to bidirectional synaptic plasticity at CA1-subiculum synapses

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.