Non-Hebbian Long-Term Potentiation of Inhibitory Synapses in the Thalamus

The thalamus integrates and transmits sensory information to the neocortex. The activity of thalamocortical relay (TC) cells is modulated by specific inhibitory circuits. Although this inhibition plays a crucial role in regulating thalamic activity, little is known about long-term changes in synaptic strength at these inhibitory synapses. Therefore, we studied long-term plasticity of inhibitory inputs to TC cells in the posterior medial nucleus of the thalamus by combining patch-clamp recordings with two-photon fluorescence microscopy in rat brain slices. We found that specific activity patterns in the postsynaptic TC cell induced inhibitory long-term potentiation (iLTP). This iLTP was non-Hebbian because it did not depend on the timing between presynaptic and postsynaptic activity, but it could be induced by postsynaptic burst activity alone. iLTP required postsynaptic dendritic Ca2+ influx evoked by low-threshold Ca2+ spikes. In contrast, tonic postsynaptic spiking from a depolarized membrane potential (−50 mV), which suppressed these low-threshold Ca2+ spikes, induced no plasticity. The postsynaptic dendritic Ca2+ increase triggered the synthesis of nitric oxide that retrogradely activated presynaptic guanylyl cyclase, resulting in the presynaptic expression of iLTP. The dependence of iLTP on the membrane potential and therefore on the postsynaptic discharge mode suggests that this form of iLTP might occur during sleep, when TC cells discharge in bursts. Therefore, iLTP might be involved in sleep state-dependent modulation of thalamic information processing and thalamic oscillations.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

Aggravation of cardiac myofibroblast arrhythmogeneicity by mechanical stress

Aims Myofibroblasts (MFBs) as appearing in the myocardium during fibrotic remodelling induce slow conduction following heterocellular gap junctional coupling with cardiomyocytes (CMCs) in bioengineered tissue preparations kept under isometric conditions. In this study, we investigated the hypothesis that strain as developed during diastolic filling of the heart chambers may modulate MFB-dependent slow conduction. Methods and results Effects of defined levels of strain on single-cell electrophysiology (patch clamp) and impulse conduction in patterned growth cell strands (optical mapping) were investigated in neonatal rat ventricular cell cultures (Wistar) grown on flexible substrates. While 10.5% strain only minimally affected conduction times in control CMC strands (+3.2%, n.s.), it caused a significant slowing of conduction in the fibrosis model consisting of CMC strands coated with MFBs (conduction times +26.3%). Increased sensitivity to strain of the fibrosis model was due to activation of mechanosensitive channels (MSCs) in both CMCs and MFBs that aggravated the MFB-dependent baseline depolarization of CMCs. As found in non-strained preparations, baseline depolarization of CMCs was partly due to the presence of constitutively active MSCs in coupled MFBs. Constitutive activity of MSCs was not dependent on the contractile state of MFBs, because neither stimulation (thrombin) nor suppression (blebbistatin) thereof significantly affected conduction velocities in the non-strained fibrosis model. Conclusions The findings demonstrate that both constitutive and strain-induced activity of MSCs in MFBs significantly enhance their depolarizing effect on electrotonically coupled CMCs. Ensuing aggravation of slow conduction may contribute to the precipitation of strain-related arrhythmias in fibrotically remodelled hearts.

Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current

BACKGROUND Membrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization. OBJECTIVE The purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes. METHODS SAP97(fl/fl) mice were generated by inserting loxP sequences flanking exons 1-3 of the SAP97 gene. SAP97(fl/fl) mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97(fl/fl) mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase-polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice. RESULTS Action potential duration was greatly prolonged in αMHC-Cre/SAP97(fl/fl) cardiomyocytes compared to SAP97(fl/fl) controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97(fl/fl) mice. CONCLUSION These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.

p.L1612P, a Novel Voltage-gated Sodium Channel Nav1.7 Mutation Inducing a Cold Sensitive Paroxysmal Extreme Pain Disorder

BACKGROUND Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. METHODS Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. RESULTS The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady-state inactivation curve (V1/2 from -61.8 ± 4.5 mV to -30.9 ± 2.2 mV, n = 4 and 7, P < 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady-state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. CONCLUSIONS The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD.

Rhythmic synaptic activity induced by mechanical injury of rat CA3 hippocampal area.

Mechanical injury of the CNS frequently results from accidents but also occurs in the course of neurosurgical interventions. A great variety of anatomical and physiological changes have been described to evolve after a brain trauma yet only little is known about processes that occur during a trauma. In the present study, I obtained whole-cell patch clamp recordings from pyramidal cells in hippocampal slice cultures while mechanically lesioning the CA3 area. Electrophysiological analysis revealed that traumatic injury massively increased excitatory and inhibitory synaptic activity in the entire CA3 region. Cutting the CA3 region induced highly rhythmic excitatory postsynaptic currents (EPSCs) that reached frequencies of around 70 Hz. Blocking voltage-dependent sodium channels with tetrodotoxin prevented the increase in synaptic activity and injury-induced neurotransmitter release in CA3 remote from the lesion site. With fast synaptic transmission blocked only neurons in the immediate vicinity of a lesion depolarized and fired action potentials upon mechanical damage. I hence suggest that mechanical injury damages the membrane and induces action potential firing in only a small population of neurons. This activity is then propagated throughout the undamaged CA3 network inducing highly rhythmic discharges. Thus mechanical brain injury initiates immediate functional changes that exceed the lesion site.

Dysfunction of Cortical Dendritic Integration in Neuropathic Pain Reversed by Serotoninergic Neuromodulation

Neuropathic pain is caused by long-term modifications of neuronal function in the peripheral nervous system, the spinal cord, and supraspinal areas. Although functional changes in the forebrain are thought to contribute to the development of persistent pain, their significance and precise subcellular nature remain unexplored. Using somatic and dendritic whole-cell patch-clamp recordings from neurons in the anterior cingulate cortex, we discovered that sciatic nerve injury caused an activity-dependent dysfunction of hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the dendrites of layer 5 pyramidal neurons resulting in enhanced integration of excitatory postsynaptic inputs and increased neuronal firing. Specific activation of the serotonin receptor type 7 (5-HT7R) alleviated the lesion-induced pathology by increasing HCN channel function, restoring normal dendritic integration, and reducing mechanical pain hypersensitivity in nerve-injured animals in vivo. Thus, serotoninergic neuromodulation at the forebrain level can reverse the dendritic dysfunction induced by neuropathic pain and may represent a potential therapeutical target.

Optical recording of calcium currents during impulse conduction in cardiac tissue

We explore the feasibility of obtaining a spatially resolved picture of Ca2+Ca2+ inward currents (ICaICa) in multicellular cardiac tissue by differentiating optically recorded Ca2+Ca2+ transients that accompany propagating action potentials. Patterned growth strands of neonatal rat ventricular cardiomyocytes were stained with the Ca2+Ca2+ indicators Fluo-4 or Fluo-4FF. Preparations were stimulated at 1 Hz, and Ca2+Ca2+ transients were recorded with high spatiotemporal resolution (50  μm50  μm, 2 kHz analog bandwidth) with a photodiode array. Signals were differentiated after appropriate digital filtering. Differentiation of Ca2+Ca2+ transients resulted in optically recorded calcium currents (ORCCs) that carried the temporal and pharmacological signatures of L-type Ca2+Ca2+ inward currents: the time to peak amounted to ∼2.1  ms∼2.1  ms (Fluo-4FF) and ∼2.4  ms∼2.4  ms (Fluo-4), full-width at half-maximum was ∼8  ms∼8  ms, and ORCCs were completely suppressed by 50  μmol/L50  μmol/LCdCl2CdCl2. Also, and as reported before from patch-clamp studies, caffeine reversibly depressed the amplitude of ORCCs. The results demonstrate that the differentiation of Ca2+Ca2+ transients can be used to obtain a spatially resolved picture of the initial phase of ICaICa in cardiac tissue and to assess relative changes of activation/fast inactivation of ICaICa following pharmacological interventions.

Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods.

Sodium/hydrogen exchangers (NHEs) are ubiquitous ion transporters that serve multiple cell functions. We have studied two mammalian isoforms, NHE1 (ubiquitous) and NHE3 (epithelial-specific), by measuring extracellular proton (H+) gradients during whole-cell patch clamp with perfusion of the cell interior. Maximal Na(+)-dependent H+ fluxes (JH+) are equivalent to currents >20 pA for NHE1 in Chinese hamster ovary fibroblasts, >200 pA for NHE1 in guinea pig ventricular myocytes, and 5-10 pA for NHE3 in opossum kidney cells. The fluxes are blocked by an NHE inhibitor, ethylisopropylamiloride, and are absent in NHE-deficient AP-1 cells. NHE1 activity is stable with perfusion of nonhydrolyzable ATP [adenosine 5'-(beta,gamma-imido)triphosphate], is abolished by ATP depletion (2 deoxy-D-glucose with oligomycin or perfusion of apyrase), can be restored with phosphatidylinositol 4,5-bisphosphate, and is unaffected by actin cytoskeleton disruption (latrunculin or pipette perfusion of gelsolin). NHE3 (but not NHE1) is reversibly activated by phosphatidylinositol 3,4,5-trisphosphate. Both NHE1 and NHE3 activities are disrupted in giant patches during gigaohm seal formation. NHE1 (but not NHE3) is reversibly activated by cell shrinkage, even at neutral cytoplasmic pH without ATP, and inhibited by cell swelling. NHE1 in Chinese hamster ovary fibroblasts (but not NHE3 in opossum kidney cells) is inhibited by agents that thin the membrane (L-alpha-lysophosphatidylcholine and octyl-beta-D-glucopyranoside) and activated by cholesterol enrichment, which thickens membranes. Expressed in AP-1 cells, however, NHE1 is insensitive to these agents but remains sensitive to volume changes. Thus, changes of hydrophobic mismatch can modulate NHE1 but do not underlie its volume sensitivity.

Complex genetic background in a large family with Brugada syndrome.

The Brugada syndrome (BrS) is an inherited arrhythmia characterized by ST-segment elevation in V1-V3 leads and negative T wave on standard ECG. BrS patients are at risk of sudden cardiac death (SCD) due to ventricular tachyarrhythmia. At least 17 genes have been proposed to be linked to BrS, although recent findings suggested a polygenic background. Mutations in SCN5A, the gene coding for the cardiac sodium channel Nav1.5, have been found in 15-30% of index cases. Here, we present the results of clinical, genetic, and expression studies of a large Iranian family with BrS carrying a novel genetic variant (p.P1506S) in SCN5A. By performing whole-cell patch-clamp experiments using HEK293 cells expressing wild-type (WT) or p.P1506S Nav1.5 channels, hyperpolarizing shift of the availability curve, depolarizing shift of the activation curve, and hastening of the fast inactivation process were observed. These mutant-induced alterations lead to a loss of function of Nav1.5 and thus suggest that the p.P1506S variant is pathogenic. In addition, cascade familial screening found a family member with BrS who did not carry the p.P1506S mutation. Additional next generation sequencing analyses revealed the p.R25W mutation in KCNH2 gene in SCN5A-negative BrS patients. These findings illustrate the complex genetic background of BrS found in this family and the possible pathogenic role of a new SCN5A genetic variant.

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

Determination of Pelvic Orientation from Ultrasound Images Using Patch-SSMs and a Hierarchical Speed of Sound Compensation Strategy

In the field of computer assisted orthopedic surgery (CAOS) the anterior pelvic plane (APP) is a common concept to determine the pelvic orientation by digitizing distinct pelvic landmarks. As percutaneous palpation is - especially for obese patients - known to be error-prone, B-mode ultrasound (US) imaging could provide an alternative means. Several concepts of using ultrasound imaging to determine the APP landmarks have been introduced. In this paper we present a novel technique, which uses local patch statistical shape models (SSMs) and a hierarchical speed of sound compensation strategy for an accurate determination of the APP. These patches are independently matched and instantiated with respect to associated point clouds derived from the acquired ultrasound images. Potential inaccuracies due to the assumption of a constant speed of sound are compensated by an extended reconstruction scheme. We validated our method with in-vitro studies using a plastic bone covered with a soft-tissue simulation phantom and with a preliminary cadaver trial.