Clozapine-induced paroxysmal discharges

PhD ThesisThe atypical antipsychotic clozapine is a widely prescribed and effective treatment for the positive and negative symptoms of schizophrenia, but reports of side effects are common. In one study EEG abnormalities were observed in 53% of patients treated with clozapine, and the absence or presence of EEG abnormalities correlated with the plasma clozapine concentration. Here, epileptiform activity was present in conventional EEG recordings from a 32 year old male patient with psychiatric illness taking clozapine for 3 weeks. Brief (ca.100ms), transient epileptiform spikes occurred at a frequency of approximately 2 per h and originated primarily in parietal cortex. One month after withdrawal of clozapine, epileptiform spikes were no longer present. An in vitro model was developed using the equivalent region of association cortex, namely 2⁰ somatosensory cortex, in normal rat brain slices to probe such activity with increased spatial and temporal resolution, and to investigate mechanisms underlying its generation. Wide band in vitro recordings revealed that clozapine (10-20µM) induced regular, frequent very fast oscillations (VFO, > 70Hz) in this region. These VFO comprised short transient high frequency discharges and were maximal in patches along layer V. The atypical antipsychotic olanzapine, but not the classical antipsychotic haloperidol, also induced prominent VFO in this region. Sharp electrode intracellular recordings revealed that there was almost no correlation between the somatic activity of layer V regular spiking (RS) pyramidal cells and field VFO, but layer V intrinsically bursting (IB) cells did correlate to some extent with the local field. Interestingly, IB cell spikelets were also weakly correlated with field VFO suggesting a role for axonal hyperexcitability in this cell type in the mechanism. Clozapine-induced VFO persisted following blockade of AMPA, NMDA, and GABAA chemical synaptic receptors, and the gap junction blockers carbenoxolone and quinine also failed to significantly attenuate the power of this activity. Although octanol abolished clozapine-induced VFO, it was not clear that this effect resulted from blockade of gap junctions as this drug also blocks spikes. In addition to VFO events, clozapine (10-20µM) also induced occasional, spontaneous transient paroxysmal discharges, similar to the EEG phenomena, in 33% (11/33 slices) of slices in vitro. Sharp electrode intracellular recordings revealed that clozapine- induced full paroxysmal discharges were associated with spikes, EPSPs and IPSPs in layer V RS and IB cells, suggesting that these events were mediated via chemical synaptic transmission in both of these cell types. Multi-electrode array recordings of local field potentials and units suggested that clozapine-induced paroxysmal events started superficially in association cortex, moved deeper and then propagated horizontally along these deep layers. The onset of clozapine-induced VFO was accompanied by a significant elevation in parvalbumin immunoreactivity, particularly in layer II-IV, where there was a greater than twofold increase in the signal, and this may be relevant to the therapeutic action of the drug

Investigating the role of fast-spiking interneurons in neocortical dynamics

PhD ThesisFast-spiking interneurons are the largest interneuronal population in neocortex. It is well documented that this population is crucial in many functions of the neocortex by subserving all aspects of neural computation, like gain control, and by enabling dynamic phenomena, like the generation of high frequency oscillations. Fast-spiking interneurons, which represent mainly the parvalbumin-expressing, soma-targeting basket cells, are also implicated in pathological dynamics, like the propagation of seizures or the impaired coordination of activity in schizophrenia. In the present thesis, I investigate the role of fast-spiking interneurons in such dynamic phenomena by using computational and experimental techniques. First, I introduce a neural mass model of the neocortical microcircuit featuring divisive inhibition, a gain control mechanism, which is thought to be delivered mainly by the soma-targeting interneurons. Its dynamics were analysed at the onset of chaos and during the phenomena of entrainment and long-range synchronization. It is demonstrated that the mechanism of divisive inhibition reduces the sensitivity of the network to parameter changes and enhances the stability and exibility of oscillations. Next, in vitro electrophysiology was used to investigate the propagation of activity in the network of electrically coupled fast-spiking interneurons. Experimental evidence suggests that these interneurons and their gap junctions are involved in the propagation of seizures. Using multi-electrode array recordings and optogenetics, I investigated the possibility of such propagating activity under the conditions of raised extracellular K+ concentration which applies during seizures. Propagated activity was recorded and the involvement of gap junctions was con rmed by pharmacological manipulations. Finally, the interaction between two oscillations was investigated. Two oscillations with di erent frequencies were induced in cortical slices by directly activating the pyramidal cells using optogenetics. Their interaction suggested the possibility of a coincidence detection mechanism at the circuit level. Pharmacological manipulations were used to explore the role of the inhibitory interneurons during this phenomenon. The results, however, showed that the observed phenomenon was not a result of synaptic activity. Nevertheless, the experiments provided some insights about the excitability of the tissue through scattered light while using optogenetics. This investigation provides new insights into the role of fast-spiking interneurons in the neocortex. In particular, it is suggested that the gain control mechanism is important for the physiological oscillatory dynamics of the network and that the gap junctions between these interneurons can potentially contribute to the inhibitory restraint during a seizure.Wellcome Trust

Underlying Mechanisms of Epilepsy

This book is a very provocative and interesting addition to the literature on Epilepsy. It offers a lot of appealing and stimulating work to offer food of thought to the readers from different disciplines. Around 5% of the total world population have seizures but only 0.9% is diagnosed with epilepsy, so it is very important to understand the differences between seizures and epilepsy, and also to identify the factors responsible for its etiology so as to have more effective therapeutic regime. In this book we have twenty chapters ranging from causes and underlying mechanisms to the treatment and side effects of epilepsy. This book contains a variety of chapters which will stimulate the readers to think about the complex interplay of epigenetics and epilepsy

Remodeling of cardiac passive electrical properties and susceptibility to ventricular and atrial arrhythmias

Coordinated electrical activation of the heart is essential for the maintenance of a regular cardiac rhythm and effective contractions. Action potentials spread from one cell to the next via gap junction channels. Because of the elongated shape of cardiomyocytes, longitudinal resistivity is lower than transverse resistivity causing electrical anisotropy. Moreover, non-uniformity is created by clustering of gap junction channels at cell poles and by non-excitable structures such as collagenous strands, vessels or fibroblasts. Structural changes in cardiac disease often affect passive electrical properties by increasing non-uniformity and altering anisotropy. This disturbs normal electrical impulse propagation and is, consequently, a substrate for arrhythmia. However, to investigate how these structural changes lead to arrhythmias remains a challenge. One important mechanism, which may both cause and prevent arrhythmia, is the mismatch between current sources and sinks. Propagation of the electrical impulse requires a sufficient source of depolarizing current. In the case of a mismatch, the activated tissue (source) is not able to deliver enough depolarizing current to trigger an action potential in the non-activated tissue (sink). This eventually leads to conduction block. It has been suggested that in this situation a balanced geometrical distribution of gap junctions and reduced gap junction conductance may allow successful propagation. In contrast, source-sink mismatch can prevent spontaneous arrhythmogenic activity in a small number of cells from spreading over the ventricle, especially if gap junction conductance is enhanced. Beside gap junctions, cell geometry and non-cellular structures strongly modulate arrhythmogenic mechanisms. The present review elucidates these and other implications of passive electrical properties for cardiac rhythm and arrhythmogenesis

Analysis of pattern dynamics for a nonlinear model of the human cortex via bifurcation theories

This thesis examines the bifurcations, i.e., the emergent behaviours, for the Waikato cortical model under the influence of the gap-junction inhibitory diffusion D₂ (identified as the Turing bifurcation parameter) and the time-to-peak for hyperpolarising GABA response γi (i.e., inhibitory rate-constant, identified as the Hopf bifurcation parameter). The cortical model simplifies the entire cortex to a cylindrical macrocolumn (∼ 1 mm³) containing ∼ 10⁵ neurons (85% excitatory, 15% inhibitory) communicating via both chemical and electrical (gap-junction) synapses. The linear stability analysis of the model equations predict the emergence of a Turing instability (in which separated areas of the cortex become activated) when gap-junction diffusivity is increased above a critical level. In addition, a Hopf bifurcation (oscillation) occurs when the inhibitory rate-constant is sufficiently small. Nonlinear interaction between these instabilities leads to spontaneous cortical patterns of neuronal activities evolving in space and time. Such model dynamics of delicately balanced interplay between Turing and Hopf instabilities may be of direct relevance to clinically observed brain dynamics such as epileptic seizure EEG spikes, deep-sleep slow-wave oscillations and cognitive gamma-waves. The relationship between the modelled brain patterns and model equations can normally be inferred from the eigenvalue dispersion curve, i.e., linear stability analysis. Sometimes we experienced mismatches between the linear stability analysis and the formed cortical patterns, which hampers us in identifying the type of instability corresponding to the emergent patterns. In this thesis, I investigate the pattern-forming mechanism of the Waikato cortical model to better understand the model nonlinearities. I first study the pattern dynamics via analysis of a simple pattern-forming system, the Brusselator model, which has a similar model structure and bifurcation phenomena as the cortical model. I apply both linear and nonlinear perturbation methods to analyse the near-bifurcation behaviour of the Brusselator in order to precisely capture the dominant mode that contributes the most to the final formed-patterns. My nonlinear analysis of the Brusselator model yields Ginzburg-Landau type amplitude equations that describe the dynamics of the most unstable mode, i.e., the dominant mode, in the vicinity of a bifurcation point. The amplitude equations at a Turing point unfold three characteristic spatial structures: honeycomb Hπ, stripes, and reentrant honeycomb H₀. A codimension-2 Turing–Hopf point (CTHP) predicts three mixed instabilities: stable Turing–Hopf (TH), chaotic TH, and bistable TH. The amplitude equations precisely determine the bifurcation conditions for these instabilities and explain the pattern-competition mechanism once the bifurcation parameters cross the thresholds, whilst driving the system into a nonlinear region where the linear stability analysis may not be applicable. Then, I apply the bifurcation theories to the cortical model for its pattern predictions. Analogous to the Brusselator model, I find cortical Turing pattens in Hπ, stripes and H₀ spatial structures. Moreover, I develop the amplitude equations for the cortical model, with which I derive the envelope frequency for the beating-waves of a stable TH mode; and propose ideas regarding emergence of the cortical chaotic mode. Apart from these pattern dynamics that the cortical model shares with the Brusselator system, the cortical model also exhibits “eye-blinking” TH patterns latticed in hexagons with localised oscillations. Although we have not found biological significance of these model pattens, the developed bifurcation theories and investigated pattern-forming mechanism may enrich our modelling strategies and help us to further improve model performance. In the last chapter of this thesis, I introduce a Turing–Hopf mechanism for the anaesthetic slow-waves, and predict a coherence drop of such slow-waves with the induction of propofol anaesthesia. To test this hypothesis, I developed an EEG coherence analysing algorithm, EEG coherence, to automatically examine the clinical EEG recordings across multiple subjects. The result shows significantly decreased coherence along the fronto-occipital axis, and increased coherence along the left- and right-temporal axis. As the Waikato cortical model is spatially homogenous, i.e., there are no explicit front-to-back or right-to-left directions, it is unable to produce different coherence changes for different regions. It appears that the Waikato cortical model best represents the cortical dynamics in the frontal region. The theory of pattern dynamics suggests that a mode transition from wave–Turing–wave to Turing–wave–Turing introduces pattern coherence changes in both positive and negative directions. Thus, a further modelling improvement may be the introduction of a cortical bistable mode where Turing and wave coexist

Preservation of Astrocytic Coupling Prevents Epileptogenesis

Antiepileptic therapies are mainly based on drugs which target neuronal function. One third of epileptic patients do not respond adequately to these treatments and, importantly, all available drugs merely suppress seizures without curing the underlying disorder. Consequentially, new strategies for the development of antiepileptogenic drugs are urgently needed. Brain inflammation contributes to a loss of gap junction-mediated coupling between astrocytes, which is a causal event in the development of temporal lobe epilepsy (TLE). In a first step we assessed the effect of XPro1595 (a selective inhibitor of soluble TNF- alpha) and Anakinra (IL-1R antagonist) in situ in acute brain slices from epileptic mice and found that astrocytic coupling was restored. To investigate this in more detail, we checked the cytokine levels in the intracortical kainate (KA) model using ELISA. The results show elevated TNF-alpha levels immediately after KA-induced status epilepticus (SE), whereas IL-1beta was less prominent. This finding prompted us to assess the effect of solTNF-alpha/TNFR1 signalling cascade on astrocytic coupling. Hence, we investigated the effect of XPro1595 i.p. injection on astrocytic coupling and the development of TLE. The results show that XPro1595 given in vivo prior to KA injection prevented the loss of astrocytic gap junction coupling and thus the development of generalised spontaneous seizures and hippocampal sclerosis (HS) were prevented. Treatment with XPro1595 in vivo after KA injection rescued astrocytic coupling, significantly decreased chronic seizure frequency in the long-term and attenuated HS-specific morphological alterations. To confirm the effect of solTNF-alpha/TNFR1 signalling cascade on astrocytic coupling, we used transgenic TNFR1 KO animals in our epilepsy model. We could show that TNFR1 KO astrocytic coupling was unaltered after SE-induction, indicating that TNFR1 activation regulates astrocytic coupling strength. Furthermore, these animals showed significantly less seizure activity, demonstrating the importance of astrocytic coupling on the progression of TLE. However, incubation of acute brain slices from wildtype mice with TNF-alpha in situ revealed no impaired astrocytic coupling. Therefore, we suggest that a second mediator, like IL-1beta or a yet unknown molecule, might be needed to induce astrocytic uncoupling. In conclusion, this study demonstrates that solTNF-alpha/TNFR1 mediates astrocytic uncoupling and plays a key role in the development of TLE-HS. Rescuing astrocytic coupling might represent a new strategy to develop antiepileptogenic therapies. The present project elucidated the therapeutic potential of targeting astrocytic proteins in epilepsy and also shed further light on the mechanisms underlying the disorder

Neuronal and astroglial correlates underlying spatiotemporal Intrinsic Optical Signal in the rat hippocampal slice

Widely used for mapping afferent activated brain areas in vivo, the label-free intrinsic optical signal (IOS) is mainly ascribed to blood volume changes subsequent to glial glutamate uptake. By contrast, IOS imaged in vitro is generally attributed to neuronal and glial cell swelling, however the relative contribution of different cell types and molecular players remained largely unknown. We characterized IOS to Schaffer collateral stimulation in the rat hippocampal slice using a 464-element photodiode-array device that enables IOS monitoring at 0.6 ms time-resolution in combination with simultaneous field potential recordings. We used brief half-maximal stimuli by applying a medium intensity 50 Volt-stimulus train within 50 ms (20 Hz). IOS was primarily observed in the str. pyramidale and proximal region of the str. radiatum of the hippocampus. It was eliminated by tetrodotoxin blockade of voltage-gated Na+ channels and was significantly enhanced by suppressing inhibitory signaling with gamma-aminobutyric acid(A) receptor antagonist picrotoxin. We found that IOS was predominantly initiated by postsynaptic Glu receptor activation and progressed by the activation of astroglial Glu transporters and Mg2+-independent astroglial N-methyl-D-aspartate receptors. Under control conditions, role for neuronal K+/Cl- cotransporter KCC2, but not for glial Na+/K+/Cl- cotransporter NKCC1 was observed. Slight enhancement and inhibition of IOS through non-specific Cl- and volume-regulated anion channels, respectively, were also depicted. High-frequency IOS imaging, evoked by brief afferent stimulation in brain slices provide a new paradigm for studying mechanisms underlying IOS genesis. Major players disclosed this way imply that spatiotemporal IOS reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. Our model may help to better interpret in vivo IOS and support diagnosis in the future

The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention

Background: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. Methods: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na/K pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. Results: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na/K ATPase elicits SD. Elevated K or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. Conclusions: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory.This work was supported by grants from the Heart and Stroke Foundation of Canada and the National Science and Engineering Research Council of Canada to RDA, an NIH grant (NS106901) to CWS, a National Research, Development and Innovation Office of Hungary grant (K1343777) and EU Horizon 2020 research and innovation program (739953) to EF and from DFG Deutsche Forschungsgemeinschaft (German Research Council) (DFG DR 323/5-1), DFG DR 323/10-1, and BMBF Bundesministerium fuer Bildung und Forschung (EraNet Neuron EBio2, with funds from BMBF 01EW2004) to JPD