Mechanisms of hyperexcitability and efficacy of antiepileptic drugs in hippocampal-entorhinal networks in the Reduced Intensity Status Epilepticus (RISE) model of chronic epilepsy

A third of epilepsy patients are resistant to anti-epileptic drug (AED) treatment leading to reduced quality of life, increased treatment costs and complexities surrounding polytherapy. The overall aim of this project was to explore dynamic network changes in the excitability and efficacy of AEDs in: acute models of epileptiform activity, chronic models of epileptogenesis and in resected human tissue, in vitro. Initial studies investigated the differences in the neuronal network excitability induced by 0[Mg]2+ in rat brain slices prepared using either a standard NaCl-based aCSF or a sucrose-based aCSF. Standard prepared slices were more excitable in comparison to sucrose-based aCSF prepared slices. Immunohistochemical investigations for parvalbumin demonstrated a reduction of interneurons in slices prepared in the standard way. There was little difference in response to combination AEDs, but this could be due to increased latency to first seizure in sucrose prepared slices. LTP was suggested to play a role in the resistance to AEDs. These results suggest sucrose prepared slices better preserve the neuronal network in vitro, and serve as a better acute model for assessing AEDs and mechanisms of resistance. Sucrose perfused slices were prepared from rodents that had undergone a refined chronic Li-pilocarpine-based model of epileptogenesis (RISE) to investigate the effects of six AED combinations on network excitability (24 hrs and 1, 5 and 12 weeks post status). Ictal-like discharges (IDs) were seen in significantly greater numbers in slices from RISE animals compared to age-matched controls. Additionally, RISE slices showed a consistently shorter latency to first seizure across all time points. Investigations exploring the efficacy of different AED combinations during epileptogenesis showed that the tiagabine and carbamazepine combination was most effective in reducing measures of ictal activity whilst the combination of lamotrigine and gabapentin was least effective. The resistance of different drug combinations was also variable depending on the stage of epileptogenesis. These findings suggest that vulnerable networks show underlying hyperexcitability even at stages when chronic behavioural seizures are not yet developed, and that the RISE model may provide insights into the variable efficacy of AEDs. In comparison to chronically epileptic rodent tissue, epileptic human tissue from the temporal lobe was not as excitable, and often required stronger ID inducing manipulations. Once IDs were initiated in vitro, inter-event intervals between seizures were longer in comparison rodent epileptic tissue. Discrepancies in excitability could be attributed to the likelihood that damage within human tissue is likely to be subtle, hence require more stimulation to induce ictal-like activity (Gabriel et al., 2004). There was a developmental trend for excitability, in response to low concentrations of the NMDA antagonist MK801 (100-300 nM), to decrease in controls and remain elevated in epileptic animals. The NOS inhibitor, 7-nitraindazole, failed to stop the induction of IDs by low concentrations of MK801. Additionally, low concentrations of MK801 had no significant effects on the frequency and amplitude of field IPSPs in control and SE latent period slices. Further investigations are required to elucidate the mechanisms of how altered excitatory drive of inhibition may promote network excitability in epilepsy Overall, my findings suggest network changes in excitability occur at stages when chronic behavioural seizures are not yet developed, and that the RISE model may provide insights into the variable efficacy of AEDs and underlying mechanisms of epileptogenesi

Funktionelle Rolle Medial Septaler Projektionen zum Parasubiculum

Oscillations are a hallmark of brain activity and can be generated by local synchronisation mechanisms. They have been implicated in the communication between brain areas. An important type of oscillations are θ oscillations (4-12 Hz), which are associated with different behaviours, such as movements and navigation, but they also play a crucial role in memory formation and retrieval. One of the major θ rhythm generators in the brain is the medial septum (MS), which with its different types of projecting neurons, innervates many cortical areas and synchronises their activity. I investigated two major projection types of the MS: GABAergic (γ-aminobutyric acid – GABA) and cholinergic (acetylcholine – ACh) projections. Both projections are known to target the medial entorhinal cortex (MEC) and hippocampus. Parvalbumin positive (PV+) projections of the MS, which are GABAergic, are known to synchronise cortical networks via disinhibition often by inhibiting interneurons. In contrast, cholinergic projections of the MS project to a wide range of cell types in the MEC and hippocampus and can have substantially different effects on the target cell (e.g. activation or inhibition). Thus, their function on a network can range from increasing activity through depolarising excitatory cells, to more inhibition of the network by activating interneurons, or even modulating synaptic integration. Previous studies have focussed on identifying projections to the hippocampus and the MEC but did not consider the parasubiculum (PaS), a major input of the MEC. In this study, we electrophysiologically characterised cells in the PaS and demonstrated layer I interneurons to be distinctly different from putative layer II interneurons. The PaS, with its strong θ rhythmic firing cells, was shown to have the highest density of MS PV+ fibres in the parahippocampal formation, suggesting that it is an important target of MS projections and yet MS inputs to the PaS are unknown. Using channelrhodopsin (ChR2), a light sensitive ion channel, expressed in the MS of PV-Cre and ChAT-Cre (choline acetyltransferase) mice in-vivo, I identified GABAergic and cholinergic MS connections to the PaS in-vitro and demonstrated cell type specific projection patterns. I found that PV+ MS projections mainly inhibit interneurons in the PaS, including layer I interneurons, representing a novel cortical target of PV+ MS cells. On the other hand, cholinergic projections depolarise layer I interneurons and have multiple effects on deeper cells of the PaS, leading to a depolarisation or hyperpolarisation. To investigate a potential role of GABAergic projections in θ generation, I recorded local field potentials (LFP) in awake head-fixed mice and entrained oscillations in the PaS by stimulating with light in the MS. In contrast, local stimulation of fibres in the PaS could not entrain oscillation, suggesting that increased activity in the PaS might be required for MS PV+ cells to entrain θ. Taken together, stimulation of PV+ cells in the MS is sufficient to drive oscillations in the PaS, likely via disinhibition in line with other areas as the MEC and hippocampus. However, novel targets in layer I could be involved via cholinergic activation and GABAergic entrainment. Whether cholinergic activation by itself can entrain θ remains to be further investigated.Oszillationen sind ein Kennzeichen von Gehirnaktivität und können durch lokale Synchronisationsmechanismen generiert werden. Sie spielen eine wichtige Rolle bei der Kommunikation zwischen Gehirnarealen. Ein wichtiger Typ von Oszillationen sind θ Oszillationen (4 − 12 Hz), welche mit verschiedenen Verhalten wie Bewegung und Navigation assoziiert sind und eine wichtige Rolle in der Gedächtnisbildung und -abrufung spielen. Einer der wichtigen θ Generatoren im Gehirn ist das Mediale Septum (MS), welches mit seinen verschiedenen projizierenden Neuronen viele kortikale Regionen innerviert. Ich habe zwei Typen von Projektionen des MS untersucht: GABAerge (γ-Aminobuttersäure – GABA) und cholinerge (Acetylcholin – ACh) Projektionen. Beide Typen projizieren zum Medialen Entohinalen Kortex (MEC) und zum Hippocampus. Parvalbumin positive (PV+) Projektionen des MS können kortikale Netzwerke via Disinhibition, durch inhibieren von Interneuronen, synchronisieren. Im Gegensatz dazu projizieren cholinerge Projektionen des MS zu verschiedensten Zelltypen des MEC und des Hippocampus und können unterschiedliche weitreichende Effekte auf Zellen haben (z.B. Aktivierung und Inhibierung). Folglich können die Konsequenzen von Aktivierung des Netzwerkes via Depolarisation von exzitatorischen Zellen, über Inhibierung des Netzwerkes via Aktivierung von Interneuronen bis hin zur Modulation von synaptischer Integration reichen. In der Vergangenheit haben Studien sich auf die Identifizierung von Projektionen zum Hippocampus und MECs fokussiert, jedoch nicht zum Parasubiculum (PaS), eines der bedeutendsten Eingänge des MEC. In dieser Studie haben wir elektrophysiologisch Zellen im PaS charakterisiert und konnten herausstellen, dass Schicht I Zellen sich von anderen vermeintlichen Interneuronen in Schicht II unterscheiden. Das PaS, mit seinen im θ Rhythmus feuernden Zellen, hat die höchste Dichte von MS PV+ Fasern im parahippocampalen Netzwerk, was es als besonderes Ziel für MS Projektionen herausstellt. Dennoch sind Projektionen vom MS zum PaS nicht untersucht worden. Mit Hilfe von Channelrhodopsin (ChR2), einem lichtsensitivem Ionenkanal, welcher im MS von PV-Cre und ChAT-Cre Mäusen exprimiert wurde, konnte ich GABAerge und cholinerge MS Verbindungen zum PaS in-vitro detektieren und Zelltyp-speziefische Projektionen identifizieren. Ich konnte herausstellen, dass PV+ MS Projektionen hauptsächlich Interneurone im PaS inhibieren. Insbesondere Schicht I Interneurone stellen ein neues kortikales Ziel von PV+ MS Zellen dar. Im Gegensatz dazu werden Schicht I Interneurone des PaS durch cholinerge MS Projektionen depolarisiert wohingegen Zellen in tieferen Schichten depolarisiert oder hyperpolarisiert werden können. Um zu zeigen, dass man mit GABAergen Projektionen θ generieren kann, nahm ich das lokale Feldpotential (LFP) in Kopffixierten Mäusen auf und fand, dass man Oszillationen mit MS-Stimulation gleichschalten kann, jedoch eine Stimulation der Fasern im PaS nicht ausreichend ist. Das weist darauf hin, dass eine erhöhte PaS-Aktivität notwendig ist, um θ Oszillationen im PaS zu generieren. Zusammenfassend zeigt sich, dass eine Stimulation der PV+ Zellen im MS ausreichend ist, um im PaS Oszillationen zu generieren. Disinhibierung im PaS ist, ähnlich wie auch im MEC und Hippocampus, ein wahrscheinlicher Mechanismus. Weiterhin könnten jedoch neue Ziele von cholinergen und GABAergen Fasern in Schicht I bei der θ Generierung involviert sein. Ob θ Oszillationen durch cholinerge Projektionen gleichgeschaltet werden kann muss jedoch noch durch weitere Studien gezeigt werden

The role of sensory experience in intrinsic biophysical diversity of glomerular interneurons of the olfactory bulb

Investigating the adaptive properties of neuronal circuits is central to understanding the operations of the brain. The mouse olfactory bulb is a structure well suited for studying these processes, since it is composed of relatively simple excitatory and inhibitory networks. Recently, it has been shown that principal neurons (mitral cells) of the olfactory bulb that participate in the same glomerular circuit exhibit similar biophysical properties based on their Ih-mediated membrane potential sag. In many cell types, including mitral cells, Ih is known to profoundly influence excitability and thus impact the input/output function of individual neurons and networks. Like principal cells, inhibitory neurons are known to exhibit Ih that influences their integrative properties. In the olfactory bulb, interneurons of the glomerular layer can receive input from one or more glomerular networks and are thought to mediate lateral or centre-surround inhibition. The regulation of Ih in these cells could thus be used as a gain-control mechanism to facilitate contrast enhancement. During my project, I therefore investigated the diversity of Ih in GAD65+ and TH+ juxtaglomerular cells belonging to the same or different glomeruli to determine the diversity of Ih-mediated membrane potential sag within and across different inhibitory circuits. I found that the two juxtaglomerular populations differed substantially in their levels of membrane potential sag. Contrary to TH+ juxtaglomerular cells, the similarity in the amount of sag recorded in GAD65+ juxtaglomerular cells was high when two neurons were found to participate in the same glomerular circuit. Furthermore, the sag amplitude of interneurons affiliated to a specific glomerular circuit was upregulated when mice were exposed to odour stimuli. This indicates that, at least for GAD65+ juxtaglomerular cells, the amount of Ih membrane sag reflects local network processing of odour information

Regulation of oestrogen receptor mRNA in rat central nervous system

The gonadal steroid l7 -ß oestradiol (E2) influences a variety of neural activities, not only those involved in aspects of reproductive physiology but also in other functions, including autonomic regulation and cognitive processes. The effects of sex steroid hormones are principally, although not exclusively, mediated by binding to their cognate intracellular receptors to regulate functions of their target cells. The expression of these receptors is an important determinant of the actions of steroid hormones. There are two principal oestrogen receptors (ER) characterised to date, ER -a and ER -ß, the products of two distinct genes.Using quantitative in situ hybridisation histochemistry we first described the distribution of neurones expressing the messenger ribonucleic acid (mRNA) moieties for both types of ER in rat brain. We then investigated whether changes in the expression of these transcripts occur in response to hormonal manipulations as well as physiological stimulation in two discrete regions: the hippocampus, involved in memory, behaviour and regulation of the hypothalamo- pituitary- adrenal (HPA) axis and the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, producing oxytocin and vasopressin, and autonomic regulation. Both ER -a and -ß mRNAs were found to be expressed in the hippocampus, while only ER -ß mRNA was localised in the PVN and SON.In the hippocampus, changes in the expression of both mRNA transcripts following gonadectomy were found in a region- and time -specific manner and displayed a sexual dimorphic pattern. However, replacement of E2 in ovariectomised rats did not restore the basal level of expression indicating that factors other than the ligand itself are involved in mediating effects of gonadectomy. Hippocampal neurones also express GR and MR, receptors by which adrenal steroid hormones act through. Because glucocorticoids might act by heterologous regulation of ER mRNA expression, the effects of decreasing or increasing corticosteroid secretion were studied.None of the adrenal manipulations changed ER -a expression, but adrenalectomy decreased ER -p mRNA expression, only in CA1, and this was prevented by corticosterone replacement. Repeated stress for 72 h had no effect on either ER -a or -ß mRNA expression in the hippocampus.The expression of ER -ß mRNA was found in regions containing oxytocin and vasopressin magnocellular neurones; expression in the PVN was low in the medial parvocellular neurones, projecting to the median eminence, but high in the ventral group of parvocellular neurones which project to brainstem and spinal cord.In the SON the greater ER-I3 mRNA signal found in female than in male rats was abolished by gonadectomy, but not restored or altered in intact male rats by E2 treatment. In the ventral parvocellular neurones, we found no sex difference in the expression of ER-E3 mRNA. Here, ovariectomy significantly increased the expression of ER -ß mRNA. The expression of ER-E3 mRNA in both the magno- and parvocellular neurones was not changed at the end of pregnancy, when oestrogen secretion is maximal. In contrast, marked changes in ER -ß mRNA expression were found in SON and PVN neurones, but not in the hippocampus, following manipulation of adrenal- corticoid secretion. First, bilateral adrenalectomy, removing gluco- and mineralo- corticoids, significantly increased ER -ß mRNA expression in the SON but not in the ventral parvocellular neurones. The effect of adrenalectomy was partially reversed by replacement with corticosterone. Stimulation of the HPA axis by repeated stress did not alter expression of ER -ß mRNA, except in the ventral parvocellular neurones where expression was significantly increased. Stimulating the magnocellular neurones by salt -loading markedly attenuated the expression of ER -p mRNA selectively in these neurones, and also in the ventral parvocellular PVN neurones.The results indicate actions of E₂ via ER -α/-ß in hippocampus, but only via ER-ß in the PVN /SON; there is a weak, regionally-specific regulation of ER-α/-ß by sex steroids. In contrast, ER-ß in magnocellular neurones may be up- regulated by gluco- and mineralo- corticoid deficiency, and down- regulated by physiological stimulation of the neurones. This would alter any ER- mediated effects of E₂ on the neurones in these state

Haemodynamic correlates of interictal and ictal epileptic discharges and ictal semiology using simultaneous scalp video-EEG-fMRI and intracranial EEG-fMRI

Interictal and ictal epileptic discharges are produced by focal and widespread dysfunctional neuronal networks. Identification and characterization of epileptic discharges underlie the diagnosis and the choice of treatment for epilepsy patients. A better knowledge of the generation, propagation and localisation of epileptic discharges, and their interaction with the physiological and pathological brain networks can be very helpful in planning epilepsy surgery and minimizing the risk of damaging the physiological brain networks. This work describes a number of methodological developments and novel applications investigating the epileptic networks in humans using EEG-fMRI. First, I implemented synchronized video recording inside the MRI-scanner during simultaneous EEG-fMRI studies, which did not deteriorate the imaging and EEG data quality. Secondly, I used video recordings to identify physiological activities to be modelled as confounds in the functional imaging data analysis for interictal activity, thus increasing the sensitivity of video-EEG-fMRI. Thirdly, I applied this modelling approach to investigate seizure related functional networks in patients with focal epilepsy. Video recordings allowed partitioning seizures into phases separating the ictal onset related functional networks from propagation related networks. Localisation of the ictal onset related networks may be useful in the planning for epilepsy surgery in a selected group of patients, as demonstrated by their comparison with intracranial-EEG recordings. Further, I investigated haemodynamic changes during preictal period which suggested recruitment of an inhibitory followed by an excitatory network prior to the ictal onset on scalp EEG. In the next step, I used simultaneous intracranial-EEG-fMRI in patients undergoing invasive evaluation, demonstrating that local and remote networks associated with very focal interictal discharges recorded on intracranial-EEG may predict the surgical outcome. Finally, I investigated the interaction of epileptic discharges with the working memory, using scalp video-EEG-fMRI, showing that the presence of epileptic activity may alter the working memory related networks. Methodological constraints, clinical applications and future perspectives are discussed

Understanding haemodynamic changes surrounding epileptic events in children

The interrelationship between cerebral haemodynamics and epileptic activity has been the subject of study for over 100 years. The overall goal of this PhD is to use and develop multimodal imaging to better understand this relationship in a paediatric population. This has important implications for the localisation of epileptic activity that can aid pre-surgical evaluation and seizure detection. The benefit of interictal epileptiform discharge (IED) suppression in clinical treatment is under debate, considering little is known about their impact on cognitive function. By applying EEG-fMRI, it was found that transient effects of IEDs were responsible for connectivity differences between patients and controls, showing the widespread impact of IEDs on BOLD signal and suggesting the importance of IED suppression for normal functional connectivity. Haemodynamic changes may occur prior to epileptic event onset. Therefore we evaluated the response function (HRF) to IEDs in paediatric focal epilepsy patients, as an HRF was created from simultaneous EEG-fMRI data and found to be beneficial in the delineation of epileptic foci. However, the underlying neurovascular changes seen in this altered HRF still needed to be explored. Therefore EEG-NIRS was utilised to interpret the mechanistic changes found in BOLD during IEDs. NIRS provides the added information of concentration changes of both –oxy and –deoxy haemoglobin rather than relative changes in deoxyhaemoglobin. To perform these experiments a new optode holder applicable to the clinical environment had to be made and tested for efficacy. The best design was a flexible optode grid, as it required no interference with the standard clinical protocol. Once tested in patients, EEG-NIRS found pre-ictal/pre-IED increases in oxygen saturation and oxyhaemoglobin concentrations, thereby corroborating with prior haemodynamic changes seen in EEG-fMRI. Therefore, by utilizing both EEG-fMRI and EEG-NIRS a greater understanding of the haemodynamic changes surrounding epileptic events in children can be obtained

EEG-fMRI in epilepsy and sleep

This thesis used simultaneous electroencephalogram (EEG) and functional magnetic resonance imaging (fMRI) to investigate both epilepsy and sleep. Initially, EEG-fMRI was used in a cohort of patients with complex epilepsy referred from a tertiary epilepsy clinic for both pre-surgical evaluation and diagnostic reasons. The results suggest a limited utility of EEG-fMRI in the epilepsy clinic with a very complex patient group. Following on, investigation of early blood oxygen level dependent (BOLD) signal changes in a group of patients with focal epilepsy demonstrated potentially meaningful BOLD changes occurring six seconds prior to interictal epileptiform discharges, and modelling less than this six seconds can result in overlap of the haemodynamic response function used to model BOLD changes. The same analysis was used to model endogenously occurring sleep paroxysms; K-complexes (KCs), vertex sharp waves (VSWs) and sleep spindles (SSs), finding early BOLD signal changes with SSs in group data. Finally, KCs and VSWs were investigated in more detail in a group of participants under both sleep deprived and non-deprived conditions, demonstrating an increase in overall activation for both KCs and VSWs following sleep deprivation. Overall, we find early BOLD changes are not restricted to pathological events and sleep deprivation can enhance BOLD responses

Functional dissection of a cortical microcircuit for spatial computation

In mammals, spatial learning and memory depend on neural processing carried out in the hippocampal formation. Interestingly, extracellular recordings from behaving animals have shown that cells in this region exhibit spatially modulated activity patterns, thus providing insights into the neural activity underlying spatial behaviour. One area within the hippocampal formation, layer II of the medial entorhinal cortex, houses cells that encode a grid-like map of space using a firing rate code. At the same time, oscillatory signals at distinct theta (4–12 Hz) and gamma (30–120 Hz) frequencies are also present in layer II, providing a substrate for a timing code. To understand how layer II of the medial entorhinal cortex produces these outputs I sought to characterise the electrical properties and functional computational architecture of its microcircuitry. The functionality of any neural circuit depends on the electrical properties of its constituent cells. Because the grid cells in layer II are likely to be stellate cells, I used the perforated patch-clamp technique to accurately assess the intrinsic excitable properties of this cell type. Compared to whole-cell recordings, these recordings indicate that some intrinsic properties of stellate cells, such as spike clustering, which is revealed to be robust, are more likely to play a functional role in circuit computation. Conversely, other intrinsic properties, such as spontaneous membrane potential fluctuations, which are confirmed to be insufficiently stable to support reliable interference patterns, are revealed to be less likely than other, more robust electrical properties to play a direct role in circuit function. The characteristic connectivity profiles of different cell types are also critical for circuit function. To investigate cell type-specific connectivity in layer II I used optogenetic stimulation in combination with in vitro electrophysiology to record synaptic activity in different cell types while selectively activating distinct subpopulations of cells with light. Using this method I found that connections between stellate cells are absent or very rare and that communication between stellate cells is instead mediated by strong feedback inhibition from fast-spiking interneurons. Dissecting oscillatory activity in neural circuits may be important for establishing functionally relevant circuit architecture and dynamics but is difficult in vivo. I accomplished this in vitro by recapitulating the interacting theta and gamma rhythms that are observed in vivo with an optogenetic method. I found that locally driving a subset of neurons in the layer II microcircuit at theta frequency with a light stimiulus produced a nested field rhythm at gamma frequency that was also evident as rhythmic inhibition onto stellate cells. Critically, these interacting rhythms closely resembled those recorded from behaving animals. In addition, I found that this thetanested gamma is sufficiently regular to act as a clock-like reference signal, indicating its potential role in implementing a timing code. To functionally dissect the circuit I performed multiple simultaneous whole-cell patch-clamp recordings during circuit activation. These recordings revealed how feedback interactions between stellate cells and fast-spiking interneurons underpin the theta-nested gamma rhythm. Together, these results suggest that feedback inhibition in layer II acts as a common substrate for theta-nested gamma oscillations and possibly also grid firing fields, thereby providing a framework for understanding how computations are carried out in layer II of the medial entorhinal cortex