Electrophysiological Models of Seizure like Activity for NO Detection in Vitro

SOUHRN Název práce: Elektrofyziologické modely epileptické aktivity pro detekci NO in vitro Vedoucí diplomové práce: MUDr. Jakub Otáhal, Ph.D. Katedra anatomie a biomechaniky FTVS UK Oddělení vývojové epileptologie FGÚ AVČR Konzultant: Mgr. Renata Konopková Oddělení vývojové epileptologie FGÚ AVČR Cíl práce: Cílem této práce je představit oxid dusnatý (NO) a jeho roli ve fyziologii a patofyziologii centrálního nervového systému (CNS) se zaměřením na epileptickou aktivitu nervové tkáně. Náš výzkum, který byl proveden in vitro na potkaních hipokampálních řezech, porovnává experimentální biochemické modely (nízká koncentrace hořčíku Mg2+ , vysoká koncentrace draslíku K+ , Carbachol, Bicuculline a 4- aminopyridine model). Takto změněné podmínky prostředí řezu by měly vyvolávat epileptickou aktivitu v nervové tkáni potkana. Konečným cílem práce je vytvoření optimálního experimentálního modelu epileptické aktivity in vitro, který je předpokladem pro další experimenty především pro optické zobrazovací techniky, jejichž pomocí jsme schopni detekovat NO. Metody: Měření bylo provedeno na řezech hippocampu laboratorních potkanů in vitro. Potkani byli dekapitováni v hluboké anestesii éterem. Mozek byl rychle exstirpován z lebky a nařezán v okysličeném (95%) a chlazeném (0,5 řC) uměle vytvořeném mozkomíšním moku s...Definition of work: Electrophysiological models of seizure like activity for NO detection in vitro Supervisor: MUDr. Jakub Otáhal, PhD. Department of Anatomy and Biomechanics FTVS CUNI Department of Developmental Epileptology CAS, Institute of Physiology Consultant: Mgr. Renata Konopková Department of Developmental Epileptology CAS, Institute of Physiology Aim: The aim of the thesis is to introduce nitric oxide (NO) and its role in physiology and pathophysiolgy of central nervous system (CNS), with the intention of epileptiform activity in the nervous tissue. Our research was realized on rat hippocampal slices in vitro and it compares the experimental biochemical models (low-Mg2+ , high-K+ , Carbachol, Bicuculline and 4-Aminopyridine model). These changes in the environment of slice should evoke the epileptiform activity in the nervous tissue of the rat. The final aim of the thesis is to create an experimental model of epileptiform activity in vitro in our setup for imaging techniques and NO detection. Methods: Experiments were performed in rat hippocampal slices in vitro. Rats were deeply anaesthetized with ether and decapitated. Brain was rapidly removed from the scull, cut in oxygenated (95%) and cooled (0, 5 řC) artificial cerebrospinal fluid (ACSF) using vibratome in coronal slices of 400µm of...Anatomie a biomechanikaFaculty of Physical Education and SportFakulta tělesné výchovy a sport

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

Hippocampal hyperexcitability is modulated by microtubule-active agent: evidence from in vivo and in vitro epilepsy models in the rat

The involvement of microtubule dynamics on bioelectric activity of neurons and neurotransmission represents a fascinating target of research in the context of neural excitability. It has been reported that alteration of microtubule cytoskeleton can lead to profound modifications of neural functioning, with a putative impact on hyperexcitability phenomena. Altogether, in the present study we pointed at exploring the outcomes of modulating the degree of microtubule polymerization in two electrophysiological models of epileptiform activity in the rat hippocampus. To this aim, we used in vivo maximal dentate activation (MDA) and in vitro hippocampal epileptiform bursting activity (HEBA) paradigms to assess the effects of nocodazole (NOC) and paclitaxel (PAC), that respectively destabilize and stabilize microtubule structures. In particular, in the MDA paroxysmal discharge is electrically induced, whereas the HEBA is obtained by altering extracellular ionic concentrations. Our results provided evidence that NOC 10 \ub5M was able to reduce the severity of MDA seizures, without inducing neurotoxicity as verified by the immunohistochemical assay. In some cases, paroxysmal discharge was completely blocked during the maximal effect of the drug. These data were also in agreement with the outcomes of in vitro HEBA, since NOC markedly decreased burst activity that was even silenced occasionally. In contrast, PAC at 10 \ub5M did not exert a clear action in both paradigms. The present study, targeting cellular mechanisms not much considered so far, suggests the possibility that microtubule-active drugs could modulate brain hyperexcitability. This contributes to the hypothesis that cytoskeleton function may affect synaptic processes, relapsing on bioelectric aspects of epileptic activity

The signaling pathways involved in the anticonvulsive effects of the adenosine A1 receptor

Adenosine acts as an endogenous anticonvulsant and seizure terminator in the brain. Many of its anticonvulsive effects are mediated through the activation of the adenosine A(1) receptor, a G protein-coupled receptor with a wide array of targets. Activating A(1) receptors is an effective approach to suppress seizures. This review gives an overview of the neuronal targets of the adenosine A(1) receptor focusing in particular on signaling pathways resulting in neuronal inhibition. These include direct interactions of G protein subunits, the adenyl cyclase pathway and the phospholipase C pathway, which all mediate neuronal hyperpolarization and suppression of synaptic transmission. Additionally, the contribution of the guanyl cyclase and mitogen-activated protein kinase cascades to the seizure-suppressing effects of A(1) receptor activation are discussed. This review ends with the cautionary note that chronic activation of the A(1) receptor might have detrimental effects, which will need to be avoided when pursuing A(1) receptor-based epilepsy therapies

Bioenergetic mechanisms of seizure control

Epilepsy is characterized by the regular occurrence of seizures, which follow a stereotypical sequence of alterations in the electroencephalogram. Seizures are typically a self limiting phenomenon, concluding finally in the cessation of hypersynchronous activity and followed by a state of decreased neuronal excitability which might underlie the cognitive and psychological symptoms the patients experience in the wake of seizures. Many efforts have been devoted to understand how seizures spontaneously stop in hope to exploit this knowledge in anticonvulsant or neuroprotective therapies. Besides the alterations in ion-channels, transmitters and neuromodulators, the successive build up of disturbances in energy metabolism have been suggested as a mechanism for seizure termination. Energy metabolism and substrate supply of the brain are tightly regulated by different mechanisms called neurometabolic and neurovascular coupling. Here we summarize the current knowledge whether these mechanisms are sufficient to cover the energy demand of hypersynchronous activity and whether a mismatch between energy need and supply could contribute to seizure control

Epileptiform bursting in the disinhibited neonatal cerebral cortex

The cerebral cortex, which include the neocortex and hippocampus, is an elaborate neuronal network communicating mainly through glutamate and gamma-aminobutyric acid (GABA). Glutamate, operating via AMPA, kainate, and NMDA receptors excites neurons, and operating via metabotropic glutamate receptors can either increase or decrease the excitation in the neuronal network. GABA, operating through GABAA and GABAB receptors, inhibits the mature neuronal network, and GABAA receptor blockade in the adult cerebral cortex leads to epileptiform bursts. In contrast, in the neonatal cerebral cortex, GABAA has been proposed to function as an excitatory neurotransmitter, and glutamatergic synapses are claimed to be underdeveloped. It is important to understand the mechanisms underlying epileptiform activity in the neonate, because epileptiform activity in the neonate can potentially damage the developing cerebral cortex. In this dissertation I explore the role of GABA in controlling epileptiform activity in the neonatal cerebral cortex. Bath application of GABAA receptor antagonists induced spontaneous generation of large-amplitude population discharges resembling interictal bursts, a form of epileptiform activity; activation of GABAA receptors reduced the amplitude of interictal bursts. Interictal bursts were mediated by glutamatergic neurotransmission, demonstrating that glutamate synapses are functional in the neonate. We conclude that GABA is inhibitory in the neonatal cerebral cortex because it serves to suppress excitatory synchronous activity. Interictal bursts in the neonatal hippocampus were generated in a temporally precise rhythm. The rhythmicity of interictal bursts was not modulated by GABAB receptors, calcium activated potassium conductances, or internally released calcium, butmanipulations that facilitate or suppress the hyperpolarization-activated cation current, Ih, increased or decreased, respectively, the frequency of the bursts. We conclude Ih plays a major role in pacing neonatal interictal bursts. Immunocytochemistry illustrated that Ih channel subunits in neonatal pyramidal neurons were distributed predominately in somata, while in the juvenile and mature hippocampus and neocortex the subunits were mostly found in GABAergic terminals and in the membrane of apical dendrites of pyramidal neurons, with diminished or no expression inside the somata. We conclude that the unique expression of Ih channel subunits in the neonatal hippocampus could contribute to the increased temporal precision of interictal bursts at this developmental stage

Valproate and 4-methyloctanoic acid, an analogue of valproate, in animal models of epilepsy

Valproic acid (VPA) is a commonly used drug for the treatment of epilepsy, bipolar disorder and migraine, yet its mechanisms of action are unknown. The neuroprotective effect of VPA has been hypothesized to be secondary to inhibition of the cAMP/protein kinase A (PKA) pathway. Here, the result show that VPA (1mM) inhibited mossy fibre long-term potentiation induced (LTP) by application of high frequency stimulation in dentate gyrus. Furthermore, VPA (1mM) inhibited enhancement of mossy fibre responses induced by application of forskolin (50 μM), consistent with an effect on the PKA pathway. Using biochemical assays, it was further demonstrated that this was not due to a direct effect on PKA, but resulted from inhibition of adenylyl cyclase. The results further show using in vitro seizure models (Pentylenetetrazole model and low- Mg2+ model) that this mechanism cannot fully explain VPA’s anti-seizure effect, but rather, by modifying synaptic plasticity, it may be more important for VPA’s antiepileptogenic and neuroprotective action. VPA therefore has distinct mechanisms of action that contribute to its diverse biological activity. In hippocampi from epileptic rats (following pilocarpine-induced status epilepticus), but not in control tissue, VPA affects short-term plasticity, indicating that VPA may have specific effects in epileptic rather than control animals. Using in vitro seizure models (Pentylenetetrazole model and low-Mg2+ model) and an in vivo status epilepticus model (the perforant pathway stimulation model), 4- methyloctanoic acid is further established that it is a more potent antiepileptic drug than VPA and provides neuroprotective effects which are similar to VPA. Furthermore, 4- methyloctanoic acid (1mM) inhibited enhancement of mossy fibre responses induced by application of forskolin (50 μM), indicating that 4-methyloctanoic acid shares the same effect as VPA on modulation of PKA

The Modulation of Epileptiform Activity in Rat Hippocampal Slices by Adenine Nucleotides

In this study the most consistent method of inducing epileptiform activity in hippocampal slices involved recording from the pyramidal cell layer of the CA3 region whilst bathing the slice in a medium containing no added magnesium and 4-aminopyridine (4AP) (50?M). The bursts produced were interictal in nature and inhibited by kynurenate (ImM) or 2-amino-5-phosphonopentanoic acid (20 and 40muM) suggesting that both NMDA and non-NMDA receptors were involved. The effect of ATP and other adenine nucleotides on the rate of spontaneous epileptiform activity was investigated. ATP and adenosine were equipotent at decreasing discharge rate at concentrations above 10muM. The depression produced by ATP was characterised by being inhibited by the A1 receptor antagonist 8-cyclopentyl, 1,3-dimethylxanthine (CPT) but resistant to adenosine deaminase (at a concentration which annulled the effect of adenosine). P2 receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and suramin) were also unable to block ATP depression of bursting. AMP depressed epileptiform activity similarly to ATP. Its effect was inhibited by CPT and 5'-adenylic acid deaminase (AMPase). AMPase also blocked the depression in discharge rate elicited by ATP. 5'-nucleotidase is involved in the metabolism of AMP to adenosine. The combination of 5'-nucleotidase and adenosine deaminase with AMP or ATP resulted in no significant change in the frequency of activity. These later results, therefore, suggest that ATP acts through metabolism to AMP. The antagonism by CPT supports the involvement of A1 receptors. The ATP analogues alpha,beta-methyleneATP and 2-methylthioATP were also tested as well as uridine triphosphate. Only alpha,beta-methyleneATP (10muM) altered the discharge rate producing an increase in the frequency of spontaneous activity which was inhibited by suramin and PPADS. These results are indicative of an excitatory P2X receptor in the hippocampal CA3 region. Several have been cloned and found expressed in the hippocampus but none with the characteristics displayed in this study. BzATP, an agonist at the P2Z receptor, decreased the discharge rate during a 10 minute perfusion. This depression was not reversed upon washing but continued to progress. The combination of BzATP and CPT produced an increase in discharge rate which was smaller than that caused by CPT alone. This suggests that A1 receptors may not be involved in producing the effect of BzATP. The alpha, omega-adenine dinucleotides Ap4A and Ap5A produced a concentration-dependent depression in discharge rate. Adenosine deaminase inhibited the effect of 1muM but not the early stages of the depression produced by 10?M Ap4A and Ap5A. CPT but not AMPase antagonised the depression in rate produced by Ap4A. This suggests that in the CA3 region of the hippocampus AP4A and Ap5A act partly by stimulating xanthine sensitive receptors directly and partly through the formation of the metabolite, adenosine. AMPase initially elevated the discharge rate to a small extent but a subsequent fall in rate occurred during the wash period which continued until a plateau was reached at which the rate remained for up to 90 minutes. IMP was ineffective. Dialysis of AMPase neither affected the enzymatic activity of AMPase nor the following depression. Boiling the enzyme rendered it inactive in that it could no longer prevent the effect of AMP. The sustained depression in activity produced by AMPase was also inhibited by denaturisation. It was proposed, therefore, that AMPase induces a form of long term depression (LTD). The induction of this LTD, as measured by an effect on discharge rate, was not altered by antagonists of A1, opioid or GABAA receptors nor inhibitors of cyclo-oxygenase, nitric oxide synthase or protein kinase

Dynamical Changes in Neuronal Network Function Underlying Epileptogenesis in the Temporal Lobe

Epilepsy is a common neurological disorder characterized by recurrent seizures. Even before the presentation of the first seizure, it is believed there is a chronic pathogenic process underlying the network, cellular and synaptic changes which result in the development of symptomatic epilepsy – termed epileptogenesis. Epileptogenesis involves several crucial, progressive steps which develop over a period of weeks, months or sometimes years: the initial precipitating assault/injury, the latent period, and spontaneous recurrent seizures (SRS). Understanding the development and progression of epileptogenesis within the temporal lobe offers a new avenue for controlling or even preventing the development of symptomatic epilepsy. Animal models of epilepsy allow researchers to investigate the pathogenic process’ of epilepsy and explore possible therapeutics, including anti-epileptic drugs. A low mortality, high morbidity model for epilepsy termed the reduced intensity status epilepticus (RISE) model was developed to study temporal lobe epilepsy. Investigation of the early stages of epileptogenesis involved ex vivo local field potential (LFP) and whole-cell patch clamp recordings of extracted brain slices from RISE animals during the latent period (weeks 2, 3, 4, 5, and 6 post-induction) and during the SRS stage of epileptogenesis (>3 months post-induction). Using several measures of brain excitability, it was found there is a marked reduction in brain excitability for RISE animals during the later stages of epileptogenesis (weeks 4 – 6 post-induction) compared to aged-matched control (AMC) in hippocampal subregions CA1 and CA3. However, there appears to be recovery to AMC or even above as animals enter SRS despite now displaying electrographic and behavioural seizures. Previous work studying the RISE model of epilepsy has uncovered a dramatic loss of the glutamatergic ionotropic AMPA receptors (AMPARs) and their associated accessory proteins in the hippocampus during the latent period which continues into SRS. Tianeptine is an atypical anti-depressant which is already known to modulate AMPAR function by increasing channel conductance and by increasing AMPAR trafficking and anchoring into the postsynaptic membrane. Control experiments confirmed this by revealing a marked increase in hippocampal gamma oscillatory power when conducting LFP experiments across all ages studied. Given the proposed mechanism of action of tianeptine, tianeptine (10µM) was then studied as a potential anti-epileptogenic drug to modify the progression of epileptogenesis within the RISE model. Ex vivo LFP and patch clamp recordings were taken and found tianeptine was able to recover the hippocampal oscillation during the early stages of epileptogenesis but had subregional differences as the epileptogenesis progressed. Under both spontaneous and kainic acid (KA) conditions, tianeptine had minimal effects on the gamma oscillations in both hippocampal subregions CA1 and CA3 during the latent period (6 weeks post-induction) compared to AMC. However, once RISE animals enter SRS, tianeptine was only capable of modulating gamma oscillations in CA1 (and not CA3) in KA conditions. Therefore, showing subregional differences in the progression of epileptogenesis. Finally, to explore seizure susceptibility, the 0 Mg2+ in vitro model of epilepsy was employed in the hippocampus. Using several measures, it was found RISE animals were more susceptible to generate seizures and seizure-like activity. Seizure susceptibility decreased as RISE animals entered later into the latent period (weeks 5 – 6 post-induction) and again increased as they entered SRS. This combines with the above findings of reduced brain excitability during the latent period which likely drives the progression of epileptogenesis into SRS. Overall, this thesis shows there are dynamical changes in neuronal network function which underlie epileptogenesis in the temporal lobe