Development and application of inhibitory luminopsins for the treatment of epilepsy

Optogenetics has shown great promise as a direct neuromodulatory tool for halting seizure activity in various animal models of epilepsy. However, light delivery into the brain is still a major practical challenge that needs to be addressed before future clinical translation is feasible. Not only does light delivery into the brain require surgically implanted hardware that can be both invasive and restrictive, but it is also difficult to illuminate large or complicated structures in the brain due to light scatter and attenuation. We have bypassed the challenges of external light delivery by directly coupling a bioluminescent light source (a genetically encoded Renilla luciferase) to an inhibitory opsin (Natronomonas halorhodopsin) as a single fusion protein, which we term an inhibitory luminopsin (iLMO). iLMOs were developed and characterized in vitro and in vivo using intracellular recordings, multielectrode arrays, and behavioral testing. iLMO2 was shown to generate hyperpolarizing outward currents in response to both external light and luciferase substrate, which was sufficient to suppress action potential firing and synchronous bursting activity in vitro. iLMO2 was further shown to suppress single-unit firing rate and local field potentials in the hippocampus of anesthetized and awake animals. Finally, expression of iLMO was scaled up to multiple structures of the basal ganglia to modulate rotational behavior of freely moving animals in a hardware-independent fashion. iLMO2 was further utilized to acutely suppress focal epileptic discharges induced by intracerebral injection of bicuculline and generalized seizures resulting from systemic administration of pentylenetetrazol. Inhibitory luminopsins have enabled the possibility of optogenetic inhibition of neural activity in a non-invasive and hardware-independent fashion. This work increases the versatility, scalability, and practicality of utilizing optogenetic approaches for halting seizure activity in vivo.Ph.D

Hyperpolarization by Activation of Halorhodopsin Results in Enhanced Synaptic Transmission: Neuromuscular Junction and CNS Circuit

Optogenetics offers a unique method to regulate the activity of select neural circuits. However, the electrophysiological consequences of targeted optogenetic manipulation upon the entire circuit remain poorly understood. Analysis of the sensory-CNS-motor circuit in Drosophila larvae expressing eHpHR and ChR2-XXL revealed unexpected patterns of excitability. Optical stimulation of motor neurons targeted to express eNpHR resulted in inhibition followed by excitation of body wall contraction with repetitive stimulation in intact larvae. In situ preparations with direct electrophysiological measures showed an increased responsiveness to excitatory synaptic activity induced by sensory stimulation within a functional neural circuit. To ensure proper function of eNpHR and ChR2-XXL they were expressed in body wall muscle and direct electrophysiological measurements were obtained. Under eNpHR induced hyperpolarization the muscle remained excitable with increased amplitude of excitatory postsynaptic synaptic potentials. Theoretical models to explain the observations are presented. This study aids in increasing the understanding of the varied possible influences with light activated proteins within intact neural circuits

Optonongenetic enhancement of activity in primary cortical neurons

It has been recently demonstrated that the exposure of naive neuronal cells to light—at the basis of optogenetic techniques and calcium imaging measurements—may alter neuronal firing. Indeed, understanding the effect of light on nongenetically modified neurons is crucial for a correct interpretation of calcium imaging and optogenetic experiments. Here we investigated the effect of continuous visible LED light exposure (490 nm, 0.18−1.3 mW/mm2) on spontaneous activity of primary neuronal networks derived from the early postnatal mouse cortex. We demonstrated, by calcium imaging and patch clamp experiments, that illumination higher than 1.0 mW/mm2 causes an enhancement of network activity in cortical cultures. We investigated the possible origin of the phenomena by blocking the transient receptor potential vanilloid 4 (TRPV4) channel, demonstrating a complex connection between this temperature-dependent channel and the measured effect. The results presented here shed light on an exogenous artifact, potentially present in all calcium imaging experiments, that should be taken into account in the analysis of fluorescence data

Optogenetic chloride loading in neurons : implications for epilepsy

PhD ThesisAltered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory, if intraneuronal chloride [Cl-]i rises. It has proved difficult, however, to separate out the role of raised [Cl-]i from other contributory factors in complex network phenomena such as epileptic pathology. To address this, in this thesis I used Halorhodopsin to load clusters of pyramidal cells artificially with Cl-, to explore the implication of raised [Cl-]i in the generation of epileptic activity and seizure initiation. Brief Halorhodopsin activation caused substantial positive shifts in the GABAergic reversal potential (EGABA). At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high frequency oscillations with evidence of out-of-phase firing. Such firing patterns can arise from quite small shifts in the mean [Cl-]i level, within heterogeneous neuronal populations. Notably, however, chloride-loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, sub-epileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that in vitro, chloride-loading has an adjunctive role in ictogenesis. Additionally, in this thesis an optogenetic approach to extrude Cl- was also explored, as a potential way to correct the raised [Cl-]i issue in epilepsy. A strategy that was proved to be successful was the combination of ArchaerhodopsinT (ArchT) hyperpolarizing effect and the opening of light activated Cl- channel (ChloC), which together provides a driving force and a conductance for an outward Cl- flow. Co-activation of the two opsins consistently induced a negative shift in EGABA indicative of Cl- extrusion mechanism, which in the future could be tested as a new optogenetic approach to control epilepsy

Using Machine Learning and Computer Simulations to Analyse Neuronal Activity in the Cerebellar Nuclei During Absence Epilepsy

Absence epilepsy is a neurological disorder that commonly occurs in children. Some studies have shown that absence seizures predominantly originate either in the thalamus or the cerebral cortex. Some cerebellar nuclei (CN) neurons project to these brain areas, as explained further in Fig. 2.6 in Chapter 2. Also, some CN neurons have been observed to show modulation during the absence seizures. This indicates that they somehow participate in the seizure and hence are referred to as "participating neurons" in this thesis. In this research, I demonstrate how machine learning techniques and computer simulations can be applied to investigate the properties and the input conditions present in these participating neurons. My investigation found a sub-group of CN neurons, with similar interictal spiking activity, spiking activity between the seizures, that are most likely to participate in seizures. To investigate the input conditions present in the CN neurons that produce this type of interictal activity, I used a morphologically realistic conductance based model of an excitatory CN projection neuron [66] and optimised the input parameters to this model using an Evolutionary Algorithm (EA). The results of the EA revealed that these participating CN neurons receive a synchronous and bursting input from Purkinje cells and bursting input with long intervals(approx. 500ms) from mossy fibre. The same interictal activity can also be produced when the Purkinje cell input to the CN neuron is asynchronous. The excitatory input in this case also had long interburst intervals but there is a decrease in excitatory and inhibitory synaptic weight. Surprisingly, a slight change in these input parameters can change the interictal spiking pattern to an ictal spiking pattern, the spiking pattern observed during absence seizures. I also discovered that it is possible to prevent a participating CN neuron from taking part in the seizures by blocking the Purkinje cell input

Neuronal Signalling Studied with Light-Activated Ion Channels to Target Interneurons, Entrain Hippocampal Gamma Oscillations and Suppress Epileptiform Activity

The versatility and the electrophysiological characteristics of the light-sensitive ion-channels channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR) make these optogenetic tools potent candidates in controlling neuronal firing in models of epilepsy and in providing insights into the physiology and pathology of neuronal network organization and synchronization. The experiments described in this thesis were designed to determine if the ChR2/NpHR system allows specific targeting and manipulation of interneuron activity in cortex and hippocampus, if it allows modulation of gamma oscillations in the hippocampal CA3 area, and if it constitutes a reliable toolbox enabling systematic analysis of epileptic neural circuits and a novel anti-epileptic treatment strategy that relies on optical activation of neurons to interrupt seizures. After successful generation of lentiviral constructs containing opsin genes driven by the interneuron-specific promoters glutamic acid decarboxylase (GAD) 67 and cholecystokinin (CCK) and the production of high-titre lentivirus, it was possible to demonstrate that both microbial opsins are expressed in neuronal cultures and rat motor cortex and hippocampus. Expression of the constructs, however, was not specific for interneurons and expression levels were low compared to the same opsins driven by the calcium calmodulin-binding kinase 2a (Camk2a) promoter: either fluorescence was only visible after immunofluorescent labelling or optical control of neural activity was not achievable despite visible fluorescence. In a separate set of experiments, stimulation of Camk2a–ChR2 with ramps of blue light induced oscillations in hippocampal area CA3. Oscillations entrained to modulated ramps over a wide range of frequencies with a frequency-dependent phase relationship. Finally, optical stimulation of halorhodopsin successfully reduced high frequency epileptic EEG activity in a tetanus toxin rat model of focal epilepsy. These results demonstrate that targeting opsins to interneurons with the GAD67 and CCK-promoters is not specific, that the CA3 network has properties that allow it to entrain and synchronize to input from the dentate gyrus, which may help explain how coherence between these two anatomically coupled networks arises, and finally, that optical inhibition of HF discharges with NpHR targeted to pyramidal neurons represents an exciting new tool to be pursued in models of epilepsy both to dissect epileptic networks and for the development of other optogenetic neuromodulation therapies

An investigation into dysfunctional feed-forward inhibition within the cortico-thalamocortical network on absence seizure generation using DREADD technology

Childhood absence epilepsy (CAE) is one of the most prevalent paediatric epilepsies, accounting for between 10-17% of all diagnosed cases of epilepsies seen in school-aged children. Absence seizures are characterized by behavioural arrest/loss of awareness and electrographic signature of spike-wave discharges (SWDs) measuring 2.5-4 Hz on an electroencephalogram (EEG). These brief episodes of impaired consciousness can occur hundreds of times a day and might increase the chance of physical injury when undertaking activities like swimming and cycling. Current treatment options are not sufficient and up to 30% of patients are pharmaco-resistant. ~60% of children with CAE have severe neuropsychiatric comorbid conditions including attention deficits, mood disorders, impairments in memory and cognition. Ethosuximide (ETX), an anti-absence epileptic drug which was first introduced almost six decades ago remains the first choice for initial monotherapy for the treatment of CAE. Large-scale clinical trials suggested that efficacy of ethosuximide is considerably lower than previous findings. Thus, safe, effective and patient specific treatment approach is imperative. For this, it is crucial first to understand the precise cellular and molecular mechanisms of absence seizures which may enable the development of novel therapeutic targets and discovery of new anti-epileptic drugs (AEDs). EEG and functional imaging evidence suggest that absence seizures are likely due to aberrant activity within the cortico-thalamocortical (CTC) network. Studies involving the genetic rodent models have shown that the cortex is the driving source for the origin of SWDs but is not capable of maintaining discharges on its own, nor is the thalamus. General consensus is that, within the CTC network, a cortical focus initiates rhythmic epileptic discharges, however, once the rhythmic oscillations are established, both the cortex and thalamus form an integrated network. Rhythmic absence-SWDs are sustained via the cortex and thalamus driving each other. Within the CTC network, feed-forward inhibition (FFI) is essential to prevent runaway excitation. FFI is mediated by fast spiking parvalbumin expressing (PV+) inhibitory interneurons in the somatosensory cortex (SScortex) and the reticular thalamic nucleus (RTN). Studies conducted in well-established stargazer mouse model of absence epilepsy with a genetic deficit in stargazin i.e. TARP [a transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein] have shown reduced expression of GluA4-AMPARs at excitatory synapses in feed-forward inhibitory (PV+) interneurons in the SScortex and RTN thalamus of the CTC network. However, the extent of this deficit in AMPARs expression impacting FFI and possibly contributing towards generation of absence-SWDs is not established via functional studies. Hence, this thesis was aimed at investigating the impact of dysfunctional feed-forward inhibitory PV+ interneurons within CTC network on absence seizure generation and behaviour. For this purpose, inhibitory and excitatory Designer Receptors Exclusively Activated by Designer Drug (DREADD) approach was utilized to silence/excite feed-forward inhibitory PV+ interneurons within the CTC network. DREADD mediated regional silencing of PV+ interneurons within the CTC network generated ETX-sensitive absence-like SWDs. Activating PV+ interneurons either prevented or suppressed pentylenetetrazole (PTZ)-induced absence-SWDs. Finally, impact of impaired FFI in γ-aminobutyric acid (GABA) levels by affecting its synthesizing enzymes (GADs) and transporter proteins (GATs) in stargazer animal model of absence epilepsy and CNO treated inhibitory Gi-DREADD animals was determined. Results indicate that upregulation of GAD65 in the SScortex of epileptic stargazers may be a consequence of absence seizures or this may have contribution in absence seizure generation. The work presented in this thesis provide an electrophysiological insight into the possible mechanism underlying the absence seizure generation. This work provides convincing evidence that dysfunctional feed-forward inhibitory PV+ interneurons within the CTC network is likely to be involved in altered excitation/inhibition balance resulting SWDs as activating these interneurons dramatically protected animals from PTZ induced absence seizures. The clinical relevance of this study is that it potentially uncovers the possibility of focally targeting PV+ interneurons within the CTC network to control absence seizures in human patients

Optogenetic investigation of cortical network dynamics in epilepsy

Ph. D. ThesisUnderstanding the cortical network properties which determine the susceptibility of cortex to the onset of seizures remains a major goal of epilepsy research. The determinants of seizure risk in cortical networks are dynamic, showing dependency on intrinsic cortical activity and environmental influences. The failure to identify reliable electrographic indicators of imminent seizure onset suggests that the contributory factors may not be electrographically obvious. A strong candidate for such a property is the activity dependent disinhibition of the excitatory network which results from increases in intracellular chloride concentration. Chloride loading has been shown previously to occur during periods of intense neuronal activity, resulting from concomitant excitatory and inhibitory synaptic transmission. To explore how network dynamics evolve from a stable healthy state to one permissive for the onset and propagation of seizures, I used an optogenetic approach to selectively interrogate dynamic changes to excitatory transmission between the principal cells of the cortical circuit following an acute ictogenic challenge, both in vitro and in vivo. Using ultra-low frequency optogenetic stimulation genetically targeted to the pyramidal cells of neocortex, I demonstrate that epileptiform activity, which develops spontaneously following an acute chemoconvulsant challenge, can both be reduced and monitored, using an active probing strategy. Delivering continuous and focal optogenetic stimulations to superficial neocortex and regions of the hippocampal formation evokes glutamatergic responses in the LFP which can be used to assay dendritic excitability in the network. At ultralow frequencies, between 0.1-0.033 Hz, optogenetic stimulation markedly reduced the rate of evolution of epileptiform activity, when delivered to neocortex or hippocampal structures, in acutely prepared adult mouse brain slices bathed in 0Mg2+ perfusate. The response evoked by these test pulses undergoes an all-or-nothing transformation observable in the LFP which reliably telegraphed the onset of ictal activity in two models of epilepsy. Using electrophysiological tools and 2-photon calcium imaging of individual dendrites, I demonstrate that this phenomenon likely reflects a reduction in the threshold for dendritic spikes. Using an anatomically realistic computational model pyramidal cell I show that this effect is reproduced by modest positive shifts in the GABAergic reversal potential in distal pyramidal cell dendrites. Finally, I report preliminary data demonstrating a potential mechanism for the diurnal modulation of seizure risk. Diurnal periodicity in seizure susceptibility have been observed longitudinal recordings from both patients and chronically epileptic experimental animals. Using the optical chloride sensor ClopHensor I examine steady-state pyramidal cell chloride concentration over the diurnal period and show that periodicity in chloride homeostasis is consistent with the phase of diurnally modulated seizure risk. In this thesis I use a range of optical and electrophysiological tools to explore the contribution of dynamic chloride concentration in pyramidal cells in determining cortical susceptibility to seizures onset. Using two acute epilepsy models I demonstrate that an assayable increase in dendritic excitability precedes ictogenesis, and demonstrate a potential mechanism by which variation in [Cl-]i can give rise to this effect. I go on to show diurnal variation in [Cl-]i in cortical pyramidal cells, and link this to circadian modulation of susceptibility to chemoconvulsants, suggesting a functional mechanism for the dynamic seizure risk observed in epileptic patients

Neural Bursting Activity Mediates Subtype-Specific Neural Regeneration by an L-type Calcium Channel

Axons are injured after stroke, spinal cord injury, or neurodegenerative disease such as ALS. Most axons do not regenerate. A recent report suggests that not all neurons are poor regenerators, but rather a small subset can regenerate robustly. What intrinsic property of these regenerating neurons allows them to regenerate, but not their neighbors, remains a mystery. This subtype-specific regeneration has also been observed in Drosophila larvae sensory neurons. We exploited this powerful genetic system to unravel the intrinsic mechanism of subtype-specific neuron regeneration. We found that neuron bursting activity after axotomy correlates with regeneration ability. Furthermore, neuron bursting activity is necessary for regeneration of a regenerative neuron subtype, and sufficient for regeneration of a non-regenerative neuron subtype. This optogenetically-induced regeneration is dependent on a bursting pattern, not simply overall activity increase. We conclude that neuron bursting activity is an intrinsic mechanism of subtype-specific regeneration. We then discovered through a reverse genetic screen that an L-type voltage gated calcium channel (VGCC) promotes neuron bursting and subsequent regeneration. This VGCC has high expression in the regenerative neuron and weak expression in the non-regenerative neuron. This suggests that VGCC expression level is the molecular mechanism of subtype-specific neuron regeneration. Together, our findings identify a cellular and molecular intrinsic mechanism of subtype-specific regeneration, which is why some neurons are able to regenerate while the majority of neurons do not. Perhaps VGCC activation or neuron activity pattern modulation could be used therapeutically for patients with nerve injury

Single unit action potential recordings in humans : implications for epilepsy

PhD ThesisSpike-sorting algorithms have been used to identify the firing patterns of isolated neurons (‘single units’) from implanted electrode recordings in patients undergoing assessment for epilepsy surgery, but we do not know their potential for providing helpful clinical information. It is important to characterize both the stability of these recordings and also their context. I therefore analysed microelectrode array recordings from four patients undergoing videotelemetry monitoring for surgical evaluation of focal neocortical epilepsies. I investigated whether units could be followed reliably over prolonged periods during the initial days postimplantation in humans. Unit specific features showed stability over at least 48 hours, including across multiple seizures. A critical consideration regarding the clinical information in these recordings is where the units are located with respect to the focus of pathology. Recent analyses have demonstrated the importance of considering seizure activity in terms of two distinct territories: the ictal core and penumbra. The pathological information in these two areas is likely very different. I isolated unit recordings from several hundred neurons across the four patients in the peri-ictal period, with reference to these regions. A key finding was that in the penumbra, spike stereotypy was maintained even during the seizure, with little alteration to population firing rate. In contrast, within ictal core territories, regions characterized by intense hypersynchronous firing, spike sorting failed as the units were incorporated into the seizure activity. Recovery of spike shape was rapid following seizure termination, and the mean firing rate returned to pre-ictal levels in the first few minutes. However, the spatiotemporal activity of units in the ictal core displayed significant alterations to firing patterns, lasting at least 30 minutes after seizure termination. These observations lay the foundation for future investigations of how these recordings may inform clinical practice.Wellcome Trus