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

Somnotate: a probabilistic sleep stage classifier for studying vigilance state transitions

Electrophysiological recordings from freely behaving animals are a widespread and powerful mode of investigation in sleep research. These recordings generate large amounts of data that require sleep stage annotation (polysomnography), in which the data is parcellated according to three vigilance states: awake, rapid eye movement (REM) sleep, and non-REM (NREM) sleep. Manual and current computational annotation methods ignore intermediate states because the classification features become ambiguous, even though intermediate states contain important information regarding vigilance state dynamics. To address this problem, we have developed "Somnotate"—a probabilistic classifier based on a combination of linear discriminant analysis (LDA) with a hidden Markov model (HMM). First we demonstrate that Somnotate sets new standards in polysomnography, exhibiting annotation accuracies that exceed human experts on mouse electrophysiological data, remarkable robustness to errors in the training data, compatibility with different recording configurations, and an ability to maintain high accuracy during experimental interventions. However, the key feature of Somnotate is that it quantifies and reports the certainty of its annotations. We leverage this feature to reveal that many intermediate vigilance states cluster around state transitions, whereas others correspond to failed attempts to transition. This enables us to show for the first time that the success rates of different types of transition are differentially affected by experimental manipulations and can explain previously observed sleep patterns. Somnotate is open-source and has the potential to both facilitate the study of sleep stage transitions and offer new insights into the mechanisms underlying sleep-wake dynamics

Indirect effects of Halorhodopsin activation: potassium redistribution, non-specific inhibition and spreading depolarisation

The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, Halorhodopsin. We recorded extracellular K+ concentration, [K+]extra, in neocortical brain slices prepared from young adult mice (both sexes) which express Halorhodopsin in pyramidal cells. Strong Halorhodopsin activation induced a pronounced drop in [K+]extra, that persisted for the duration of illumination. Pharmacological blockade of K+ channels reduced the amplitude of this drop, indicating that it represents K+ redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl- directly, and K+ secondarily. When the illumination period ended, a rebound surge in extracellular [K+] developed over tens of seconds, partly reflecting the previous inward redistribution of K+, but additionally driven by clearance of Cl- coupled to K+ by the potassium-chloride co-transporter, KCC2. The drop in [K+]extra during light activation leads to a small (2-3mV) hyperpolarization also of other cells that do not express Halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of Halorhodopsin causes cortical spreading depolarisations (CSDs), both in vitro and in vivo. This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K+]extra is low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally

Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice.

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools for remote control of targeted cell populations using chemical actuators that bind to modified receptors. Despite the popularity of DREADDs in neuroscience and sleep research, potential effects of the DREADD actuator clozapine-N-oxide (CNO) on sleep have never been systematically tested. Here, we show that intraperitoneal injections of commonly used CNO doses (1, 5, and 10 mg/kg) alter sleep in wild-type male laboratory mice. Using electroencephalography (EEG) and electromyography (EMG) to analyse sleep, we found a dose-dependent suppression of rapid eye movement (REM) sleep, changes in EEG spectral power during non-REM (NREM) sleep, and altered sleep architecture in a pattern previously reported for clozapine. Effects of CNO on sleep could arise from back-metabolism to clozapine or binding to endogenous neurotransmitter receptors. Interestingly, we found that the novel DREADD actuator, compound 21 (C21, 3 mg/kg), similarly modulates sleep despite a lack of back-metabolism to clozapine. Our results demonstrate that both CNO and C21 can modulate sleep of mice not expressing DREADD receptors. This implies that back-metabolism to clozapine is not the sole mechanism underlying side effects of chemogenetic actuators. Therefore, any chemogenetic experiment should include a DREADD-free control group injected with the same CNO, C21, or newly developed actuator. We suggest that electrophysiological sleep assessment could serve as a sensitive tool to test the biological inertness of novel chemogenetic actuators