The most widely accepted hypotheses suggest that general anaesthetics interrupt conscious processes in the brain by decreasing synaptic excitation or by potentiating synaptic inhibition, especially in the neocortex. The putative transmitters in the neurological systems that generate neocortical arousal include acetylcholine, glutamate and γ-aminobutyrate (GABA). The primary objective here was to determine the neuronal mechanisms by which anaesthetics may obtund this arousal. The majority of the investigations were carried out on pyramidal neurons in layers IV and V of guinea pig neocortex (in vitro slices), using intracellular recording and pharmacological, including microiontophoretic, techniques. Bath applications of structurally dissimilar anaesthetics, isoflurane - a halogenated ether, and Althesin - a steroidal preparation, in concentrations of 0.5-2.5 minimum alveolar concentration (MAC) and 10-1300 μM, respectively, produced a small hyperpolarization (3-5 mV) which was associated with an increase in input conductance (10-30%). The lower concentrations (0.5-1.5 MAC and 10-200 μM) of these agents which are most relevant to the production of unconsciousness did not significantly affect the passive membrane properties. However, they produced striking decreases in spontaneous activities and the repetitive spike firing evoked by orthodromic (electrical) stimulation or intracellular current injections. Because the observed changes in membrane properties could not explain the reduction in neuronal excitability, the effects of anaesthetics were investigated extensively on excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). The application of isoflurane or Althesin induced a dose-dependent, reversible depression in the amplitude of EPSPs, with EC₅₀s of 1 MAC and ~50 μM, respectively. The IPSPs also were reduced in a dose-dependent manner. In order to eliminate possible shunting of the EPSPs by the GABA-activated Cl-conductance that produces the IPSP in the observed EPSP-IPSP sequence, a GABA[symbol omitted]-antagonist, bicuculline, was additionally applied. Despite this IPSP-blockade, the anaesthetics strongly depressed the EPSPs as well as epileptiform activities evoked by subpial electrical stimulation. In cognizance of the possibility that a postsynaptic attenuation of responsiveness to transmitter substances may be involved in the EPSP depression, the neuronal sensitivities to acetylcholine, glutamate. and GABA were determined. Anaesthetic administration markedly reduced the depolarizations and associated conductance changes evoked by dendritic applications of acetylcholine, glutamate and N-methyl-D-aspartate (NMDA). The hyperpolarizing responses to somatic applications of GABA were not affected significantly whereas the depolarizing effects observed with its dendritic application were slightly depressed. Same degree of selectivity also was evident from the lower EC₅₀s for the isoflurane- and Althesin-induced depressions of responses to acetylcholine compared with glutamate. Under in vitro conditions of hypomagnesia the responses to acetylcholine were totally blocked and the order of depression in the responses to GABA and glutamate was reversed; this may be of importance in the mechanism for the known increase in anaesthetic requirements in clinical syndromes associated with hypomagnesaemia. Because the genesis of synaptic transients is affected by Ca²⁺ influx or disposition, the interactions of anaesthetics were investigated on spike afterhyperpolarizations (AHPs). The AHPs which are produced specifically by a Ca²⁺ -activated K⁺ -conductance were suppressed by the anaesthetics in a dose-dependent manner under conditions where contaminating IPSPs had been blocked by bicuculline. Since the passive membrane properties were unaffected, an interference with a transmembrane Ca⁺ -influx may be involved in the anaesthetic actions. The effects of anaesthetics on glutamate-induced and voltage-dependent increases in intraneuronal Ca²⁺ ([Ca²⁺]i) were determined in cultured hippocampal neurons with a Ca-sensitive probe (Fura-2) and microspectro- fluorometric techniques. Isoflurane application depressed the increases in [Ca²⁺]i. produced by application of glutamate under conditions where its actions would be favoured at NMDA- and quisqualate-subtypes of receptors. K⁺ -induced increases in [Ca²⁺]i also were reduced by application of isoflurane, probably due to actions on voltage-dependent Ca-channels in the membrane. These investigations have provided evidence for the first time that excitatory transmitter actions in neocortex are selectively depressed by anaesthesia. A plausible mechanism would include suppression of the postsynaptic Ca-conductances associated with the AHPs and glutamatergic, as well as cholinergic interactions at pre- and post-synaptic sites on neurons involved in neocortical arousal. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

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The brain is a complex system which consists of billions of neuron cells and gives rise to diverse neural dynamics spatially and temporally. Spontaneous neural activities construct the foundation for various cognitive processing. However, caused by the limitation spatiotemporal resolution and coverage of recording methods in experiments, the organization of spatiotemporal dynamics of the self-organized brain activity remains largely unknown. Current experimental technique can optically image population voltage transients generated by pyramidal neurons across cortical layer 2/3 of the mouse dorsally with a genetically encoded voltage indicator. Such data provided unique opportunities to investigate the structure- dynamics relationship to elucidate the mechanisms of spontaneous brain activity. The aim of this thesis is to develop a systematic understanding of spatiotemporal mechanism in the mouse cortex by analyzing voltage imaging data, in collaboration with neuroscientist Dr. Knöpfel from the Imperial College London. Local oscillation properties such as duration, amplitude and oscillation forms were studies on the cortex-wide scale and be compared among brain states. Wakefulness modulated the excitability of the neural activity which influenced the duration of the oscillation and the transition of different half wave types. Relatively larger amplitude of parietal cortex reflected stronger neural activity determined by structural hierarchy. Motifs of the oscillations showed consistency in different brain states which indicated typical pathways of the wave propagations. Dynamical properties of various waves and their interactions in sedated mice were investigated. Based on phase velocity fields, there were only a small number of large-scale, cortex-wide plane wave and synchrony (standing wave) patterns during Up-Down states. Interactions of local sources and sinks can generate saddles, and interactions of local wave patterns with large plane waves can induce a change of their wave propagating direction. Local wave patterns emerged at preferred spatial locations. Specifically, sources were predominantly found in cortical regions with high cumulative input through the underlying connectome. The findings revealed the principled spatiotemporal dynamics of Up-Down states and associated them with the large-scale cortical connectome. Waking from deep anesthesia to consciousness increased the number of local wave patterns and made the spatiotemporal dynamics more complex. Although the active state increased the wave propagation speeds, the average speed decreased because of the interaction and collapse of wave patterns. Not affected by the brain states, the two principal modes with the highest variance remained stable. The first mode represented the large waves spreading across the cortex forward or backward while the second mode corresponded to the waves propagating in opposite direction in the frontal and parietal cortex. An infra-slow frequency of the wave number might reflect the bold flow and oxygenation. The characterizations presented in this thesis can be used to predict and guide measurement and analysis of large-scale brain activity. The analysis of cortex-wide neural dynamical patterns builds foundation for further investigation of their functional implications.

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abstract: LKB1/STK11 is a serine/threonine kinase first identified in C.elegans as a gene important for cell polarity and proliferation. Mutations in LKB1 are the primary cause of Peutz-Jegher’s cancer syndrome, an autosomal dominantly inherited disease, in which patients are predisposed to benign and malignant tumors. Past studies have focused on defining LKB1 functions in various tissue types, for example LKB1 regulates axonal polarization and dendritic arborization by activating downstream substrates in excitatory neurons of the developing neocortex. However, the implications of LKB1, specifically in the developing cortical inhibitory GABAergic interneurons is unknown. LKB1 deletion was achieved by using Cre-lox technology to induce LKB1 loss in cells localized in the medial ganglionic eminence (MGE) that express Nkx2.1 and generate cortical GABAergic neurons. In this research study it is suggested that LKB1 plays a role in GABAergic interneuron specification by specifically regulating the expression of parvalbumin during the development of fast-spiking interneurons. Preliminary evidence suggest LKB1 also modulates the number of Nkx2.1-derived oligodendrocytes in the cortex. By utilizing a GABAergic neuron specific LKB1 deletion mutant, we confirmed that the loss of parvalbumin expression was due to a GABAergic neuron autonomous function for LKB1. These data provide new insight into the cell specific functions of LKB1 in the developing brain. / Dissertation/Thesis / Masters Thesis Biology 2019

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