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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

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.

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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This thesis provides insights into the mechanism by which thalamocortical axons (TCAs) approach the cortex from their origin in the thalamus. Previous studies suggested that the reciprocal projections from the prethalamus and the ventral telencephalon guide TCAs to descend through the prethalamus and cross the diencephalic-telencephalic boundary (DTB), after which TCAs navigate through permissive corridor cells in the ventral telencephalon and cross the pallial-subpallial boundary (PSPB) before reaching their final targets in the cortex. The ‘Handshake Hypothesis’ proposed that pioneer axons from cortical preplate neurons guide TCAs into corresponding cortical areas. However, there is a lack of convincing evidence on whether TCAs need any guidance to cross the PSPB. In the current study, Adenomatous polyposis (Apc) gene is conditionally deleted from the cortex, by using Emx1Cre-APCloxP recombination technology. Apc is widely expressed in the nervous system including the cortical plate of the cortex and regulates axonal growth and neuronal differentiation. Deleting Apc may block neurite extension and/or affect the formation of attractive or repulsive cues in the cortex. By using DiI tracing as well as L1 immunohistochemistry techniques, I showed that in the Apc mutants cortical axons are absent and that TCAs initially navigate into the ventral telencephalon normally but fail to complete their journey into the cortex. They stop as they approach the PSPB, although the PSPB doesn’t seem to be directly affected by the mutation of Apc in the cortex. Additionally, Ig-Nrg1 (Neuregulin-1), the secreted protein that was suggested to play long-range roles in attracting TCAs towards the cortex, is present in the Apc mutant. This implies that Ig-Nrg1 is not sufficient for guiding TCAs into the cortex, and that additional guidance factors are needed. Moreover, my in vitro explant culture experiments show that the mutant cortex neither repel nor inhibit thalamic axonal outgrowth, indicating that the failure of TCAs in reaching the cortex is not due to the change of repulsive cues secreted by the mutant cortex. It rather indicates that the guidance factors for TCAs are likely to function through cell-cell contact mediated mechanisms. The Apc mutant cortex lacks these guidance factors, which might be the cortical axons. In conclusion, my data reveal a choice point for TCAs at the PSPB. Guidance factors from the cortex are needed for TCAs to cross the PSPB, which are absent in the Apc mutant. TCAs may need the direct contact with cortical axons and use them as an axonal scaffold to navigate into the cerebral cortex.

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Abstract (for Chapter 2) Objective: The aim was to test the hypothesis that the order is a significant phylogenetic grouping in terms of quantifiable gyrification indices. Method: The gyrification index (GI) was measured from serial sections of the brain of twenty five different mammalian species, representing the different orders i.e. primates, carnivores, artiodactyls and rodents. Image J analysis was used to measure the contours of the cerebral cortex and the GI was calculated using three different methods of analysis i.e. complete vs outer; gyral vs sulcal and outer vs inner surface contours. The measurements were then computed against the brain weights of each species within the order. Results: An increasing GI correlates with an increasing brain weight in all the mammalian orders. Each order has its own specific allometric patterns that are significantly different from the other orders examined. The artiodactyls were the mammals with the most gyrencephalic brains, these species being significantly more gyrencephalic than all other mammals when species of similar brain weights are compared. The North American beaver has an atypically lissencephalic brain for its size, differing from the trend for increased gyrencephaly found in the other rodent species examined. Conclusions: Our results show definite trends and patterns specific to each order. So it would seem that the order is a significant phylogenetic grouping in terms of this neural parameter, from which we can predict with a reasonable degree of certainty, the GI of any species of a particular order, if we know the brain weight. Abstract (for Chapter 3) The mammalian order has proven to be a significant phylogenetic grouping in terms of gyrification from which we can predict with a reasonable degree of certainty, the GI of any species of a particular order, if we know the brain weight. We have attempted in the present study to identify potential causes for gyrification at the class level by investigating relationships at the level of the order. It appears that clues to the extent and pattern of gyrification in the different mammalian orders might be related to the bones that constitute the braincase. The external surface areas of the bones of the cranial vault of seventeen different mammalian species were measured using a microscribe digitiser. These values were plotted against brain weight from which we could then calculate residual values, determining if there was more or less external cranial vault area than expected for the size of the brain. These residuals were then plotted against the gyrification indices determined in a previous study for the species examined. Results indicated that for the primates and artiodactyls the skull may potentially be considered as a limiting factor on the expansion of the cerebral cortex; however, the carnivore and rodent orders show conflicting results which suggest that the relative surface area of the skull appears to have no effect on the quantitative extent of gyrencephaly. These inconclusive findings suggest that causes contributing to the quantitative extent of gyrification across mammals may be multifactorial, and more parameters may need to be included in the analysis to arrive at an answer.