MEG responses to the perception of global structure within glass patterns

The perception of global form requires integration of local visual cues across space and is the foundation for object recognition. Here we used magnetoencephalography (MEG) to study the location and time course of neuronal activity associated with the perception of global structure from local image features. To minimize neuronal activity to low-level stimulus properties, such as luminance and contrast, the local image features were held constant during all phases of the MEG recording. This allowed us to assess the relative importance of striate (V1) versus extrastriate cortex in global form perception.

Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro

The application of mechanical insults to the spinal cord results in profound cellular and molecular changes, including the induction of neuronal cell death and altered gene expression profiles. Previous studies have described alterations in gene expression following spinal cord injury, but the specificity of this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile stresses on cultured spinal cord cells from E15 Sprague-Dawley rats, using the FX3000 Flexercell Strain Unit. We examined cell morphology and viability over a 72 hour time course. Microarray analysis of gene expression was performed using the Affymetrix GeneChip System, where categorization of identified genes was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. Changes in expression of 12 genes were validated with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).

Biochemical correlates of cognition: exploring the relationships between blood, brain and behaviour

This multi-modal investigation aimed to refine analytic tools including proton magnetic resonance spectroscopy (1H-MRS) and fatty acid gas chromatography-mass spectrometry (GC-MS) analysis, for use with adult and paediatric populations, to investigate potential biochemical underpinnings of cognition (Chapter 1). Essential fatty acids (EFAs) are vital for the normal development and function of neural cells. There is increasing evidence of behavioural impairments arising from dietary deprivation of EFAs and their long-chain fatty acid metabolites (Chapter 2). Paediatric liver disease was used as a deficiency model to examine the relationships between EFA status and cognitive outcomes. Age-appropriate Wechsler assessments measured Full-scale IQ (FSIQ) and Information Processing Speed (IPS) in clinical and healthy cohorts; GC-MS quantified surrogate markers of EFA status in erythrocyte membranes; and 1H-MRS quantified neurometabolite markers of neuronal viability and function in cortical tissue (Chapter 3). Post-transplant children with early-onset liver disease demonstrated specific deficits in IPS compared to age-matched acute liver failure transplant patients and sibling controls, suggesting that the time-course of the illness is a key factor (Chapter 4). No signs of EFA deficiency were observed in the clinical cohort, suggesting that EFA metabolism was not significantly impacted by liver disease. A strong, negative correlation was observed between omega-6 fatty acids and FSIQ, independent of disease diagnosis (Chapter 5). In a study of healthy adults, effect sizes for the relationship between 1H-MRS- detectable neurometabolites and cognition fell within the range of previous work, but were not statistically significant. Based on these findings, recommendations are made emphasising the need for hypothesis-driven enquiry and greater subtlety of data analysis (Chapter 6). Consistency of metabolite values between paediatric clinical cohorts and controls indicate normal neurodevelopment, but the lack of normative, age-matched data makes it difficult to assess the true strength of liver disease-associated metabolite changes (Chapter 7). Converging methods offer a challenging but promising and novel approach to exploring brain-behaviour relationships from micro- to macroscopic levels of analysis (Chapter 8).

Cortical oscillatory activity associated with the perception of illusory and real visual contours

We used magnetoencephalography (MEG) to examine the nature of oscillatory brain rhythms when passively viewing both illusory and real visual contours. Three stimuli were employed: a Kanizsa triangle; a Kanizsa triangle with a real triangular contour superimposed; and a control figure in which the corner elements used to form the Kanizsa triangle were rotated to negate the formation of illusory contours. The MEG data were analysed using synthetic aperture magnetometry (SAM) to enable the spatial localisation of task-related oscillatory power changes within specific frequency bands, and the time-course of activity within given locations-of-interest was determined by calculating time-frequency plots using a Morlet wavelet transform. In contrast to earlier studies, we did not find increases in gamma activity (> 30 Hz) to illusory shapes, but instead a decrease in 10–30 Hz activity approximately 200 ms after stimulus presentation. The reduction in oscillatory activity was primarily evident within extrastriate areas, including the lateral occipital complex (LOC). Importantly, this same pattern of results was evident for each stimulus type. Our results further highlight the importance of the LOC and a network of posterior brain regions in processing visual contours, be they illusory or real in nature. The similarity of the results for both real and illusory contours, however, leads us to conclude that the broadband (< 30 Hz) decrease in power we observed is more likely to reflect general changes in visual attention than neural computations specific to processing visual contours.

A general linear model for MEG beamformer imaging

A new general linear model (GLM) beamformer method is described for processing magnetoencephalography (MEG) data. A standard nonlinear beamformer is used to determine the time course of neuronal activation for each point in a predefined source space. A Hilbert transform gives the envelope of oscillatory activity at each location in any chosen frequency band (not necessary in the case of sustained (DC) fields), enabling the general linear model to be applied and a volumetric T statistic image to be determined. The new method is illustrated by a two-source simulation (sustained field and 20 Hz) and is shown to provide accurate localization. The method is also shown to locate accurately the increasing and decreasing gamma activities to the temporal and frontal lobes, respectively, in the case of a scintillating scotoma. The new method brings the advantages of the general linear model to the analysis of MEG data and should prove useful for the localization of changing patterns of activity across all frequency ranges including DC (sustained fields). © 2004 Elsevier Inc. All rights reserved.

Role of protein kinase C and NF-kappa B in proteolysis-inducing factor-induced proteasome expression in C2C12 myotubes

Proteolysis-inducing factor (PIF) is a sulphated glycoprotein produced by cachexia-inducing tumours, which initiates muscle protein degradation through an increased expression of the ubiquitin–proteasome proteolytic pathway. The role of kinase C (PKC) in PIF-induced proteasome expression has been studied in murine myotubes as a surrogate model of skeletal muscle. Proteasome expression induced by PIF was attenuated by 4alpha-phorbol 12-myristate 13-acetate (100 nM) and by the PKC inhibitors Ro31-8220 (10 muM), staurosporine (300 nM), calphostin C (300 nM) and Gö 6976 (200 muM). Proteolysis-inducing factor-induced activation of PKCalpha, with translocation from the cytosol to the membrane at the same concentration as that inducing proteasome expression, and this effect was attenuated by calphostin C. Myotubes transfected with a constitutively active PKCalpha (pCO2) showed increased expression of proteasome activity, and a longer time course, compared with their wild-type counterparts. In contrast, myotubes transfected with a dominant-negative PKCalpha (pKS1), which showed no activation of PKCalpha in response to PIF, exhibited no increase in proteasome activity at any time point. Proteolysis-inducing factor-induced proteasome expression has been suggested to involve the transcription factor nuclear factor-kappaB (NF-kappaB), which may be activated through PKC. Proteolysis-inducing factor induced a decrease in cytosolic I-kappaBalpha and an increase in nuclear binding of NF-kappaB in pCO2, but not in pKS1, and the effect in wild-type cells was attenuated by calphostin C, confirming that it was mediated through PKC. This suggests that PKC may be involved in the phosphorylation and degradation of I-kappaBalpha, induced by PIF, necessary for the release of NF-kappaB from its inactive cytosolic complex.

Stopping the clock on proteomic degradation by heat treatment at the point of tissue excision

The effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and prevent proteome degradation has been evaluated. Mouse brains were bisected immediately following excision, with one hemisphere being heat treated followed by snap freezing in liquid nitrogen while the other hemisphere was snap frozen immediately. Sections were cut by cryostatic microtome and analyzed by MALDI-MS imaging and minimal label 2-D DIGE, to monitor time-dependent relative changes in intensities of protein and peptide signals. Analysis by MALDI-MS imaging demonstrated that the relative intensities of markers varied across a time course (0-5 min) when the tissues were not stabilized by heat treatment. However, the same markers were seen to be stabilized when the tissues were heat treated before snap freezing. Intensity profiles for proteins indicative of both degradation and stabilization were generated when samples of treated and nontreated tissues were analyzed by 2-D DIGE, with protein extracted before and after a 10-min warming of samples. Thus, heat treatment of tissues at the time of excision is shown to prevent subsequent uncontrolled degradation of tissues at the proteomic level before any quantitative analysis, and to be compatible with downstream proteomic analysis.

The spatial distribution and temporal dynamics of brain regions activated during the perception of object and non-object patterns

Both animal and human studies suggest that the efficiency with which we are able to grasp objects is attributable to a repertoire of motor signals derived directly from vision. This is in general agreement with the long-held belief that the automatic generation of motor signals by the perception of objects is based on the actions they afford. In this study, we used magnetoencephalography (MEG) to determine the spatial distribution and temporal dynamics of brain regions activated during passive viewing of object and non-object targets that varied in the extent to which they afforded a grasping action. Synthetic Aperture Magnetometry (SAM) was used to localize task-related oscillatory power changes within specific frequency bands, and the time course of activity within given regions-of-interest was determined by calculating time-frequency plots using a Morlet wavelet transform. Both single subject and group-averaged data on the spatial distribution of brain activity are presented. We show that: (i) significant reductions in 10-25 Hz activity within extrastriate cortex, occipito-temporal cortex, sensori-motor cortex and cerebellum were evident with passive viewing of both objects and non-objects; and (ii) reductions in oscillatory activity within the posterior part of the superior parietal cortex (area Ba7) were only evident with the perception of objects. Assuming that focal reductions in low-frequency oscillations (< 30 Hz) reflect areas of heightened neural activity, we conclude that: (i) activity within a network of brain areas, including the sensori-motor cortex, is not critically dependent on stimulus type and may reflect general changes in visual attention; and (ii) the posterior part of the superior parietal cortex, area Ba7, is activated preferentially by objects and may play a role in computations related to grasping. © 2006 Elsevier Inc. All rights reserved.

Neuronal network pharmacodynamics of GABAergic modulation in the human cortex determined using pharmaco-magnetoencephalography

Neuronal network oscillations are a unifying phenomenon in neuroscience research, with comparable measurements across scales and species. Cortical oscillations are of central importance in the characterization of neuronal network function in health and disease and are influential in effective drug development. Whilst animal in vitro and in vivo electrophysiology is able to characterize pharmacologically induced modulations in neuronal activity, present human counterparts have spatial and temporal limitations. Consequently, the potential applications for a human equivalent are extensive. Here, we demonstrate a novel implementation of contemporary neuroimaging methods called pharmaco-magnetoencephalography. This approach determines the spatial profile of neuronal network oscillatory power change across the cortex following drug administration and reconstructs the time course of these modulations at focal regions of interest. As a proof of concept, we characterize the nonspecific GABAergic modulator diazepam, which has a broad range of therapeutic applications. We demonstrate that diazepam variously modulates ? (4–7 Hz), a (7–14 Hz), ß (15–25 Hz), and ? (30–80 Hz) frequency oscillations in specific regions of the cortex, with a pharmacodynamic profile consistent with that of drug uptake. We examine the relevance of these results with regard to the spatial and temporal observations from other modalities and the various therapeutic consequences of diazepam and discuss the potential applications of such an approach in terms of drug development and translational neuroscience.

Frontoparietal control of spatial attention and motor intention in human EEG

Relations between spatial attention and motor intention were investigated by means of an EEG potential elicited by shifting attention to a location in space as well as by the selection of a hand for responding. High-density recordings traced this potential to a common frontoparietal network activated by attentional orienting and by response selection. Within this network, parietal and frontal cortex were activated sequentially, followed by an anterior-to-posterior migration of activity culminating in the lateral occipital cortex. Based on temporal and polarity information provided by EEG, we hypothesize that the frontoparietal activation, evoked by directional information, updates a task-defined preparatory state by deselecting or inhibiting the behavioral option competing with the cued response side or the cued direction of attention. These results from human EEG demonstrate a direct EEG manifestation of the frontoparietal attention network previously identified in functional imaging. EEG reveals the time course of activation within this network and elucidates the generation and function of associated directing-attention EEG potentials. The results emphasize transient activation and a decision-related function of the frontoparietal attention network, contrasting with the sustained preparatory activation that is commonly inferred from neuroimaging.

A systematic exploration of perceptual and semantic differences in category-specific object-processing using magnetoencephalography

In a series of experiments, we tested category-specific activation in normal parti¬cipants using magnetoencephalography (MEG). Our experiments explored the temporal processing of objects, as MEG characterises neural activity on the order of milliseconds. Our experiments explored object-processing, including assessing the time-course of ob¬ject naming, early differences in processing living compared with nonliving objects and processing objects at the basic compared with the domain level, and late differences in processing living compared with nonliving objects and processing objects at the basic compared with the domain level. In addition to studies using normal participants, we also utilised MEG to explore category-specific processing in a patient with a deficit for living objects. Our findings support the cascade model of object naming (Humphreys et al., 1988). In addition, our findings using normal participants demonstrate early, category-specific perceptual differences. These findings are corroborated by our patient study. In our assessment of the time-course of category-specific effects as well as a separate analysis designed to measure semantic differences between living and nonliving objects, we found support for the sensory/motor model of object naming (Martin, 1998), in addition to support for the cascade model of object naming. Thus, object processing in normal participants appears to be served by a distributed network in the brain, and there are both perceptual and semantic differences between living and nonliving objects. A separate study assessing the influence of the level at which you are asked to identify an object on processing in the brain found evidence supporting the convergence zone hypothesis (Damasio, 1989). Taken together, these findings indicate the utility of MEG in exploring the time-course of object processing, isolating early perceptual and later semantic effects within the brain.

The neurotoxicity of acrylamide

The experiments described in this thesis compared conventional methods of screening for neurotoxins with potential electrophysiological and pharmacological tests in an attempt to improve the sensitivity of detection of progressive distal neuropathy. Adult male albino mice were dosed orally with the neurotoxicant acylamide and subjected to a test of limb strength and co-ordination and a functional observational battery. These methods established a no observable effect level of 10 mg/kg. A dose of 200 mg/kg resulted in abnormalities of gait and reduced limb strength and/or co-ordination. Analysis of the in vitro 'jitter' of the latency of trains of action potentials evoked at a frequency of 30 Hz in the mouse phrenic nerve/hemidiaphragm preparation showed this technique to be unsuitable for detection of the early phases of acrylamide induced peripheral neuropathy (l00 mg/kg). The evoked and spontaneous twitch responses of the hemidiaphragm preparation following in vitro exposure to the organophosphorous anticholinesterase compound ecothiopate were altered by in vivo pre treatment with acrylamide. Acrylamide caused an increase in the time course of the potentiation of stimulated twitches and a decrease in the maximum potentiation. Spontaneous twitches were reduced in amplitude and frequency. These effects occurred at an acrylamide dose level insufficient to cause clinical signs of neuropathy. Investigations into the mechanisms underlying these observations yielded the following observations. Analysis of miniature endplate potentials at this dose level indicated prolongation of the life of acetylcholine in the synaptic cleft but the implied decrease in cholinesterase activity could not be demonstrated biochemically or histologically. The electrical excitability of the nerve terminal region of phrenic motor nerves was reduced following acrylamide although a possible compromise of antidromic action potential conduction could not be confirmed. There was no histopathological evidence of neuropathy at this dose level. Further exploration of this phenomenon is desirable in order to ascertain whether the effect is specific to acrylamide and/or ecothiopate and to elucidate the mechanisms behind these novel observations.

The delivery of bioactive proteins using biodegradable microspheres

In this project, antigen-containing microspheres were produced using a range of biodegradable polymers by single and double emulsion solvent evaporation and spray drying techniques. The proteins used in this study were mainly BSA, tetanus toxoid, F1 and V, Y. pestis subunit vaccines and the cytokine, interferon-gamma. The polymer chosen for use in the vaccine preparation will directly determine the characteristics of the formulation. Full in vitro analysis of the preparations was carried out, including surface hydrophobicity and drug release profiles. The influence of the surfactants employed on microsphere surface hydrophobicity was demonstrated. Preparations produced with polyhydroxybutyrate and poly(DTH carbonate) polymers were also shown to be more hydrophobic than PLA microspheres, which may enhance particle uptake by antigen presenting cells and Peyer's patches. Systematic immunisation with microspheres with a range of properties showed differences in the time course and extent of the immune response generated, which would allow optimisation of the dosing schedule to provide maximal response in a single dose preparation. Both systematic and mucosal responses were induced following oral delivery of microencapsulated tetanus toxoid indicating that the encapsulation of the antigen into a microsphere preparation provides protection in the gut and allows targeting of the mucosal-associated lymphoid tissue. Co-encapsulation of adjuvants for further enhancement of immune response was also carried out and the effect on loading and release pattern assessed. Co-encapsulated F1 and interferon-gamma was administered i.p. and the immune responses compared with singly encapsulated and free subunit antigen.

Mechanisms of ion channel activation in primary cultured and clonal cell lines

The effects of hypotonic shock upon membrane C1 permeability of ROS 17/2.8 osteoblast-like cells was investigated using the patch-clamp technique. Hypotonic shock produced cell swelling that was accompanied by large amplitude, outwardly rectifying, currents that were active across the entire physiological range of membrane potentials (-80 to +100 mV). At strong depolarisations (> +50 mV) the currents exhibited time-dependent inactivation that followed a monoexponential time course. The currents were anion selective and exhibited a selectivity sequence of SCN- > I > Br- > Cl- > F- > gluconate. Current activation was unaffected by inhibitors of protein kinase (A (H-89) and tyrosine kinase (tyrphostin A25), and could not be mimicked by elevation of intracellular Ca2+ or activation of protein kinase C. Similarly, disruption of actin filaments by dihydrocytochalsin B, or generation of membrane tension by dipyridamole failed to elicit significant increases in cell chloride permeability. The mechanism of current activation is as yet undetermined. The currents were effectively inhibited by the chloride channel inhibitors NPPB and DIDS but resistant to DPC. A Cl- conductance with similar characteristics was found to be present in mouse primary cultured calvarial osteoblasts. The volume-sensitive Cl- current in ROS 17/2.8 cells was inhibited by arachidonic acid in two distinct phases. A rapid block that developed within 10 s, preceding a slower developing inhibitory phase that occurred approximately 90 s after onset of arachidonate superfusion. Arachidonic acid also induced kinetic modifications of the current which were evident as an acceleration of the time-dependent· inactivation exhibited at depolarised potentials. Inhibitors of cyclo-oxygenases, lipoxygenases and cytochrome P-4S0 were ineffectual against arachidonic acid's effects sugtgesting that arachidonic acid may elicit it's effects directly. Measurements of cell volume under hypotonic conditions showed that ROS 17/2,8 cells could effectively regulate their volume, However, effective inhibitors of the volume-sensitive CI" current drastically impaired this response suggesting that physiologically this current may have a vital role in cell volume regulation, In L6 skeletal myocytes, vasopressin was found to rapidiy hyperpolarise cells. This appears to occur as the result of activation of Ca2+ -sensitive K+ channels in a process dependent upon the presence of extracellular Ca2+.

Task classification and decision processes in monitoring behaviour

Task classification is introduced as a method for the evaluation of monitoring behaviour in different task situations. On the basis of an analysis of different monitoring tasks, a task classification system comprising four task 'dimensions' is proposed. The perceptual speed and flexibility of closure categories, which are identified with signal discrimination type, comprise the principal dimension in this taxonomy, the others being sense modality, the time course of events, and source complexity. It is also proposed that decision theory provides the most complete method for the analysis of performance in monitoring tasks. Several different aspects of decision theory in relation to monitoring behaviour are described. A method is also outlined whereby both accuracy and latency measures of performance may be analysed within the same decision theory framework. Eight experiments and an organizational study are reported. The results show that a distinction can be made between the perceptual efficiency (sensitivity) of a monitor and his criterial level of response, and that in most monitoring situations, there is no decrement in efficiency over the work period, but an increase in the strictness of the response criterion. The range of tasks exhibiting either or both of these performance trends can be specified within the task classification system. In particular, it is shown that a sensitivity decrement is only obtained for 'speed' tasks with a high stimulation rate. A distinctive feature of 'speed' tasks is that target detection requires the discrimination of a change in a stimulus relative to preceding stimuli, whereas in 'closure' tasks, the information required for the discrimination of targets is presented at the same point In time. In the final study, the specification of tasks yielding sensitivity decrements is shown to be consistent with a task classification analysis of the monitoring literature. It is also demonstrated that the signal type dimension has a major influence on the consistency of individual differences in performance in different tasks. The results provide an empirical validation for the 'speed' and 'closure' categories, and suggest that individual differences are not completely task specific but are dependent on the demands common to different tasks. Task classification is therefore shovn to enable improved generalizations to be made of the factors affecting 1) performance trends over time, and 2) the consistencv of performance in different tasks. A decision theory analysis of response latencies is shown to support the view that criterion shifts are obtained in some tasks, while sensitivity shifts are obtained in others. The results of a psychophysiological study also suggest that evoked potential latency measures may provide temporal correlates of criterion shifts in monitoring tasks. Among other results, the finding that the latencies of negative responses do not increase over time is taken to invalidate arousal-based theories of performance trends over a work period. An interpretation in terms of expectancy, however, provides a more reliable explanation of criterion shifts. Although the mechanisms underlying the sensitivity decrement are not completely clear, the results rule out 'unitary' theories such as observing response and coupling theory. It is suggested that an interpretation in terms of the memory data limitations on information processing provides the most parsimonious explanation of all the results in the literature relating to sensitivity decrement. Task classification therefore enables the refinement and selection of theories of monitoring behaviour in terms of their reliability in generalizing predictions to a wide range of tasks. It is thus concluded that task classification and decision theory provide a reliable basis for the assessment and analysis of monitoring behaviour in different task situations.