A novel apparatus/protocol designed for optogenetic manipulation and recording of individual neurons during a motivation and working memory task in the rodent

Innovative molecular tools allow neuroscientists to study neural circuitry associated with specific behaviors. Consequently, behavioral methods must be developed to interface with these new molecular tools in order for neuroscientists to identify the causal elements underlying behavior and decision-making processes. Here we present an apparatus and protocol for a novel Go/No-Go behavioral paradigm to study the brain attention and motivation/reward circuitry in awake, head-restrained rodents. This experimental setup allows: (1) Painless and stable restraint of the head and body; (2) Rapid acquisition to simple or complex operant tasks; (3) Repeated electrophysiological single and multiple unit recordings during ongoing behavior; (4) Pharmacological and viral manipulation of various brain regions via targeted guide cannula, and; (5) Optogenetic cell-type specific activation and silencing with simultaneous electrophysiological recording. In addition to the experimental advantages, the head-restraint system is relatively inexpensive and training parameters can be easily modulated to the specifications of the experimenter. The system runs on custom LabView software. In summary, our novel apparatus and protocol allows researchers to study and manipulate components of behavior, such as motivation, impulsivity, and reward-related working memory during an ongoing operant behavioral task without interference from non task-related behaviors. For more information on the custom apparatus, software or to collaborate please visit www.neuro-cloud.net/nature-precedings/dolzani

Polar group exchange receptor-ligand engineering of protein kinase B's pleckstrin homology domain

A diverse range of cellular functions are influenced by the activity of protein kinase B (PKB, also known as Akt). PKB is involved in a number of physiological pathways, perturbation of which can lead to pathological conditions. PKB activation has been shown to involve binding of its pleckstrin homology (PH) domain to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 generated on the inner face of the plasma membrane upon receipt of an external hormonal signal. To study PKB independently of other inositol-phospholipid binding proteins which recognise the same lipid signals, their mutual dependence on the same ligand must be broken. It is proposed that this can be achieved by mutating PKB‟s PtdInsPn binding PH domain to accept an unnatural ligand. By altering the inositol-phospholipid head-group to include an additional carboxylic acid substituent, its binding to the wild-type PH domains should be inhibited through steric compression. Disruption of a salt-bridge in the wall of the binding pocket by point mutation of glutamate 17 to alanine, E17A, introduces an additional positively charged pocket into the PH domain of PKB, which should bind with the tailored lipid, resulting in a polar group exchange receptor-ligand pair. To this end, a novel 2-O-acetic acid functionalised analogue of Ins(1,3,4,5)P4 was synthesised. The key steps were the protection/deprotection of myo-inositol‟s hydroxyls, enabling selective functionalisation of the 2-hydroxyl, and phosphorylation of the 1,3,4,5-O positions. Using an allyl ether as a masked form of acetic acid allowed a divergent synthetic strategy to be employed to access additional 2-O analogues. Both the wild-type and the E17A mutant of the PH domain were expressed in order to assess ligand binding, and determine whether an ion pair exchange receptor-ligand interaction occurred. Intrinsic tryptophan fluorescence spectrometry was initially used, but was unsuccessful due to denaturing problems arising from isolating the protein. Isothermal titration calorimetry suggested binding, but protein concentration could not be increased high enough for the binding energy to become significantly greater than the energy of mixing. The analogues were also tested as potential inositol poly-phosphate phosphatase catalytic (IPPc) domain inhibitors

Advances in quantitative microscopy

Microscopy allows us to peer into the complex deeply shrouded world that the cells of our body grow and thrive in. With the emergence of automated digital microscopes and software for anlysing and processing the large numbers of image that they produce; quantitative microscopy approaches are now allowing us to answer ever larger and more complex biological questions. In this thesis I explore two trends. Firstly, that of using quantitative microscopy for performing unbiased screens, the advances made here include developing strategies to handle imaging data captured from physiological models, and unsupervised analysis screening data to derive unbiased biological insights. Secondly, I develop software for analysing live cell imaging data, that can now be captured at greater rates than ever before and use this to help answer key questions covering the biology of how cells make the decision to arrest or proliferate in response to DNA damage. Together this thesis represents a view of the current state of the art in high-throughput quantitative microscopy and details where the field is heading as machine learning approaches become ever more sophisticated.Open Acces

Optogenetic control of genetically-targeted pyramidal neuron activity in prefrontal cortex

A salient feature of prefrontal cortex organization is the vast diversity of cell types that support the temporal integration of events required for sculpting future responses. A major obstacle in understanding the routing of information among prefrontal neuronal subtypes is the inability to manipulate the electrical activity of genetically defined cell types over behaviorally relevant timescales and activity patterns. To address these constraints, we present here a simple approach for selective activation of prefrontal excitatory neurons in both in vitro and in vivo preparations. Rat prelimbic pyramidal neurons were genetically targeted to express a light-­activated nonselective cation channel, channelrhodopsin-­2, or a light-­driven inward chloride pump, halorhodopsin, which enabled them to be rapidly and reversibly activated or inhibited by pulses of light. These light responsive tools provide a spatially and temporally precise means of studying how different cell types contribute to information processing in cortical circuits. Our customized optrodes and optical commutators for in vivo recording allow for efficient light delivery and recording and can be requested at www.neuro-­cloud.net/nature-precedings/baratta

Breaking the seismic wall: how to improve gravitational wave detectors at low frequency

The era of gravitational-wave astronomy was enabled by the incredible sensitivity of the LIGO and VIRGO detectors. However, they are still plagued by technical noises at frequencies below 30 Hz, driven in part by the limitations of the seismic isolation of the detector. To detect gravitational waves at low frequency, the isolation performance must be improved to reduce these technical noises. To improve the performance of seismic isolation systems, I have developed HoQI a new interferometrically sensor, that can be applied to both the isolation tables and suspensions. HoQI has a resolution a factor 1000 higher than sensors currently used in LIGO and I have quantified the level of non-linearity present in the sensor and shown this to not be a limiting factor. HoQIs impact on the performance of the seismic isolation system has also been quantified, through the use of a accurate model of an Advanced LIGO isolation platform that I have developed. Using the model I have shown that using using HoQI, the expected isolation platform motion can be reduced by a factor of 70 at 0.1 Hz and a factor of 10 at 2 Hz. I have shown that the control filters used in this model can be improved by up to 70% by designing them using particle swarm optimisation

An economic evaluation of the training and technology program, Oak Ridge, Tennessee, 1966-1970

Training and Technology (TAT), a skill development program at Oak Ridge, Tennessee, has been training people for entry level skills since 1966. This study utilized data collected by TAT in 1972. The surveyed covered 472 graduates who were trained between 1966 and 1971, from areas as diverse as rural Appalachia and Chicago. This study compared the trainees according to whether they had rural or urban backgrounds; their sociodemographic characteristics; attitudes toward work, coworkers, and mobility; and post-training experiences including income. Initially, tabular comparisons were made. In the second portion of the analysis, multiple regression was utilized to examine the effect of (1) rural or urban background and (2) training year on hourly wage. Hourly wage was used as a proxy for income because virtually all trainees worked 40 hours per week and tenure on the job was unknown. Variables which were controlled in the analysis were age, education, training area, number of jobs since training, employment status prior to entering training, training year, years since training, and rural and urban background. Hourly wage was the independent variable. Regressions were run for the total group and each individual training year. The regressions were run twice, once including and then excluding the rural and urban background variables. Regressions were then run for the rural and urban groups separately. Findings in the tabulated profiles showed that rural trainees were, on the average, older, the rural trainees were nearly all white while half of the urban trainees were nonwhite, and more rural trainees had graduated from high school but fewer had gone to college. Rural trainees had fewer post-training adjustment problems but did experience greater difficulty in adapting to shift work. In other characteristics the trainees were quite similar. The regression findings indicated consistently that being male, white, employed at entry to training and holding a training related job contributed positively to wages. These and the other independent variables included in the analysis explained a higher percent of the variation in wages of the rural trainees than they did of urban trainees\u27 wages. However, there appeared to be little overall difference between the rural and urban groups in their wage success and the effects of various background characteristics on these wages

'A lot to answer for': the English legacy of the Situationist International

This thesis draws attention to the overlooked period of English avant-garde activity that arose in response to the Situationist International (SI, 1957-1972). I attempt to consolidate the continued literary, political and artistic relevance of the disparate manifestations of English Situationist practices, while reflecting more broadly on the Anglicisation of Continental avant-garde traditions. The thesis contributes also to the ongoing re-evaluation of the SI through its peripheral and international permutations. The thesis presents an historical narrative in four stages. The first follows the formation of the Surrealist Group in England and its transformation into Mass- Observation. This activity in the Thirties is both prolepsis to and prehistory of the English Situationist tradition, which begins at the thesis’s second stage, in the early Sixties, with the novelist and cultural organiser Alexander Trocchi, one of the first British members of the SI. The third stage introduces the short-lived English Section of the SI and its immediate precursor and successor groups of the late Sixties, all of whom undertook a cultural translation of the SI’s project. The thesis’s final stage identifies contemporary manifestations of what I argue is an English Situationist poetics, which exists independently of self-identifying avant-garde groups. Interwoven through my historical narrative are investigations of three recurring problems: the problem of ‘Englishness’, and its perceived incompatibility with Continental avant-gardism; the problem of influence, whereby neo-avantgardism is dismissed as the hollow repetition of early twentieth century formations; and the Situationist problem of the radical aesthetic object’s subsumption to a commodity economy. I offer readings of novels, films and visual materials; of tracts, manifestos and journal debates; and of theorists (especially Rancière) whose perspectives on history and political aesthetics are productively dissonant with those of the SI. Anglicised Situationist practices, I propose, are valuable precisely for their contestation of and movement through Situationist theory