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Prospective Memory (PM) - memory for a delayed intention - is essential for functioning in everyday life. Event-based PM relies on a cue (eg a postbox) to trigger recall of an intention (eg mail a letter). In the laboratory a typical event-based PM paradigm comprises a pre-designated PM cue which is embedded in an ongoing computerized task. Through a series of four articles I address the impact of methodology, pharmacology, and genetics on event-based PM in a laboratory setting. Article I examines the effect of varying PM cue frequency. Increasing cue frequency improves performance and causes an alteration in strategy towards the task. Article IV introduces a novel paradigm which differentiates the attention-based (prospective) and memory-based (retrospective) PM components, specifically measuring both accuracy and reaction time for these components. Article II concentrates on pharmacologic manipulations, focusing on the indirect noradrenergic agonist modafinil and the cholinergic against nicotine, and how their beneficial effects interact with cue frequency. When cue frequency is high (20%) modafinil facilitates performance, but not when cue frequency falls to 5%. Conversely, nicotine improves performance when cue frequency is low, but not when high. This pharmacologic double dissociation lends further support to the behavioural data, indicating activation of different attentional processes when cue frequency changes. Article III explores the cognitive consequences of possessing the ε4 variant of the Apolipoprotein E gene in younger adults; and extends the argument for early-age cognitive dominance of ε4 carriers to include superior PM performance. Additionally Article III reveals that nicotine again improves PM performance, but critically only for ε4 carriers. Finally, Article IV shows that non-ε4 carriers' PM performance remains unaffected by an increased nicotine dosage. These articles contribute to understanding PM and the complex interaction between the methods used to measure it, the drugs used to influence it, and individual differences.

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The mammalian neocortical areas arise from graded/regionalized gene expression from the earliest stages of corticogenesis. However, little is known about the establishment of the human motor cortex, despite being a common site of perinatal damage causing cerebral palsy. Affymetrix gene chip analysis of human neocortical tissue from 8-12.5 post-conceptional weeks (PCW) previously discovered genes expressed in gradients along the anterior-posterior human neocortex. Aims: i) Identify putative anterior/posterior molecular determinants of the developing human neocortex. ii) Test the hypothesis that the anterior pole of the human neocortex as an early site of the developing motor cortex generating corticofugal neurones and understand their development. iii) Establish a human in-vitro regionalisation model and study potential regulatory mechanisms of some corticofugal neurone-related genes. Results and Conclusion: Graded expression of some putative anterior/posterior markers was confirmed in the human neocortex by real-time PCR (8-12 PCW). Among them, the corticofugal neurone-related genes (transcription factor CTIP2, axon guidance molecule ROBO1 and its downstream signalling molecule SRGAP1) were up-regulated anteriorly. Their spatiotemporal expression patterns were examined further by tissue in-situ hybridization and immunohistochemistry, and compared with other corticofugal neurone-related transcription factors that showed no/opposite expression gradients (FEZF2 and SOX5) and various laminar-enriched markers previously established in humans (ER81, TBR1, TBR2, GAP43 and Synaptophysin) or validated in the current project (SATB2 and NURR1). Layer V was shown to arise as early as 12 PCW in humans. Prominent medullary pyramids containing corticospinal fibres strongly expressed both ROBO1/SRGAP1 by 14-17 PCW, when a distinct ROBO1/SRGAP1-positive Layer V emerges. Co-expression of the three genes in anterior neocortex might mark the site of the emerging human motor cortex, which is the predominant origin of corticofugal neurones such as corticospinal/corticopontine projection neurones. Dissociated neocortical cell cultures were established from anteriorly-/posteriorly-derived foetal neocortex that maintained regional intrinsic molecular identities. This in-vitro model of regionalization allowed gene regulation study to be performed in which a preliminary investigation of fibroblast growth factor signalling in controlling the expression of corticofugal neurone-related genes was initiated.

There is ample evidence that attention to stimuli can facilitate perception under different experimental tasks. For example, human observers are faster and more accurate at detecting an object in a visual scene when they know in advance its location, motion or colour. Previous electrophysiological studies on attentional modulation have characterized the effects of attention on firing rates and oscillatory activity. They have also studied how attention can change basic neuronal integration properties, such as the size of the classical receptive field or its summation area. However, the cellular mechanisms underlying this response modulation are not clear. In this thesis, I investigate which neurotransmitters and receptors contribute to attentional modulation in the primate brain. I use pharmacological manipulations on neurons in the primary visual cortex (V1) while monkeys perform a visual spatial attention task. The contribution of two main neuromodullatory systems, the cholinergic and glutamatergic system, to visual attention is examined. Findings reveal that the amount of attentional modulation in V1 is augmented when the cholinergic system is pharmacologically enhanced. This effect is mediated by the activation of muscarinic, but not nicotinic, receptors. Glutamatergic NMDA receptor activation also leads to enhanced attentional modulation in V1, although the effects are largely restricted to improved response reliability and less to increased response gain. We note that both attention and acetylcholine can alter basic neuronal coding, namely integration properties of V1 neurons. Our findings show that acetylcholine affects contextual integration through muscarinic receptors, while nicotinic receptors affect the gain with no changes in the integration. Additional investigations of the cholinergic (and gabaergic) mechanisms in extrastriate area (area MT) are conducted, as ACh may result in improved direction selectivity computations similar to those reported in the attention literature. It appears that acetylcholine does not increase neuronal sensitivities as measured by a sharpening in the motion--‐ direction tuning curve. Instead, it improves response reliability to optimal stimulus features. Taken together, these findings may have implications to neuromodulatory accounts of visual attention.

Cortical processing of even the most elementary visual stimuli can result in the propagation of information over significant spatiotemporal scales. To fully understand the impact of such phenomena it is essential to consider the influence of both the neural circuitry beyond the immediate retinotopic location of the stimulus, including pre-cortical areas, and the temporal components of stimulus driven activity that may persist over significant periods. Two computational modelling studies have been performed to explore these phenomena and are reported in this thesis. I) The plexus of long and short range lateral connections is a prominent feature of the layer 2/3 microcircuit in primary visual cortex. Despite the scope for possible functionality, the interdependence of local and long range circuits is still unclear. Spatiotemporal patterns of activity appear to be shaped by the underlying connectivity architecture and strong inhibition. A modelling study has been conducted to capture population activity that has been observed in vitro using voltage sensitive dyes. The model demonstrates that the precise spatiotemporal spread of activity seen in the cortical slice results from long range connections that target specific orientation domains whilst distinct regions of suppressed activity are shown to arise from local isotropic axonal projections. Distal excitatory activity resulting from long range axons is shaped by local interneurons similarly targeted by such connections. It is shown that response latencies of distal excitation are strongly influenced by frequency dependent facilitation and low threshold characteristics of interneurons. Together, these results support hypotheses made following experimental observations in vitro and clearly illustrate the underlying mechanisms. However, predictions by the model suggest that in vivo conditions give rise to markedly different spatiotemporal activity. Furthermore, opposing data in the literature regarding inter-laminar connectivity give rise to profoundly different spatiotemporal patterns of activity in cortex. 2) The second computational modelling study considers simple moving stimuli. These stimuli are implicated in the 'motion streak' phenomenon whereby the movement of a visual feature can give rise to trajectory information that is not explicitly present. Published experimental data of an in vivo study in the cat has shown that a single small light square moving stimulus elicits activity in populations of neurons in primary visual cortex that are selective for orientations parallel to stimulus trajectory (Jancke 2000). In more recent, unpublished data, this work is extended to consider long term persistent cortical activity that is generated by similar stimuli. These data indicate that following initial cortical activation that appears to result directly from the stimulus, iso-orientation domains display persistent activity. Furthermore, initial activity is broadly tuned with respect to orientation whilst later activity is strongly selective for orientations that are parallel to the stimulus trajectory. Currently the generative processes involved have not been clearly defined. Hence the proposed thesis will contribute to a more complete understanding of the mechanisms responsible for such cortical representations of moving visual stimuli. More specifically this will be achieved by a large scale mean field model that will enable a thorough investigation of the anatomical and electrophysiological elements concerned with the observed spatiotemporal dynamic behaviour and will represent a significant region of cortex. In conjunction, an existing computational model of the retina will be integrated. In doing so this thesis will offer the notion that certain cortical representations are inextricably linked with earlier stages of the visual pathway. As such consideration of retinal processing is fundamental to the understanding cortical functions and failure to do so can only result in erroneous conclusions.

This thesis concentrates on the induction of the immediate early genes zif268 and c-fos in a functional and dysfunctional brain network. Initial studies focused on the creation of a task to allow the study of immediate early gene activation after working memory. Previous studies using such a paradigm have compared the animals performing the experimental task with poorly matched control groups. Experiments in this thesis attempted to rectify this. A group of experiments using the water maze found decreases in Zif268 activation in the experimental group as compared to control groups, mainly in the hippocampus and some parahippocampal areas. This was believed to have arisen because of a streamlining of the brain network in the Working Memory group. An increase in c-Fos immunoreactivity was seen in the Working Memory group as compared to controls in prefrontal regions. Structural equation modelling analysis was performed, which allows immediate early gene counts to be used to analyse networks of brain regions. In the Working Memory group connections were seen between the parahippocampal regions and the subiculum that progressed via the hippocampus, indicating that the hippocampus was still engaged by the task. In the control group analysed no such hippocampal pathway was found. This water maze task was then used to study zif268-EGFP activation in a novel transgenic rodent model, where the Zif268 promoter drives the expression of a fluorescent protein EGFP. Activation of both EGFP and Zif268 immunoreactivity was seen in the CA1 region of the animals performing the control task. No EGFP activation was seen in this region in the Working Memory group even though EGFP expression was seen in other regions. The GFP protein was also able to be seen under direct visualisation in the CA1 and dentate gyrus region of control animals. Concerning the dysfunctional brain, gene expression was analysed in the retrosplenial cortex after NMDA lesions of the anterior thalamic nuclei. Previous research has shown that lesions produce a dramatic hypoactivity in the protein products of the immediate early genes c-fos and zif268. Microarray analysis of retrosplenial tissue revealed that as well as decreases in expression of genes related to repair and cell adhesion/neurogenesis, an increase in c-fos mRNA was seen in the lesion hemisphere of the brain. This pattern of expression is opposite to that of the protein. Possible reasons for this are discussed.

This thesis explores the influence of brain regions within the "extended hippocampal memory system" on the activity of the retrosplenial cortex in the rat. One of the first goals was to use lesion studies to improve the understanding of the vulnerability of the retrosplenial cortex, especially in the context of diencephalic and temporal lobe amnesia. The second was to assess what are the brain areas within the temporal lobe involved in object recognition and how they interact. These two objectives were made possible by visualising immediate-early gene expression. By combining this technique with lesions, distal effects of different lesions (hippocampus, mammillothalamic tract and fornix) on the activity of the retrosplenial cortex were measured. For object recognition, the immediate-early gene imaging enabled the assessment of normal brain activity in rats associated with behavioural discrimination of novelty. The lesion studies provide information about the specific and common vulnerability of the retrosplenial cortex, as all three distal lesions resulted in a decrease of immediate-early gene activity in the retrosplenial cortex. In addition, these findings unify diencephalic amnesia with temporal amnesia, and emphasize the need to study networks or systems instead of individual structure. The immediate-early gene/object recognition experiment implicated the caudal part of the perirhinal cortex (and Te2) and of the hippocampus in object recognition, and highlighted the importance of mapping brain region relationships within a connected system. Taken together, these experiments provide clear support for the concept of an extended hippocampal memory system, but also show how this system may interact with other structures involved in different forms of memory. The findings underlie the potential afforded by use of immediate-early gene expression techniques in animal studies.

Virtually all studies on the molecular regulation of axonal growth have been carried out on axotomised neurons that regenerate axons in culture and are dependent on neurotrophic factors not only for survival but also for axonal growth. The technical difficulty of obtaining and culturing early, newly differentiated neurons that initiate axonal growth for the first time in culture has meant that developmentally relevant <italic>de novo</italic> axonal growth has been almost ignored. Numerous studies have shown that axonal regeneration from embryonic and postnatal neurotrophic factor-dependent neurons is regulated by a variety of signalling pathways that influence the assembly and stability of key components of the cytoskeleton in growth cones. Depending on neuron type, these can include MEK, PI3 kinase, GSK3, NF-kB and calcium signalling. The extent to which these pathways are important for <italic>de novo</italic> axonal growth, and how their importance changes as axonal growth becomes responsive to neurotrophic factors during early development is not known. I have used the experimentally advantageous placode-derived sensory neurons of the chicken embryo to study the molecular basis of <italic>de novo</italic>, neurotrophic factor independent axonal growth and to compare this neurotrophic factor-dependent axonal growth at later stages of development. These neurons can be dissected from the earliest stages in their development and cultures can be established in which neurons extend axons for the first time. Previous work has shown that at this stage of development, axonal growth is independent of neurotrophic factors and its rate is correlated with target distance. For example, neurons of the nodose ganglion have the most distant targets, the fastest axonal growth rate and survive longest before becoming dependent on the neurotrophic factor BDNF for survival, whereas neurons of the vestibular ganglion have the nearest targets, slowest axonal growth rate and survive for the shortest time before acquiring BDNF dependence. My initial studies focused on the role of the extracellular calcium-sensing receptor (CaSR), a G protein coupled receptor that has recently been shown to regulate axonal growth from sympathetic neurons during the stage when neurons are innervating their targets. I found that during the stage of development when the earliest axons of placode-derived sensory neurons are growing to their targets, nodose ganglion neurons (which have the fastest axon growth rates) express the highest levels of the CaSR, and vestibular neurons (which have the slowest axon growth rates) express the lowest levels of the CaSR. Experimental manipulation of CaSR activation in cultured nodose neurons at the stage in development when their axons are normally growing to their targets markedly affects axon growth rate (enhancing activation increases growth rate whereas reducing activation has the opposite effect). In contrast, similar manipulations of CaSR activation in cultured vestibular neurons have no effects on axonal growth rate. These findings suggest that the CaSR plays an important role in the regulation of <italic>de novo </italic> axonal growth rate. Manipulating CaSR activation in older, BDNF-dependent nodose neurons at the stage in development when these neurons are innervating their targets also demonstrated a role for the CaSR in promoting axonal growth at this stage. Having demonstrated a role for the CaSR in promoting axonal growth at these two successive stages of development, I then characterised the intracellular signalling pathways that mediate the effects of the CaSR on axonal growth at these stages. Using Western blot analysis and pharmacological inhibitors of PI3-kinase, GSK3 and MEK1/2, I discovered a clear switch in the signalling pathways that are involved in promoting axon elongation between early BDNF-independent stages of <italic>de novo</italic> axon growth to later BDNF-dependent stages of axon growth. Whereas PI3-kinase signalling plays a pivotal role in transducing CaSR-enhanced, neurotrophin-independent axon growth, GSK3 signalling plays a major role in transducing the growth enhancing effects of CaSR activation on BDNF-promoted axonal growth from older BDNF-dependent nodose neurons. My findings suggest that PI3-Kinase and GSK3 signalling are not linked in developing nodose neurons, but are regulated independently of each other. Furthermore, Western analysis also suggests the operation of a novel activation mechanism of GSK3 in axon growth in BDNF-dependent nodose neurons that involves tyrosine phosphorylation of GSK3 rather than serine phosphoryation following CaSR activation. In all, my studies have revealed several novel and unexpected aspects of regulation of axonal growth by the CaSR during the early stages of neuronal development.

Neocortical and hippocampal synaptic long-term potentiation (LTP) initially requires the AMPA receptor subunit GluR1, while late-phase LTP depends upon nitric oxide synthase (NOS). To investigate whether this was relevant to in vivo ED potentiation, GluR1 and/or NOS knockout mice were deprived of all whiskers but the D1 to induce barrel cortex synaptic potentiation, which was quantified by single unit recordings. In deprived cortex, D1 whisker responses potentiated approximately 40% less in GluR1 and NOS3 knockout mice than wild-type mice. Potentiation in the NOS1 knockout was influenced by gender; female NOS1 knockout potentiation was similar to wild-types, yet was absent in male NOS1 knockout mice. The ED potentiation in GluR1 knockout mice was dependent upon NOS, supporting LTP studies. However, NOS1 was more important for potentiation. Thus, while potentiation occurred in the GluR1/NOS3 double knockout mice, it was completely absent in the GluR1/NOS1 double knockout. To determine the interaction between GluR1 and NO activity in memory, behavioural studies examined their impact on spatial and contextual memory. The results partly confirmed earlier findings that retention of contextual fear conditioning was sensitive to GluR1 deletion. However, this was only the case in male GluR1 knockout mice. Female GluR1 KO mice were unimpaired. In a spatial radial arm watermaze task, GluR1 knockout mice acquired the location of a submerged platform more slowly than wild-types. Nevertheless, spatial reference memory was comparable to wild-type mice at the end of training and was not influenced by gender. In contrast to predictions, GluR1-indepdnent reference memory was not dependent upon NOS. Therefore while emotional learning requires GluR1 in male mice, spatial reference memory can form in its absence in both genders and is insensitive to NOS antagonism.