Models of stimulus-response functions have been used for decades in an attempt to understand the complex relationship between a sensory stimulus and the neural response that it elicits. A popular model for characterising auditory function is the spectrotemporal receptive field (STRF), originally due to Aertsen and Johannesma (1980); Aertsen et al. (1980, 1981). However, the STRF model predicts auditory cortical responses to complex sounds very poorly, presumably because the model is linear in the stimulus spectrogram and thus incapable of capturing spectrotemporal nonlinearities in auditory responses. Ahrens et al. (2008a) introduced a multilinear framework, which captures neuron-specific nonlinear effects of stimulus context on spiking responses to complex sounds. In such a framework, contextual effects are interpreted as nonlinear stimulus interactions that modulate the input to a subsequent STRF-like linear filter. We derive various extensions to this framework, and demonstrate that the nonlinear effects of stimulus context are largely inseparable, and fundamentally different for near-simultaneous and delayed non-simultaneous sound energy. In two populations of neurons, recorded from the mouse auditory cortex and thalamus, we show that simultaneous sound energy provides a nonlinear positive (amplifying) gain to the subsequent linear filter, while non-simultaneous sound energy provides a negative (dampening) gain. We demonstrate that this structure is largely responsible for providing a significant increase in the predictive capabilities of the model. Using this framework, we show that nonlinear context dependence differs between cortical fields, consistent with previous studies (Linden et al., 2003). Furthermore, we illustrate how such a model can be used to probe the nonlinear mechanisms that underly the ability of the auditory system to operate in diverse acoustic environments. These results provide a novel extension to the study of receptive fields in multiple brain areas, and extend existing understanding of the way in which stimulus context drives complex auditory responses.

Centrosomes are the major microtubule organising centre of most cells. A centrosome consists of a pair of perpendicular centrioles surrounded by a cloud of pericentriolar material. Centrioles must be duplicated once per cell cycle to ensure each daughter cell inherits the correct number of centrioles. In somatic cells this process is controlled by centriole replication proteins including SAS-6 and Plk4. Centrioles have an unusual life-cycle in early mouse development. Both the sperm and the egg lack centrioles. Therefore the first few divisions in mouse embryos take place without centrioles. New centrioles are then formed ‘de novo’ in early developing embryos. In this thesis the molecular basis for this unusual scenario is examined. Firstly, a transgenic mouse model was used to confirm centrioles are formed de novo at the mouse blastocyst stage. This model was then used to examine the influence of centriole emergence on microtubule organisation. Secondly, overexpression of the centriole replication protein Plk4 was found to drive precocious formation of centriole-like structures in all stages of oocyte and embryo examined, which cause abnormal spindles. However, this does not appear to affect embryo development or chromosome segregation. Finally, overexpression of the downstream centriole component SAS-6 was also found to drive the formation of foci in embryos but strikingly, SAS-6 fails to arrange into centriole-like foci in immature or mature unfertilised oocytes. These experiments document the presence of an inducible de novo centriole formation pathway in mammalian oocytes and embryos, and show that the pathway is more resistant to activation prior to fertilisation.

The optic vesicles, the primordia of the eyes, arise by evagination of a single neural plate domain called the eye field. In this thesis, we provide a cellular mechanism involved in the first steps of eye morphogenesis by high-resolution 4-dimensional live imaging in the zebrafish. We show that as neurulation proceeds, eye field cells acquire apico-basal (AB) polarity and adopt a pseudo- stratified neuroepithelial organisation early as compared to other tissues of the neural plate. As a consequence, eye field cells show reduced convergence to the midline and they bulge out from the lateral walls of the forebrain. Early neuroepithelial organisation is first apparent at the margin of the eye field, where cells are in contact with the basal lamina, and is dependent on the extracellular matrix protein (ECM) Lamininl. Cells in the core of the eye field localise polaritry proteins in a polarized manner too and contribute gradually to the marginal layer by elongating and intercalating in between the marginal cells as the bilateral expansion of the eye field takes place. As the optic vesicle further expand laterally during subsequent stages of evagination, eye field cells shorten again along their apico-basal axis. This results in the lateral displacement of their apical surface relative to the basal lamina, and in the completion of optic vesicle evagination.

The thesis describes experiments on the role of Epidermal Growth Factor receptor (ErbB1) in limiting axonal regeneration. Pharmacological inhibitors of ErbB1 are known to improve neurite outgrowth from neurons grown in vitro on various inhibitory substrates such as CNS myelin, chondroitin sulphate proteoglycans (CSPGs) and fibrinogen (Koprivica et al., 2005). However, the hypothesis that ErbB1 is involved in the signalling mechanisms from various CNS inhibitory molecules has been challenged in papers form the Logan laboratory (Ahmed et al., 2010) which concluded that pharmacological blockers disinhibit neurite outgrowth by acting off-target to ErbB1. The first stage of this project was to use primary sensory neurons from ErbB1 knockout mice to test the hypothesis that ErbB1 is involved in inhibition of neurite outgrowth in vitro in the presence of CNS myelin, a Toll-Like Receptor 3 ligand (Poly I:C) and CSPGs. Through a series of experiments that have been described in this thesis, it was shown that ErbB1 antagonists act on-target and that ErbB1 signalling causes inhibition of neurite outgrowth of neurons cultured in the presence of Poly I:C, CNS myelin or CSPGs. Moreover, buffering calcium was shown to abolish the inhibitory effects caused by ErbB1 signalling, indicating that calcium is essential for the activation of the receptor. Other molecules that may be associated with ErbB1 signalling leading to inhibition of neurite outgrowth include PTEN and novel Sulfatase enzymes- Sulf1 and Sulf2. Using immunohistochemistry, Sulf1 and Sulf2 were shown to be expressed by a large variety of neurons. Also, ErbB1 expression detected immunohistochemically was predominantly localised to neurons. The thesis also describes preliminary observations on the efficacy of an ErbB1 antagonist or a dominant-negative ErbB1 lentivirus in improving axonal regeneration following an optic nerve crush or spinal cord injury in adult rats. In conclusion, ErbB1 signalling following injury is associated with regeneration failure.

The wild cheetah population is rapidly declining, and the captive population is not self-sustaining. This is of great concern for cheetah conservation and the latter might indicate underlying captive welfare concerns. This research measured the behaviour and personality of cheetahs held in zoo exhibits in the UK and beyond, to investigate the effects of social group housing and personality on the behaviour and reproductive success of captive cheetahs. Behavioural observation indicated that the natural social groupings of wild male cheetahs can be replicated in captivity. Group-housed males displayed frequent affiliative behaviours and few instances of aggression. Females, naturally solitary in the wild, might also be safely housed in groups since overt aggression was seldom recorded. However pacing behaviour, typically associated with poor welfare, was more prevalent in unnatural-type groups. Relatedness appears to be an important factor in captive cheetah social interactions. A new method for correcting indices of association, developed in this research, allowed association indices to be compared for dyads housed in different sized exhibits. Related individuals were observed in proximity more frequently, and displayed higher rates of affiliative interactions, than unrelated individuals. These findings may have welfare implications in the event that captive individuals are separated for management purposes. Social group housing and personality can affect captive cheetah reproductive success. The personality profiles of individuals in successful breeding pairs were more divergent than those of individuals in unsuccessful pairs. In addition, it appears that zoos housing their cheetahs in social groups that occur in wild populations have better institutional breeding success than those housing their cheetahs in unnatural-type groups. This research uncovers some of the factors which may contribute to the poor reproductive success of the captive cheetah population, and offers recommendations for improvements to current cheetah management practices.

An essential part of plant innate immunity is the perception of pathogen-associated molecular patterns (PAMPs) through surface-localised pattern-recognition receptors (PRRs). EFR and FLS2 are well characterised PRRs, which recognise the bacterial elongation factor Tu and flagellin, respectively. A variety of downstream responses upon PAMP perception have been described. However, it is still poorly understood how EFR- and FLS2-dependent signalling is mediated. Here, I used two different approaches in order to identify novel components of both signalling pathways. First, I characterised seven candidate genes of a predicted flagellin-dependent gene expression network, which are predicted to regulate each other’s expression in a flagellin-dependent manner, and therefore, are potentially involved in the FLS2 signalling pathway. Further characterisation revealed that mutation of at least three of the seven candidate genes was affecting flg22-mediated signalling. In addition, I characterised a predicted protein phosphatase 2C (PP2C), which had been identified in a yeast two-hybrid screen with the EFR cytoplasmic domain and was therefore referred to as PIE (PP2C-interacting with EFR). Using Co-IP experiments, I confirmed that PIE associates with EFR in planta. Furthermore, PIE also associates with FLS2 and BIK1, a co-regulator of both PRRs, in planta. Interestingly, PIE dissociates from both the EFR and FLS2 complexes upon PAMP perception. PIE expressed in planta and in E. coli exhibits protein phosphatase activity and we showed that PIE dephosphorylates EFR, FLS2 and BIK1 in vitro. As expected from bio-informatic predictions, I confirmed that PIE is phosphorylated upon PAMP perception. Furthermore, I demonstrated that EFR and FLS2 kinase activities are partially required for PIE phosphorylation. Both PIE overexpression and pie knock-down lines display reduced PAMP-triggered responses, indicating that an optimal PIE expression level is required for proper induction of signalling. All together, these results imply that PIE is a novel regulator of the EFR and FLS2 signalling pathway.

Epigenetic modifications of DNA and histones play a key role in governing the expression of genes essential during neural development. Histone deacetylase (HDAC) enzymes exert their functions by directly promoting changes in neuronal gene expression through the interaction with chromatin remodelling proteins and specific transcription factors. In cortical neurons, S-nitrosylation of HDAC2 results in transcriptional activation of genes that support neuronal survival and dendritogenesis (Nott et al., 2008). Bead array analysis identified brahma (Brm), a subunit of the ATP-dependent chromatin remodelling complex Brm/Brg-associated factor (BAF), as one of the genes regulated by HDAC2 S-nitrosylation. Expression of mutant form of HDAC2 that cannot be S-nitrosylated decreased Brm protein levels, leading to defects of radial neuronal migration and cortical laminar pattern. Thus, HDAC2 S-nitrosylation is necessary for the correct neuron radial migration during cortical development. HDACs are recruited to chromatin and exert their transcriptional regulatory functions via interaction with other nuclear factors, as part of multiprotein complexes. To identify the binding partners of HDAC2 in cortical neurons, whole sample mass spectrometry analysis was performed. Our analysis revealed a direct interaction of HDAC2 with nucleosome remodelling and histone deacetylase (NuRD) repressor complex. The core ATPase subunit of the NuRD complex includes a family of mutually exclusive chromodomain helicase DNA binding (CHD) proteins, named CHD3, CHD4 and CHD5. In embryonic cortex CHD4 is expressed in proliferating neuronal progenitors and maintained in postmitotic neurons, whereas homologous CHD3 and CHD5 are confined to postmitotic neurons. CHD3, CHD4 and CHD5 are associated with the promoters of neuronal genes indicating that they directly regulate transcriptional programs during neural development. Importantly, down-regulation of CHD3 subunit specifically expressed in postmitotic neurons, results in cortical radial migration defects, confirming the role of NuRD in cortical development. These studies highlight the importance of HDAC2 S-nitrosylation and ATP-dependent chromatin remodelling complexes in cortical development and cortical radial migration.

Intercellular junctions are composed of tight, gap and adherens junctions and have been shown to play many roles in embryonic morphogenesis. I have been studying the role that intercellular junctions play in zebrafish development. First I have studies the organisation of apical neuroepithelial junctions in the developing brain. By quantifying size and segmental patterning of the junctional arrangement in the hindbrain and elsewhere I have described a level of organisation that is characteristic of compartment boundaries. I describe a distinct pattern of apical junctions in both boundary and non-boundary regions. This pattern appears to be in part regulated by the Notch signalling pathway since apical junctions distribution is different in hdac1-/- zebrafish, which have deficient Notch-Delta signalling. Second I have established that boundary cells prevent the exchange of small molecular weight, gap junction permeable dyes between adjacent CNS compartments, suggesting that the compartments are separate developmental units. Third I have studies the role of gap junction mediated intercellular communication in the propagation of calcium waves and in the coordination of cell divisions in the zebrafish blastocyst. Calcium activity is cyclical, with cells producing a greater number of calcium transients and intercellular waves during cytokinesis than during interphase. In control conditions distinct waves of cell divisions spread across the embryo in an animal to vegetal progression. I show that both the calcium activity and the cell division waves require gap junction communication because pharmacological blockade of coupling reduces the frequency of calcium activity and disrupts cell division waves.

The neurokinin-1 receptor knock-out (NK1R-/-) mouse is hyperactive and shows deficits in attentional processing, and has recently been put forward as a model of attention deficit hyperactivity disorder (ADHD). Acute amphetamine, a first-line treatment for ADHD and a drug of abuse, paradoxically reduces the hyperactivity of NK1R-/- mice, and the characteristic amphetamine-stimulated increase in striatal dopamine efflux seen in wild-type animals is attenuated in NK1R-/- mice. The research presented in this thesis centres on the behavioural and molecular responses of NK1R-/- mice to amphetamine. Over the course of this work, a fundamental deficit in the striatal cholinergic neuroanatomy of NK1R-/- and wild-type mice was discovered. While the responses of NK1R-/- mice to amphetamine were normal compared with wild-types, NK1R-/- mice were found to have a reduced number of cholinergic interneurones in the lateral striatum, independent of treatment, and an increased number of cholinergic neurons in the nucleus basalis. In the striatum, the reduction in cell numbers was specific to cholinergic interneurones, which all express the NK1 receptor in wild-type animals and play a key role in regulating striatal dopamine release. The number of striatal parvalbumin-expressing interneurones, which do not express NK1 receptors, did not differ between NK1R-/- and wildtype mice. The present results demonstrate that global disruption of the NK1 receptor results in significant alterations in central cholinergic system neuroanatomy, which may contribute to the previously observed impaired dopaminergic response to amphetamine. The results presented in this thesis provide support for emerging evidence that deficits in cholinergic transmission play a role in the pathophysiology of ADHD, and could have significant implications for future ADHD research.

Previous studies have reported that the adult periodontal ligament (PDL) may contain progenitor/stem cells that function as precursors for periodontal tissue regeneration, although the ability of this population to differentiate into the multiple lineages present in the PDL is not yet certain. In addition, although Enamel Matrix Derivative (EMD; Emdogain®), derived from the enamel matrix of developing teeth) has been used extensively to help re-build new periodontal tissue, its effect on bone regeneration remains inconclusive and its effect on PDL blood vessels and nerve cell development not yet known, because it comprises a heterogeneous mixture of proteins. EMD has recently been separated into two main fractions: Fraction C, containing proteins < 6 kDa (mainly the tyrosine-rich amelogenin peptide TRAP); and Fraction A, containing proteins > 6 KDa (including the full-length amelogenin, sheathlins and a leucine-rich amelogenin peptide LRAP). The present study examined the effects of EMD Fractions on multi-lineage differentiation pathays of PDL cells in vitro. The results of the present study have shown that that Fraction C and Fraction A differentially regulate multilineage specification of PDL cells. Thus, Fraction C was found to up-regulate chondrogenic, vasculogenic, angiogenic, neurogenic and gliogenic genes and ‘terminal’ differentiation, whereas Fraction A was found to stimulate osteogenic genes and terminal osteogenic differentiation in vitro; both fractions suppressed adipogenesis. Moreover, the TRAP and LRAP peptides of Fraction C and Fraction A, respectively, were found to be at least partly responsible for the differential activities of these two fractions. In addition, at least some components in these EMD Fractions bound to and were internalized into PDL cells, most probably by receptor-mediated endocytosis. These findings thus demonstrate that the PDL contains cells with multi-lineage differentiation potential and that the components of EMD have differential effects on the diverse activities on the heterogeneous cells present in the PDL.