Kallmann's syndrome (KS) is characterised by the association of anosmia (a loss of sense of smell) and hypogonadotrophic hypogonadism. Defects of anosmin-1, a protein encoded by KAL-1, underlie the X-linked inherited form of KS. Anosmin-1 comprises an N-terminal cysteine rich domain and a whey acidic protein like (WAP) domain, followed by four consecutive fibronectin type HI (Fnlll) like repeats. In vivo and in vitro studies revealed that anosmin-1 functions as an adherent extracellular matrix protein involved in cell adhesion and neurite outgrowth, actions dependent on an interaction of Fnin domains with heparan sulphate (HS). However, the binding characteristics and kinetics between C-terminal Fnin domains of anosmin-1 and HS have not hitherto been systematically investigated. Although anosmin-1's N-terminal WAP domain shares homology with serine protease inhibitors, its putative protease ligand is still unknown. In this thesis: 1. A Drosophila S2 cell expression system was used to express full length and truncated domain specific versions of anosmin-1. The full-length (PIWF4), truncated (PIWF1, with single FnlH-1 domain) protein, and their C172R WAP domain mutants were purified using Ni-NTA affinity chromatography. The high yield of PIWF1 protein enables the generation of an anti-anosmin-1 antibody. 2. A HS competitive assay showed that anosmin-1 was S2 cell surface associated through a Fnin domain-HS interaction. A solid phase ELISA assay indicated purified anosmin-1 bound to HS in a dose-dependent manner. Surface plasmon resonance (SPR) revealed real-time kinetic interactions with K& = 2 nM.

Axonal loss and remyelination are major pathological substrates implicated in the balance between recovery and fixed disability that occurs following optic neuritis and other forms of relapse in multiple sclerosis. Optic neuritis is an ideal model for studying the functional consequences of a single demyelinating lesion. Several quantitative measures of vision can assess function of the anterior visual pathway, and electrophysiological testing can provide measures of axonal integrity and myelination. These measures are highly reproducible and permit the functional relevance of any structural changes to be determined. The studies presented in this thesis applied three retinal imaging techniques and three optic nerve MRI techniques to investigate the extent and functional significance of axonal loss and remyelination following optic neuritis. Two of the retinal imaging measures, optical coherence tomography (OCT) and scanning laser polarimetry, demonstrated functionally relevant neuroaxonal loss in patients with incomplete recovery following optic neuritis. In acute optic neuritis patients, OCT detected retinal nerve fibre layer (RNFL) thinning after three months and quantified acute peripapillary RNFL swelling in bulbar cases. MRI-detected optic nerve atrophy correlated well with OCT RNFL thinning supporting the hypothesis that axonal loss is the major substrate of atrophy. Furthermore, there was evidence from the atrophy study of retrograde degeneration from the optic nerve to RNFL to macula. Optic nerve diffusion tensor imaging demonstrated increased mean diffusivity and reduced fractional anisotropy in nerves affected by optic neuritis compatible with a process of axonal disruption or loss. Optic nerve magnetisation transfer ratio in affected optic nerves correlated with both visual evoked potential latency and RNFL thickness suggesting that MTR may not be an exclusive marker of myelination alone and also reflects co-existent axonal loss. Anterior visual pathway imaging may be useful in monitoring therapies that aim to prevent axonal loss and enhance remyelination in optic neuritis.

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Following sciatic nerve transection in adult rats, a proportion of injured dorsal root ganglion (DRG) neurons die, through apoptosis, over the following 6 months. Axotomy also has effects on expression of neurotrophins (NGF, BDNF, and NT-3) and their receptors (trkA, trkB, trkC, and p75NTR) in DRG as shown by in situ hybridisation or Northern blotting. Previous groups showed that administration of neurotrophin-3 (NT-3) to the proximal stump or intrathecally appears to prevent neuronal loss and functional impairment after axotomy. This thesis tests the hypothesis that (1) Axotomy may cause certain cells to differentiate into DRG neurons and NT-3 may stimulate this process. (2) Systemic NT-3 may produce morphological and biochemical changes in DRG that may assist regeneration. During the course of the study, the 4th and 5th lumbar DRGs were examined up to 8 weeks after right sciatic nerve transection and ligation. Stereology was used to estimate neuronal numbers, and morphological and immunohistochemical techniques were used to examine the incidence of neuronal apoptosis. Antibodies for p-III tubulin, trkA, trkC and CGRP were applied to characterise nestin-immunoreactive cells. Real-time quantitative PCR was used to investigate the effects of axotomy and systemic NT-3 on the mRNA expression of neurotrophins, their receptors and nestin in injured DRGs at various time points. In addition, the effects of axotomy and NT-3 treatment on neuronal genes were investigated by microarray. The results obtained suggest that: (1) Axotomy led to 16% neuronal loss in L4 and L5 DRGs 4 weeks after injury administration of NT-3 systemically for 4 weeks prevented neuronal loss, but did not reduce neuronal apoptosis. The appearance of nestin- and p-III tubulin- immunoreactive cells in these DRGs suggests an axotomy-initiated replacement mechanism, which was enhanced by systemic NT-3 treatment. (2) The changes of neurotrophin and neurotrophin receptor mRNA suggest a higher overall responsiveness of DRG neurons to neurotrophins after axotomy and NT- 3 treament. (3) Microarray data showed the up-regulation of a few genes relevant to neuronal lineage commitment of progenitor cells following axotomy and that some signalling pathways were activated after axotomy and NT-3 administration. NT- 3 may stimulate axonal regeneration after axotomy.

In vivo electrophysiological recordings of neuronal ensemble single-unit and local field potential (LFP) activity were performed in the hippocampus and medial prefrontal cortex (mPFC) of isoflurane-anaesthetised rats to exannine the role of the endogenous cannabinoid system in regulating neuronal excitability. These data suggest that KA-induced hyper-excitability in the rat hippocampus can be suppressed by inhibition of endocannabinoid metabolism (partly via a CB1 receptor-mediated mechanism), the application of a non-selective CB receptor agonist, and in part, by administration of a phytocannabinoid. These observations may have future applications in treating hyper-excitable disorders.