No description available.

No description available.

No description available.

No description available.

No description available.

Despite great progress in understanding of how experience modifies cortical circuitry in primary visual cortex (VI), the underlying physiological and molecular mechanisms still remain to be understood fully. Although some of the molecules associated with the critical period for ocular dominance plasticity in cats have been examined, the role of downstream signalling molecules that form pathways with receptor subunits has received scant attention. The present study demonstrates using optical imaging of intrinsic signals that sensory experience is not required for initial establishment of ocular dominance column layout and iso-orientation domains but is required for maintenance of these properties its absence leads to their eventual breakdown. Animals were sacrificed and VI was removed and homogenised, followed by immunoblotting for quantification of protein expression. The immunoblotting findings point to a set of proteins (including NR2A, PSD-95, aCaMKQ, NR2B and GABAAala) that are regulated developmentally and the effects of dark-rearing indicate that sensory activity regulates mechanisms associated with both excitatory (NR2A and NR2B) and inhibitory (GABAAala) transmission and synaptogenesis (synaptogenesis) so as to maintain a homeostatic balance. Pattern or form information is necessary in both eyes to maintain normal maps in both eyes while differences in illumination between two eyes did not affect ocular dominance and orientation maps in normally reared or in dark-reared cats subsequently exposed to light. Monocular deprivation (MD) for 2 days and 7 days resulted in similar depression of deprived eye responses. In contrast, potentiation of non-deprived eye responses was almost double in magnitude after 7 days compared to 2 days of MD. The immunoblotting findings demonstrate that MD regulates signalling molecules (PSD-95, aCaMKH and synGAP) downstream of NMD A receptors and GluRl subunit it appears that different mechanisms are activated depending on the nature of sensory experience.

In this study, the expression of several sensory neuron specific/predominant genes, and the effects of neurotrophic factors upon them were studied in embryonic, postnatal and adult mouse sensory neurons. In the embryonic mouse, NGF/TrkA signalling was shown to be essential for the expression of mRNAs encoding substance P and the sodium channels Navl.8 and Navl.9 in DRG and trigeminal ganglia. Differential regulation of the two isoforms of calcitonin gene related peptide (CGRP) mRNA was apparent in the DRG with a requirement of NGF/TrkA signalling for expression of a, but not p CGRP. This was not reflected in the trigeminal ganglia. Postnatally, experiments revealed that NGF/TrkA signalling within the DRG and trigeminal ganglia is 1) essential for expression of SP, ocCGRP, pCGRP, Navl.8, Navl.9 mRNAs, 2) possibly required for expression of the neuropeptide galanin and the capsaicin receptor vanilloid receptor 1 (VR1) mRNAs, 3) not required for pituitary adenylate cyclase-activating peptide (PACAP) mRNA. Conversely, within the nodose ganglia, expression of Navl .8 and Navl .9 mRNAs did not require NGF/TrkA signalling. No regulation of all aforementioned genes by neurotrophin-3 (NT-3) was observed in trigeminal, nodose or dorsal root ganglia. In the adult mouse, DRG cultures were utilised to study gene regulation by the neurotrophic factors NGF, artemin and macrophage stimulating protein (MSP). Expression of SP, aCGRP, pCGRP, Navl.8, Navl.9 and VR1 mRNAs all showed a decrease following 96 hours in culture that was inhibited by presence of MSP (50ng/ml), NGF (lOng/ml) or artemin (lOng/ml). PACAP, galanin, damage induced neuronal endopeptidase (DINE) and activating transcription factor 3 (ATF3) mRNAs increased over time, but neurotrophic factors could impede such increases. No axotomy or neurotrophic factor-induced effects were observed for P2X3, Navl.6 or Navl.7 mRNAs. Interestingly the additional presence of leukaemia inhibitory factor (LIF) opposed NGF, MSP and artemin-induced effects on PCGRP, SP, VR1 and galanin mRNAs, whilst enhancing effects on PACAP and DINE transcripts.

Endocytosis proteins contain a number of different specialised domains which control protein-protein interactions at specific steps. A variety of these domains were used in a proteomic screen using GST-pull down assays from nerve terminal lysates to investigate whether Ca²⁺ controlled any specific interactions. Many interactions appeared to occur only in the presence of Ca²⁺, however this was attributed to non-specific binding due to the presence of ZnSO₄­. Two new interactions were identified that bind SH3 domains only in the absence of Ca²⁺, the adaptor protein caskin I and a protein known as “similar to KIAA0856”. Since SH3 domain containing proteins had Ca²⁺-dependent interactions, these were examined in more detail. Most proteins bound to SH3 domains only in the absence of Ca²⁺. These proteins included caskin I, synaptojanin, amphiphysin I, dynamin I and synapsin I. However an unidentified 90 kDa band bound only in the presence of Ca²⁺ suggesting Ca²⁺ positively and negatively regulates interactions. These interactions were characterised further by examining the amount of Ca²⁺ required to stimulate or inhibit the above associations. Ca²⁺ binding of endophilin II has been proposed to control its protein-protein interactions. A number of complementary techniques were used to examine the Ca²⁺ affinity of SH3 domains including endophilin. These techniques included ⁴⁵Ca²⁺ overlay assays, tyrosine and tryptophan fluorescence and equilibrium dialysis. From all of the fusion proteins investigated the SH3 domains appeared to bind ⁴⁵Ca²⁺ however the full length proteins did not. Tyrosine and tryptophan fluorescence showed a Ca²⁺ dependent fluorescence shift suggesting a conformational change on the addition of Ca²⁺, meaning that Ca²⁺ may bind to the SH3 domains. Finally equilibrium dialysis showed that no endophilin GST fusion protein bound to Ca²⁺. From these results it is still open to question whether endophilin binds Ca²⁺.

This thesis is devoted to examining the role of Apc protein in the development of the mouse telencephalon. As <i>Apc^-/-</i> embryos die at gastrulation, a conditional knock-out approach was used to examine Apc’s role in cortical development. Mutant mice with conditional (flowed) alleles of <i>Apc</i> were crossed to a strain in which Cre expression is driven by the <i>Emx1 </i>promoter (<i>Emx1^Cre</i>), allowing specific deletion of <i>Apc </i>in the dorsal telencephalon. The current work presents a detailed description and possible explanations of Apc’s functions in the developing cerebral cortex. Conditional knock-out of <i>Apc </i>in the cortex led to severe developmental defects in this region, showing that Apc is required for normal development of the cerebral cortex. The deletion of <i>Apc </i>leads to over-activation of the canonical Wnt pathway and disregulation of PCP Wnt pathway. I examined the expression of regional markers in the residual dorsal telencephalon in conditional <i>Apc </i>mutant embryos. Expression of Foxg1 is lost, showing that the mutant dorsal telencephalon loses telencephalic identify from E12.5. My data show that Apc is important for proper patterning of the cortex probably mostly by antagonizing the canonical Wnt pathway. Measurement of cell-cycle times revealed that in the absence of Apc, S-phase and cell cycle length remains similar to controls. Deletion of <i>Apc </i>in the dorsal telencephalon leads to defects in maintenance of the size of the progenitor pool and regulation of apoptosis. The conditional Apc deletion has a pleiotropic effect on cortical development. Developmental defects found include disregulation of Wnt signalling, loss of cell polarity, adhesion defects, cell identify defects, and disregulation of apoptosis. Assignment of these defects to particular functions of Apc is not completely clear yet.

Dynamin 1 is a 96kDa GTPase and is one of eight proteins known to undergo coordinated dephosphorylation upon nerve terminal stimulation and subsequent rephosphorylation. They are collectively referred to as the dephosphins and are all dephosphorylated by calcineurin and at least three are rephosphorylated by cyclin-dependent kinase 5. Phosphomemetic peptides were used to investigate the specific role of dynamin 1 phosphorylation in synaptic vesicle endocytosis. The peptide mimicking dephosphorylated dynamin 1 was nearly four times more effective at inhibiting synaptic vesicle endocytosis than the peptide mimicking phosphorylated dynamin 1, indicating that there is an essential role for these phosphorylation sites in endocytosis. Experiments to probe the function of the phosphorylation sites identified syndapin 1 as a stimulation dependent binding partner to this region of dynamin 1. The interaction was further characterised by immunoprecipitation and GST-pulldown assays. Dynamin 1 can also be regulated by Ca²⁺ either directly or indirectly through Ca²⁺-dependent binding partners. Dynamin 1 is published to bind to the cytosolic C-terminus of the integral vesicle protein synaptophysin in a Ca²⁺-dependent manner. This thesis shows that the over-expression of the C-terminus of synaptophysin-eGFP in cerebellar granule neurites inhibits vesicle recycling and that a peptide designed from this region of synaptophysin also inhibits vesicle recycling in isolated nerve terminals. GST-pulldowns with the C-terminus of synaptophysin extracted dynamin 1, in the presence of Ca²⁺, but also a more prominent band shown by western blot to be amphiphysin 1. Amphiphysin 1 was confirmed to bind to the C-terminus of synaptophysin independently from dynamin 1. Amaphiphysin 1 may interact with synaptophysin, in nerve terminals, linking synaptophysin to a potential role in synaptic vesicle endocytosis.