Glutamate release onto a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is the primary mechanism of fast synaptic transmission in the mammalian brain. Previous studies have revealed that at the cerebellar mossy-fibre to granule cell synapse, the AMPAR mediated synaptic current con sists of a fast-rising and a slow-rising component. The aim of this thesis is to examine the properties of release, diffusion and receptor activation underlying these two components. Two plausible mechanisms could underlie the slow-rising current: spillover of glutamate from neighbouring synaptic contacts and prolonged local release of glutamate via a narrow fusion pore. Using simulations of glutamate diffu sion and receptor activation, I show that lowering the diffusion coefficient of glutamate in the synaptic cleft (Dgjut), which is unknown but can be modulated with macromolecules, has different effects on currents mediated by these two mechanisms. Recordings of the effect of perfusion of dextran (43 kDa) are consis tent with the spillover model and also indicate that Dgiut is approximately 3-fold lower than in free solution. I show using simulations that linear diffusion cannot alone account for the acceleration of the decay of the synaptic current observed at this synapse in lower release probability, but that it can result from non-linear activation of AMPARs. Evidence is presented that diffusion is linear and only one vesicle is released per active zone. In addition, I have extended the diffusion-reaction model of the synapse to develop a framework for examining properties of synaptic AMPARs using glutamate uncaging. I demonstrate that certain kinetic properties of synap tic receptors can be measured using this technique and derive a kinetic model based on preliminary data. Together, these data fill shortcomings in our understanding of synaptic func tion. Based on the present results, I construct a model of synaptic transmission that explains previous observations.

This thesis is an investigation of the behaviour of radial progenitors in the hindbrain of the teleost fish Danio rerio, the zebrafish, during embryonic neurogenesis. These progenitors have many of the characteristics of radial glial cells described in other systems. Recent studies from several laboratories worldwide have shown that radial glia play an important role in the patterning and neurogenesis of the mammalian forebrain. However, despite the enormous interest in the subject, investigations into neurogenic radial glia have to date been largely limited to the mouse telencephalon. No similar study has yet investigated embryonic neurogenic radial glia in other vertebrates, and very few have been carried out in brain regions other than the forebrain. In this study I used the zebrafish as a model system to identify and characterise radial glia and progenitors in the hindbrain, and examine the role of these cells in neurogenesis in this brain region. Radial progenitor cells in the hindbrain ventricular zone (VZ) were found to express glial fibrillary acidic protein (GFAP) during embryonic development, and some of these cells also expressed markers of proliferation such as PH3. Fatemapping these radial glia-like cells using single-cell labelling techniques, I demonstrated that the major neurogenic event in the zebrafish hindbrain after 48hpf is direct differentiation of progenitors into neurons, without cell division. Very few cell divisions were recorded, and those few that were observed were without exception symmetric, neuron-pair terminal mitoses. In contrast to previous studies of mammalian neurogenesis, no asymmetric cell divisions were observed in my experiments. I then examined the behaviour of VZ progenitors as a population, by taking advantage of the Tg(zFoxD3:GFP) stable transgenic line. In this line, a subpopulation of hindbrain VZ progenitors expresses GFP during development, and I recorded timelapse movies of hindbrain neurogenesis, in transgenic embryos, between 34hpf and 50hpf This revealed the radial migration of GFP-expressing newborn neurons from VZ into the mantle layer, and how these cells utilise multiple GFAP-expressing radial processes as a substrate for this migration. These timelapse videos are the first description of radial migration in a vertebrate brain in vivo.

No description available.

No description available.

No description available.

No description available.

In the brain, classical (canonical) transient receptor potential (TRPC) channels are thought to be involved in different aspects of neuronal development. This study investigated the postnatal developmental expression profile of the different TRPC subunits in rat cerebellum for the first 6 weeks after birth to establish potential links between cellular development and TRPC channel expression as well as TRPC expression in diseased human cerebellar tissue.

Noxious stimuli have a profound influence on motor behaviour, but there is a paucity of information regarding how somatosensory information is transformed into a motor output. The cerebellum is the largest sensorimotor control structure within the brain and previous electrophysiological studies have demonstrated that climbing fibres, one of its major sources of afferent input, are activated in response to a noxious pinch stimulus. This thesis investigates the anatomical pathways by which nociceptive information may reach the cerebellum, with a particular focus upon the olivo-cerebellar system, the sole source of climbing fibres.

In the cerebellar cortex, forms of both long-term depression (LTD) and long-term potentiation (LTP) can be observed at parallel fibre (PF) - Purkinje cell (PC) synapses. A presynaptic variant of cerebellar LTP can be evoked in PCs by raised frequency stimulation (RFS) of parallel fibre at 4-16Hz for 15s. This form of LTP is dependent on protein kinase A (PKA) and nitric oxide (NO), and can spread to distant synapses. Application of an extracellular NO scavenger, cPTIO, was found to prevent the spread of LTP to distant PF synapses in rat cerebellar slices. G-substrate may be an important mediator of the NO-dependent pathway for LTD. 8-16Hz RFS of PFs without a high concentration of calcium chelator in the postsynaptic cell evokes LTD. In cerebellar slices from wild-type and transgenic, G-substrate knockout mice, 8Hz RFS was applied to PFs, with a low concentration of postsynaptic calcium chelator. In PCs from wild-type mice, LTD predominated, whereas in those from transgenic mice LTP predominated. The ascending axon (AA) segment of the granule cell axon forms synapses with PCs as well as the PF segment. PPF and fluctuation analysis of EPSCs in rat PCs confirmed that the release sites of AA synapses have a greater probability of transmitter release than PF synapses. Furthermore, AA release sites have greater mean quantal amplitude than PF synapses, which is not due to a different type of postsynaptic receptor. AA synapses were found to have limited capacity to undergo the presynaptic variant of LTP, and were potentiated less than PF synapses in the presence of the PKA activator, forskolin. AA synapses also did not undergo the postsynaptic form of LTP, nor LTD induced by conjunctive stimulation of climbing fibre and PF.

- / -