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Methods used for studying neurite outgrowth in culture range from attachment of dissociated neurons or explants to coated substrata, to embedding cells or tissues in three-dimensional matrices. Here I describe a new technique in which isolated chick embryo dorsal root ganglia (DRG) can be cultured floating on the surface of serum-free culture medium. The utility of the preparation for studies of neurite growth at the cellular level, and at the level of pattern formation during development is demonstrated. 1. The quantitative effects of neurotrophic factors in regulating neurite outgrowth can be measured using this new preparation. The extent of outgrowth from floating DRG was compared with that from ganglia adhered to laminin, or to polylysine. This preparation provides a substrate-independent assay for neurotrophic factors which offers a number of advantages over existing bioassays. It is also potentially useful in the search for novel neurotrophic molecules. 2. The movement of neuronal growth cones is not well understood. Cytoskeletal dynamics and cell-substratum adhesion are involved. Since the growth of neurites from floating ganglia occurs in the absence of a conventional substratum it was thought that this preparation might provide new insight into the mechanism of growth cone movement and neurite elongation. Experiments were carried out using the drugs colcemid and nocodazole which affect microtubules and cytochalasin B which affects actin filaments. Colcemid and nocodazole inhibited neurite outgrowth from both floating and adhered ganglia. In contrast cytochalasin B did not inhibit outgrowth from floating ganglia although it completely inhibited outgrowth from ganglia adhered to laminin. 3. During the development of sensory innervation, segmental differences arise in the rates of growth of axons invading limb and non-limb regions. Growth rates of axons invading developing limb are faster. It is not known how these different axonal growth rates are controlled.

This thesis examines mechanisms of regulation of neuropeptide gene expression <I>in vivo</I> in some neurosecretory hypothalamic neurones of the rat. In particular, the influence of neural pathways, acting via receptors and subsequent regulation of genetic transcription factors was measured and second messenger pathways were directly manipulated and the effects of their mutation on gene expression were measured. The magnocellular neurones of the supraoptic nucleus (SON) are known to be directly (i.e. non-synaptically) osmosensitive. Fos, the protein product of the immediate early gene <I>c-fos</I>, has been used as a marker of neuronal activation and its expression is induced in these neurones by increasing systemic hyperosmolarity. The effects of acute, direct hyperosmotic stimulation, via a microdialysis probe, on Fos expression in supraoptic nucleus magnocellular neurones was investigated. Fos expression was detected by immunohistochemistry (IHC). However, direct hyperosmotic stimulation failed to induce Fos expression in the magnocellular neurones in the SON but did induce Fox expression in glial cells within the SON and in the ventral glial lamina, as detected with double IHC. A receptor-mediated stimulus, cholecystokinin, applied directly to the SON did induce Fos expression in magnocellular neurones. These results indicate that Fos induction requires synaptically-mediated activation and consequent specific changes in cellular secondary messenger systems. Therefore Fos is not always induced when neurones are otherwise defined as active. In rats, chronic morphine administration into a lateral cerebral ventricle, over 5 days, results in the magnocellular oxytocin neurones developing dependence as demonstrated by the effects of administration of the opioid antagonist, naloxone, which results in their withdrawal excitation.

Activation of the N-methyl-D-aspartate(NMDA) receptor is important in both physiological and pathological phenomena, including synaptogenesis, long-term potentiation, neuroexcitotoxicity and epilepsy. Although implicated in postnatal formation of synaptic connections, over-activation of the NMDA receptor in the neonate, as a result of say a hypoxic-ischaemic insult, can lead to neuronal damage. Antagonists for specific sites on the NMDA receptor complex may therefore prove to be novel therapeutic agents for preventing excitotoxic neuronal damage. I have therefore investigated the ontogeny of the NMDA receptor complex as well as modulation of the receptor in both adult and neonatal rat brain tissue. Radioligand binding studies were performed using high-affinity NMDA receptor antagonists. [<SUP>3</SUP>H]3-((±)-2-carboxypiperazin-4-yl)propyl-1-phosphonoate ([^3H]CPP) and [^3H]D-2-amino-5-phosphonopentanoate([^3H]D-AP5), both competitive antagonists which bind to the NMDA neurotransmitter recognition site, and [^3H]dizocilpine a non-competitive antagonist which binds to a site within the lumen of the NMDA-associated ion channel, were used. Optimal experimental conditions were established for the binding of each ligand to membranes prepared from rat brain tissue. This allowed reliable and reproducible measurements to be made under different modulatory conditions. Using mature tissue the binding of [^3H]CPP was compared with that of [^3H]AP5 in the presence of compounds active at the glycine modulatory site including glycine, 7-chlorokynurenate and 3-amino-1-hydroxypyrrolid-2-one(HA-966). Differential effects were seen on the binding of each ligand. The findings provide further evidence for the hypothesis that the NMDA receptor exists in more than one conformational state, these being regulated by glycine. [^3H]CPP and [^3H]dizocilpine were used to investigate the ontogeny of their respective binding sites. Each ligand bound to membranes prepared at various postnatal ages between birth and adulthood. The binding of both ligands was detectable at the earliest ages examined, increasing with postnatal age.

The effect of phosphorylation on Ca<SUP>2+</SUP> current, in the presence and in the absence of 5-HT<SUB>1A</SUB> receptor activation, has been studied here using a whole-cell voltage-clamp technique in acutely dissociated adult rat DR neurones. In various neuronal preparations, transmitters have been shown to reduce voltage-dependent Ca<SUP>2+</SUP> currents. In DR neurones, the inhibitory action of 5-HT is incomplete, usually around 60%, and is accompanied by a dramatic slowing of the activation. 8-OH DPAT, a highly specific 5-HT<SUB>1A</SUB> agonist, also partially and reversibly reduces high-voltage activated (HVA) Ca<SUP>2+</SUP> currents and slows the activation time. It appears that the effect is G-protein mediated and it is believed that the G-protein acts directly on the Ca<SUP>2+</SUP> channel proteins, rather than through a freely diffusible second messenger. The action of 8-OH DPAT and GTP-γ-S on both the amplitude and the activation of the current can be removed by a large depolarising prepulse. A variety of data indicate that protein phosphorylation regulates the efficacy of synaptic transmission, both by controlling the release of neurotransmitters from the presynaptic nerve terminal and by modulating the sensitivity of receptors in the postsynaptic membrane. Presumably, at rest, some kind of phosphorylation/dephosphorylation balance exists. Evidence has been given that phosphorylation is a necessary step in ion channel functioning. It appears that inhibition of specific phosphatases, could potentiate already activated ion channel currents.

The medial prefrontal cortex (mPFC} and hippocampus (HPC} are central to executive control, spatial learning and working memory. In order to enable behavioral control, the function of the mPFC and HPC is tuned by complex interplay between excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmitter systems. This thesis has employed lesions, pharmacological and optogenetic methodologies to investigate how the relationship between excitation and inhibition within the adult mouse mPFC and HPC affects cognition, using a battery of touchscreen-based operant assays: the automated spatial array task (ASAT), Spatial Reversal (SR) and Visual Discrimination (VD). Behavioral testing following excitotoxic lesions showed that the HPC was strongly implicated in the performance of both spatial tests (ASAT and SR), while removal of the mPFC had only marginal effects on learning with several trends that did not reach significance. Additionally, in VD, effects were only present as trends towards an involvement of the mPFC in formations of new stimulus-reward relationships. Interdependent processing spanning the mPFC and HPC while not directly assessed can be considered likely to explain complex changes in task performance. Using the described assays helped validate their application to test mPFC and HPC function in mice.