Effetti di nicotina e colina sull'omeostasi del calcio e sull'espressione dei recettori per le neurotrofine in culture neuronali della corteccia di ratto

Nicotine is known to induce effects at molecular and cellular levels and to affect the whole organism and its behaviour. Nicotine binds to a group of receptors, nicotinic acetylcholine receptors (nAChRs). They are protein structures composed of five subunits, which are arranged to form a cation channel through the cell membrane. Many different subunits exist and they can assemble in a variety of combinations giving rise to many forms of nAChRs. When a ligand, such as nicotine, binds to nAChRs the permeabiliy of the channels may change. Different nAChRs show differences in their permeability to various cations. The homomeric alpha7 subunit containing nAChR shows the highest fractional Ca2+ current among nAChRs in the nervous system. Choline, the degradation product of the natural nAChR ligand acetylcholine, is a full and selective agonist of alpha7 nAChRs. Some nAChRs can mediate fast synaptic transmission, but modulatory functions of nAChRs are thought to be more common. Presynaptically localized, nAChRs may be involved in modulation of neurotransmitter release. At postsynaptic and somal sites nAChRs may have modulatory effects both at synaptic and cellular level. Many of the modulatory effects in which nAChRs are thought to be involved require calcium signals, i.e. changes of intracellular calcium concentration. Since alpha7 nAChRs have a high permeability for Ca2+, they are thought to be involved in many of these effects. When the environment changes, a cell can try to adapt to the new situation. Two examples of neuroadaptation are changes of intracellular calcium concentration [Ca2+]i and changes of protein levels. Both nAChRs and neurotrophic factors, such as neurotrophins and their receptors (NTRs), can be involved in these two forms of neuroadaptation. The first part of this work aims to investigate the role of nAChRs on calcium signals and is based upon different imaging studies, such as fluorescence immuno-cytochemistry and calcium imaging. The second part is based on Western blot studies and aims to analyse a possible role of nAChRs on expression levels of NTRs. In both parts a special emphasis is put on alpha7 nAChRs and in both parts primary cell cultures from rat cerebral cortex were used. The results obtained in the first part of the present study indicate that alpha7 nAChRs may be involved in calcium signals in cortical cell cultures and that choline may be selective for alpha7 nAChRs, since it induced a trend of increased [Ca2+]i only in cells expressing alpha-bungarotoxin binding sites, known to correspond to alpha7 nAChRs. The second part of this study indicates an interaction between alpha7 nAChRs and one NTR, i.e. TrkB, on an expression level basis. This conclusion is drawn from the observation that choline exposure of cell cultures attenuated TrkB levels. However, in order to clarify the involvement of alpha7 nAChRs in these observations more analysis have to be done.