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|Autori: ||Colavita, Michelangelo|
|Titolo: ||Dynamics of hippocampal networks revealed by voltage sensitive dye imaging|
|Abstract: ||In order to better understand brain functioning we need to investigate all the structural domains present in it, from single cell to interconnected entire brain regions. However, while our knowledge in terms of single/few cells functioning is vast, very little is known about neuronal networks, which are interacting collections of neurons functionally related to the same task. Moreover, the balanced and concerted activity of excitatory and inhibitory networks plays a key role for proper cortical computations. However, while exist several tools to record excitatory networks activity, this is not the case for inhibitory networks. Voltage sensitive dye imaging (VSDI) is a technique that allows the recording of neuronal activity by mean of proportional emission of fluorescence according to changes in membrane potential. The advantage of using VSDI over other recording techniques using electrodes is that VSDI allows not invasive recording of neuronal activity from hundreds of sites at the same time.
During the last decades, VSDI has been widely used both in vitro and in vivo and to investigate both single cells and excitatory network activities. However, by using VSDI, investigations on excitatory networks activity have been mainly performed by quantifying fluorescence emission in defined regions of interest at time-fixed points, while inhibitory activity has been evaluated only at single cell level. The former approach misses several information of the dynamics of spreading of glutamatergic transmission because does not consider for example how fast a signal propagates and in which direction. The latter approach instead, does not allow the monitoring of network inhibitory events, which would be very important considering the extensive spatial spreading of interneurons within cortical areas.
During my doctoral course I aimed at studying in detail excitatory and inhibitory neuronal networks in the CA1 area of mouse hippocampus with VSDI.
To study excitatory networks more comprehensively, in collaboration with a team of mathematicians, we developed a mathematical algorithm that allowed measuring the velocity and the direction of spreading of the VSDI signal and it represents a new method to determine an optical flow. After successful validation of the algorithm with surrogate data to test its accuracy, we analysed two set of experiments in which network excitatory activity has been manipulated either by increasing Schaffer s collaterals stimulation intensity or by blocking GABAergic transmission with the GABAA receptor antagonist picrotoxin in order to increase the depolarization in the CA1 region of the hippocampus. The results of these manipulations significantly decreased signal velocity whereas picrotoxin application significantly modified the direction of spreading, making the depolarization-mediated VSDI signal less dispersed compared to control.
Using VSDI I was able to fully characterize GABAA receptor-mediated hyperpolarizing signals in all the CA1 sublayers (field IPSPs), thus providing a new way of monitoring inhibitory events at network level. Moreover, I found that the activation of mGluR5 receptors induced an increase in a long-lasting manner of the VSDI-recorded field IPSPs, with duration and magnitude that relied on the specific CA1 sublayer considered.
Overall, my work shows new methodologies and new findings that may represent a step forward in the quest for a better understanding of neuronal networks, both excitatory as well as inhibitory, which hopefully can contribute to reduce the gap of knowledge between single cell activity and behaviour.|
|In||Area 06 - Scienze mediche|
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