How do TTX and AP5 affect the postrecovery neuronal network activity synchronization

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Slide 1 : How do TTX and AP5 affect the post-recovery neuronal network activity synchronization? F. Esposti, M.G. Signorini, J. Lamanna, F. Gullo, E. Wanke federico.esposti@polimi.it
Slide 2 : Aim of the work Studying the topologic property variations of in vitro cortical neuronal networks on MEA supports in particular conditions: development: from 6 to 12 days in vitro (div) after Tetrodotoxin (TTX) administration after D-2-amino-5-phosphonovalerate (AP5) administration How? Analyzing the spontaneous activity spatial distribution of murine dissociated cortical neuronal networks on MEA devices as an index of self-organization of the network
Slide 3 : Methods The topology of bursts is analyzed in terms of number of channels involved in the burst. This approach allows to obtain an immediate index of the connectivity of the network. Starting from the Raster Plot, a classic spatio-temporal representation of the network spiking activity, we located bursts with an easy spiking frequency-based algorithm. Once bursts were singled out, we classified them as Global bursts if they involved more than 25% of MEA channels and as Local if they involved between 10% to 25% of them (under 10% it wasn’t classified as burst).
Slide 4 : Data MEA data were recorded at the Wanke lab of the Università degli Studi di Milano-Bicocca, Dipartimento di Biotecnologie e Bioscienze Data recorded from electrodes were filtered using a 100Hz – 2kHz band-pass filter and recorded in a PC. For the analysis that follows we chose three cultures: The first (6 div) was recorded for 7 hours with no perturbation. A grand total of 411 bursts were recorded. The second one (10 div) was recorded for 6 hours: the first 2 with no perturbation and then other 4 hours of activity recovery after a bath of TTX (1µM) (with a 30 min. wait after washing out). A grand total of 3375 bursts were recorded. The last one (12 div) was recorded for 2.5 hours: the first 1 hour and half with no perturbation and then other 1 hour of activity recovery after a bath of AP5 (10µM) (with a 30 min. wait after washing out). A grand total of 1345 bursts were recorded.
Slide 5 : Results: network development Network topology comparison in 6 div, 10 div and 12 div cultivations. Data are compared over an hour of recording. As notable, local bursting activity appears after the 6 div. This phenomenon can be traced back to the connectivity reduction that clashes with network maturation after the first week in vitro as reported from, e.g., (Shefi et al., Phys. Rev. E 66, 021905, 2002) or (Mukai et al., El. Eng. In Japan, 145, 4, pp. 1481-1489, 2003).
Slide 6 : The total burst activity, as expectable, increases from day in vitro 6 to 12 Both TTX and AP5 use, after washing out, increases total burst number: ?TTX = +66%, ?AP5 = +24% Results: drugs administration 1
Slide 7 : The TTX burst increase is an intensive Global activity explosion The AP5 modest burst increase is mainly a Local activity rising and do not essentially change the ratio between the local and global burst number. Results: drugs administration 2
Slide 8 : The presence of a localized activity, particularly in cultures older than 1 week in vitro, besides sustaining the idea of a connectivity reduction in developing neuronal networks, supports the modeling approach that employs Small World network like topologies. The burst number increase during development is completely understandable considering the increasing in number and type of synaptic and extra-synaptic neurotransmitter receptors. Differences in AP5 and TTX long-time effects probably rise from their different chemical properties. AP5 interferes with bursting activity derived from NMDA receptors activation. NMDAr are involved in activity-dependent synapse long term potentiation and long term depression. It is not astonishing that the effect on a mature cultivation (12 div) is tiny and limited to a silence - induced bursting increase. Discussion 1
Slide 9 : TTX induces a complete synapse activity blockage due to Na channels obstruction. The complete network silencing causes an impressive bursting increase. Furthermore, the bursting topology is completely twisted. The global activity explosion could be related with a persistent TTX effect on inhibitory synapses, causing an effective network synchronization. This effects can be observed also in vivo in TTX poisoning cases (e.g. puffer fish ingestion) where this kind of global network (cortical) synchronization causes epileptic seizures before the death. At the moment we are deepening these preliminary considerations comparing the local / global characterization in TTX and AP5 administration cases with linear and non-linear correlation indexes among bursts. Discussion 2
Slide 10 : Thank you

 


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