INFORMACIÓN, COMUNICACIÓN Y EDUCACIÓN AMBIENTAL

In addition to neuronal culture methodology described in Chapter 4(section 4.2), further spontaneous firing activity characterisation and development of sponta- neous activity is presented in this section. Due to the sensitive nature of neuronal cultures on Biochips (as previously discussed), handling and recording were kept to a minimum for the first two weeks after seeding cells on MEAs to avoid cell deaths and detachments. Hence, recordings before day 12 were not performed and the cultures were only disturbed for mandatory media change. Electrophysiolog- ical activities were recorded on day 12 for the first time for comparison purpose and only after day 18 for experiments and regular recordings.

It is typical for a rat hippocampal cultures to exhibit spontaneous synchro- nised burst of activities without external stimuli [3]. As cultures mature, they exhibit rich synchronised bursting activity [200]. Random and uncorrelated fir- ing patterns become less frequent, and the activity patterns are dominated by a network wide bursting patterns during development. This development of syn- chronous bursting behaviour implies that wider connections are also explored, possibly as a consequence of the rapid chemical synaptogenesis during the second week in culture [201].

Figure7.1shows two raster plots of a hippocampal culture, recorded at 12 DIV and 18 DIV. The recordings were performed for 5 minutes. The raster plots make it easier to observe the electrophysiological activity of the whole network repre- sented by a dot for each spike. As anticipated, significant random firing appears on both plots, and the subsequent recordings. However, the firings became more synchronised with spontaneous bursts of events as the culture matured - indicated by the presence of highly synchronous bursting patterns at 18 DIV recording as shown by red vertical rectangles in Figure 7.1 (DIV18) in comparison to DIV12. Due to the highly interconnected network, synchronous behaviour became more evident as the cultures matured. Some of the channels were saturated (indicated in Figure 7.1) which could be due to blocking of channels due to cellular debris caused by dead cell structures. Such channels showed continuous firing which is not plausible in reality due to the required refractory period necessary for neu-

Figure 7.1: Electrophysiological activity of a dissociated hippocampal network recorded at DIV12 and DIV18 during the development phase. Continuously fir- ing saturated channels are highlighted with a dotted red horizontal rectangle. Bursting patterns are indicated by a red vertical rectangles.

Figure 7.2: (A)Raw voltage fluctuations recorded from a single channel. Each vertical line underneath the waveform indicates a detected activity as a spiking event. (B)Snapshot of a real time activity laid out in MEA layout where each pixel is an electrode.

rons to recover before firing again. The presence of such channels is inevitable in live cultures and were filtered out in further analysis. Single channel electrophys- iological activity can also be observed as a series of voltage fluctuations shown in Figure 7.2(A). Each vertical line underneath the waveform indicates a spik- ing event as shown in Figure 7.2(A) and the firing pattern resembles the general firing pattern of the network wide bursting firing (Figure 7.1) where a bursting firing is followed by a quiescence period. Figure 7.2(B) on the other hand shows a snapshot of network-wide activity in real time for the culture, represented with a colour map, where each pixel represents an electrode laid out in a 64 x 64 grid mimicking the actual MEA layout.

Furthermore, Peristimulus time histogram (PSTH) analysis (Figure7.3(A,B)) clearly shows an emergence of firing patterns characterised as highly synchronised bursting patterns from day 12 to day 18. The PSTH bin size was set to 1000ms, and shows the firing rate measured at every second of the 5 minute recording. Figure 7.3(A) at day 12 does show synchronous patterns as oscillating firing behaviour with some instances of highly synchronous patterns observed at around 130 seconds and 160 seconds (note the ms scale on the plot), but the general firing was still rather random without discernible network wide bursts. Also, the

Figure 7.3: Activity characterisation of a spontaneously firing rat hippocampal network. (A) PSTH plot at day12(DIV12). (B)PSTH plot at day 18(DIV18) (C) Mean bursting of a spontaneously firing network measured at day 12,18 and 21. [95% confidence interval ]

firing rate was much lower than at DIV18. This indicates a culture which is yet to mature. When the culture reached day 18, the culture started to mature indicated by clearly visible highly synchronous firing patterns, where the firing

rate was almost double that observed at day 12 (Figure 7.3(B)). The network was bursting regularly followed by a period of quiescence. This behaviour was prevalent all throughout the period of the culture up to week 4. After DIV32, the culture began to degrade and eventually died few days after. Hence, the experiments were performed between day 21 to day 32.

To further characterise the spontaneous activity of the network, the mean bursting rate was computed for the same culture over three stages that corre- spond to (a) an early stage(DIV12); (b) maturing stage(DIV18); and (c), matured stage(DIV21), as shown in Figure 7.3(C). Not surprisingly, the development of bursting activity followed the pattern as anticipated from the PSTH plots (Figure

7.3(A,B)). There was a clear increment in the bursting activity from day 10 to day 18. Even though, the bursting activity increases with maturity, at around day 21 the bursting activity plateaued as reported in the previous studies [8,200,202]. From week 3-4, the bursting profiles become increasingly narrow hence shorter burst - this change is attributed to the development of GABAergic neurotrans- mission(inhibitory) which was observed to occur after a delay of 1-2 weeks as compared to the glutamatergic (excitatory) system [114].