Energía generada por la biomasa (H f )

During ischaemia there was an increase in the frequency o f sPSCs. Figure 4.3A

is an example trace showing the increase in sPSCs occurring in the build up to the

anoxic depolarization (AD), which happens after 6-7 minutes. Figure 4.3B shows 1

second long expansions o f the full trace taken at one minute intervals as the ischaemic

episode progressed, showing the increase in sPSC frequency. Because the time between

applying the ischaemia solution and the AD occurring varied between slices (between 5

and 7 minutes), to average data from different cells when quantifying the change o f

sPSC rate, I aligned the date either on the AD or on the start o f ischaemia, as follows.

Figure 4.4 shows the mean properties o f the ischaemic evoked sPSCs in 4 cells,

with the data aligned to superimpose the AD for each cell at time zero. The frequency o f

sPSCs increased with each subsequent minute, until 2.5 minutes before the AD when it

peaked at 48.0 ± 11.2Hz, before decreasing to 37.6 ± 8.0Hz ju st before the AD,

before the AD with the values just before the AD with a paired t-test; Figure 4.4A). The

amplitude o f events also increased as the AD was approached, peaking at 71.1 ± 12.4pA

2 minutes before the AD, significantly larger than the 40.5 ± 3.6pA before ischaemia

solution was applied (p=0.05; see Figure 4.3 where the amplitude increase is clearly

visible, and Figure 4.9B, where the amplitudes o f events before ischaemia are

quantified). The events then remained around 65pA in amplitude until the AD occurred.

The event decay time remained fairly constant and near its control value throughout

ischaemia (Figure 4.4C).

Next, for the same data as Figure 4.4, I aligned the records at the time when

ischaemia solution was applied. Figure 4.5 shows a specimen part o f the current trace 4

minutes after the start o f ischaemia (Figure 4.5A), together with the alteration in sPSC

frequency, amplitude and decay time (Figure 4.5B-D) that occurred as the ischaemic

episode progressed. The changes seen in ischaemia are similar to those in Figure 4.4,

although the decrease in sPSC frequency before the AD seen in Figure 4.4A is not

evident when the data are aligned as in Figure 4.5.

To test the effect o f drug treatments on the sPSCs produced in ischaemia, I

aligned the data from the start o f ischaemia (as in Figure 4.5). The sPSC frequency in

different conditions was measured for the period between 3.5-4 minutes (which for

brevity is referred to as 4 minutes in the rest o f the chapter) after the application of

ischaemia solution, just before the peak increase o f sPSC frequency that occurred at 4-5

minutes (Figure 4.5B). Measuring at 4 minutes was a compromise between choosing a

time at which a dramatic increase o f sPSC frequency had occurred, and minimising the

amount o f time consuming data analysis. Data are presented in figures with the format

o f Figure 4.5, i.e. a 1 second sample recording o f sPSCs taken 4 minutes into the

ischaemic episode showing individual events, and plots against time (with each point

amplitude and decay time for the first 4 minutes o f ischaemia. All figures are the

average o f data fi-om 4 cells. Tables 4.1, 4.2 and 4.3 summarise the sPSC frequency,

amplitude and decay time respectively, after 4 minutes o f ischaemia, for the various

manipulations that are described in more detail below, and compare the values

statistically w ith the values obtained after 4 minutes o f ischaemia with no drugs added.

Figures 4.6 and 4.9 summarise the effects o f the same manipulations on the properties

o f sPSCs seen in non-ischaemic solution.

4.3.3 sPSCs in ischaemia are predominantly mediated via GABAa receptors

W hen lOpM GABAzine was present throughout the ischaemic episode then the

frequency o f sPSCs reached only 0.36 ± 0.12Hz after 4 minutes (Figure 4.7), which is

only 1.3% o f the fi-equency reached after 4 minutes in control ischaemia solution, i.e.

28.4 ± 5.4Hz (Figure 4.5B; p=0.01). This reduction is similar to the reduction o f sPSC

frequency produced by GABAzine in non-ischaemic solution (Figures 4.1 and 4.6), and

suggests that the majority o f miniature events measured during ischaemia are

GABAergic in origin. (Note that, as indicated on Figure 4.7A, in this experiment

GABAzine was applied at the same time as the ischaemia solution, accounting for the

decrease in the frequency o f events seen on initially applying the ischaemia solution due

to the block o f sIPSCs). The average amplitude o f the events remaining (after 4 minutes

ischaemia) was approximately halved, from 50.1 ± 5.5pA at 4 minutes in control

ischaemia to 30.8 ± 5.3pA in GABAzine (close to the amplitude o f events recorded in

GABAzine in non-ischaemic solution, i.e. 20.5 ± 4.5pA). Decay times were too variable

to interpret, owing to the small number o f events measured. From now on, since 99% o f

events were abolished when GABAa receptors were blocked during ischaemia, all

detected events will generally be assumed to be sIPSCs, except in zero calcium solution