GANANCIA OBTENIDA DE LA IMPLEMENTACIÓN DE MEDIDAS DE AHORRO

extracellular calcium entry

Blocking action potentials with TTX had no effect on the frequency or

amplitude o f sPSCs during ischaemia. This suggests that action-potential induced

depolarisation o f presynaptic terminals was not necessary for the generation o f

spontaneous events. This is in contrast to work by Djali and Dawson (2001), who

analysed the release o f GABA from hippocampal slices using HPLC analysis o f the

superfusate. They exposed hippocampal slices to half an hour o f glucose deprivation

and sodium azide to block oxidative phosphorylation, and then analysed the

composition o f the superfusate. In the presence o f IpM TTX the amount o f GABA

released was decreased by 87%. However, these recordings were taken over a 30 minute

period, and there is no indication o f the time at which the TTX-dependent GABA

release was occurring, nor from which hippocampal area; m y recordings were focused

ischaemia. In addition, Djali and Dawson (2001) monitored GABA release throughout

the depth o f the slice. M y recordings were from C A l pyramidal cells near the surface o f

the slice so some o f the inhibitory inputs to the cells could have been damaged during

the slicing process (consistent with this, TTX had no effect on the number o f non-

ischaemic IPSCs).

Blocking vesicular release using concanamycin treatment reduced ischaemia-

evoked sIPSC frequency by 88%, and removing external calcium reduced it by 97%.

This demonstrates that the majority o f GABA release in the early stages o f ischaemia is

from vesicles, and that this vesicular release is dependent on external calcium influx

(although a potential effect o f removing external calcium on depleting internal stores o f

calcium cannot be excluded, see below).

The external calcium-dependence o f release is in contrast to that reported for

early GABA and glutamate release in hypoxia (Fleidervish et al., 2001; Katchman and

Hershkowitz, 1993b). These groups reported that sIPSCs and sEPSCs in hypoxia

occurred at normal rates in zero external calcium solution (and that sEPSCs were

greatly reduced if intracellular calcium stores were depleted). However, in both these

studies calcium was just removed from the extracellular solution, and no EGTA was

added to chelate trace extracellular calcium. When examining sEPSCs in hypoxia

Katchman and Hershkowitz (1993b) applied cadmium to block voltage-dependent

calcium channels, which also had no effect on the frequency o f sEPSCs, again

suggesting that they were independent o f external calcium entry. It is unlikely that

intracellular calcium stores have been significantly depleted in m y experiments due to

the removal o f external calcium, as zero calcium solution was only present for 2

minutes before ischaemia and then for the duration o f the ischaemic episode, i.e. a total

o f 6 minutes for the data I measured at 4 minutes into ischaemia. Paltauf-Doburzynska

endothelial cells were only depleted by 30% after a 7 minute exposure to calcium-free

external solution. The difference in calcium dependence (sIPSCs being Ca^-dependent

in my ischaemia experiments and sEPSCs being Cao independent in the hypoxia

experiments using cadmium o f Katchman and Hershkowitz (1993b)) may be due to a

difference in the mechanism o f the energy deprivation-induced release o f

neurotransmitter from glutamatergic and GABAergic terminals. Alternatively, it could

reflect a difference between the effects o f energy deprivation induced by hypoxia and

ischaemia (which will be considered in more detail in Chapter 5).

W hat triggers the calcium-dependent release o f GABA during ischaemia? One

explanation is as follows. Oxygen and glucose deprivation cause a rundown o f ATP

levels and the cessation o f ATP-dependent processes, including the Na^-K^-ATPase,

within neurons. This depolarises the presynaptic terminal and leads to the opening o f

voltage-sensitive calcium channels. The subsequent calcium influx causes vesicles to

fuse with the presynaptic membrane and release GABA into the synaptic cleft. W ork by

del Castillo and Katz (1954) at the neuromuscular junction demonstrated that the rate o f

spontaneous vesicular release increases with the depolarisation level o f the presynaptic

terminal. This fits with the increase in frequency o f sIPSCs seen as the ischaemic

episode progresses: the longer the energy deprivation lasts, the more depolarised the

presynaptic terminal becomes and more vesicles are released.

There is a second possible explanation for the increase in frequency o f sIPSCs in

the build up to the AD. Engel et al. (2001) showed that blocking GABA breakdown by

GABA transaminase, using y-vinyl-GABA (GVG), led to an increase in the level o f

GABA in presynaptic terminals and to an increase in the frequency and amplitude o f

sIPSCs. They attributed this increase in frequency and amplitude to increased vesicular

filling and turnover, so causing more (and more easily detectable) spontaneous release

to both increased synthesis and decreased degradation (Madl and Royer, 2000; see

Section 3.4.3 for a detailed explanation), so there may be increased filling o f vesicles

and increased spontaneous release. However, this will not continue indefinitely as

vesicle filling is an ATP-dependent process and ATP levels fall in ischaemia. Indeed,

the frequency o f sIPSCs decreases slightly (although not significantly) in the 2 minutes

ju st preceding the AD (Figure 4.4).