Intracellular ATP Influences Synaptic Plasticity in Area CA1 of Rat Hippocampus via Metabolism to Adenosine and Activity-Dependent Activation of Adenosine A₁Receptors

Abstract

The extent to which brain slices reflect the energetic status of the in vivo brain has been a subject of debate. We addressed this issue to investigate the recovery of energetic parameters and adenine nucleotides in rat hippocampal slices and the influence this has on synaptic transmission and plasticity. We show that, although adenine nucleotide levels recover appreciably within 10 min of incubation, it takes 3 h for a full recovery of the energy charge (to Ն0.93) and that incubation of brain slices at 34°C results in a significantly higher ATP/AMP ratio and a threefold lower activity of AMP-activated protein kinase compared with slices incubated at room temperature. Supplementation of artificial CSF with D-ribose and adenine (Rib/Ade) increased the total adenine nucleotide pool of brain slices, which, when corrected for the influence of the dead cut edges, closely approached in vivo values. Rib/Ade did not affect basal synaptic transmission or paired-pulse facilitation but did inhibit long-term potentiation (LTP) induced by tetanic or weak theta-burst stimulation. This decrease in LTP was reversed by strong theta-burst stimulation or antagonizing the inhibitory adenosine A 1 receptor suggesting that the elevated tissue ATP levels had resulted in greater activity-dependent adenosine release during LTP induction. This was confirmed by direct measurement of adenosine release with adenosine biosensors. These observations provide new insight into the recovery of adenine nucleotides after slice preparation, the sources of loss of such compounds in brain slices, the means by which to restore them, and the functional consequences of doing so.

The Purine Salvage Pathway and the Restoration of Cerebral ATP: Implications for Brain Slice Physiology and Brain Injury

“…We found that, despite the attenuation in ATP levels, EC was close to values recorded in vivo (typically ~0.95) [23,77]. This suggests that a new metabolic equilibrium had been achieved amongst the remaining adenine nucleotides.…”

“…2) [23]. We additionally examined the energy charge (EC) [76] of the slices, which conveys the energetic state of a cell and is given by the following equation:…”

Measurement of purine release with microelectrode biosensors

“…Since their inception, brain slices have been criticised for their ATP levels, which are typically 50% lower than values observed in vivo. We have recently addressed this issue [70] to investigate whether the reduction in ATP reflects loss of salvageable metabolites during brain slice preparation, analogous to injury to the intact brain in vivo, or whether this reflects a fundamental metabolic handicap that brain slices suffer from.…”

“…6), which raised the threshold for the induction of long-term potentiation. This release seemed to be independent of prior release of ATP as it was insensitive to the ecto-ATPase inhibitor POM1 [71], but sensitive to a combination of equilibrative nucleoside transporters (ENTs) inhibitors [70]. In these experiments, sensors were invaluable as they allowed the kinetics of enhanced adenosine release to be established, which would not have been possible through the use of receptor antagonists.…”

“…However, lack of a rapid and convenient measurement system in whole blood has hindered largerscale studies aimed at establishing the diagnostic value of Concentration of adenosine in nanomolar' indicates that the signal recorded reflects adenosine and metabolites such as inosine. Reproduced with permission from [70] purines more clearly. In this section, we briefly review three clinical areas in which purinergic signalling is involved and where purine-based diagnostics may ultimately have a valuable role.…”