ATP-induced synaptic potentiation in hippocampal slices

356

Brain Research, 491 (1989) 356-359 Elsevier

BRE 23587

ATP-induced synaptic potentiation in hippocampal slices A. Wieraszko and T.N. Seyfried Department of Biology, Boston College, MA 02167 (U.S.A.) (Accepted 13 March 1989) Key words: Adenosine triphosphate; Long-term potentiation; Hippocampal slice

The purpose of this study was to investigate the influence of different adenosine triphosphate (ATP) concentrations (ranging from 400 nM to 250/~M) on hippocampal potentials recorded from pyramidal neurons. ATP applied at a concentration of 400 nM induced a 100% increase in the size of the population spike (potentiation). The potential started to increase 30-60 s after ATP application, reached a maximum after 20 rain, and remained potentiated for longer than 1.5 h. Washing the slices with fresh Ringer solution did not reverse the effect. ATP applied at a concentration of 50-150/~M, temporarily depressed the potential. This depression, however, was transient, as the potential gradually recovered by itself and reached a value higher than that observed before ATP application. ATP applied at the concentration of 250/~M caused a long-lasting depression of the potential. The potential was not restored by washing the slices, but recovered after addition of 0.7 #M 3,4-diaminopyridine. These data show a concentrationdependent mode of ATP action on hippocampal neurons and suggest a role for ATP in regulating synaptic efficiency. Since the first demonstration of a stimulationdependent release of adenosine triphosphate (ATP) from nervous tissue 11, there has been a growing interest in the role of ATP in synaptic transmission 21' 25,2s The release of ATP following electrical or chemical stimulation was demonstrated in the motor cortex 31 and vas deferens of rats 17 and in the electric organ of Torpedo marmorata 13. We recently demonstrated a presynaptic, calcium-dependent release of ATP from hippocampal slices following electrical stimulation of Schaffer collaterals 3°. Furthermore, this stimulation induced a stable increase in the size of the population spike; a phenomenon known as a long-term potentiation (LTP) 26. The concentration of extracellular ATP in the chamber following electrical stimulation reached the nM level 3°. The objective of the present study was to determine if exogenous ATP, applied at nM concentrations, could influence synaptic responses recorded from pyramidal neurons following stimulation of Schaffer collaterals. Fifteen male C57BL/J6 (B6) mice were used in the present investigations. The slices were prepared as described previously 29. Animals were decapitated and both hippocampi were removed and sliced using

a manual tissue chopper 6. The slices, 500 ~ M thick, were placed in a stationary slice chamber as described elsewhere 29 and were maintained in a glucose-Ringer solution at 20 °C 29. In some experiments, calcium concentration was increased from 3.1 nM to 3.3 mM to compensate for the calcium chelating properties of ATP 2°. One hour later a twisted bipolar stimulating electrode (62/~m wire) was placed in the region of the Schaffer collateralscommissural projection where they enter the regio superior field. A recording electrode was guided into the pyramidal cell layer. In some of the experiments, the slices were stimulated antidromically by placing the stimulation electrode on the alvear path. Experiments were conducted only on slices that displayed robust potentials, characteristic for this system. The slices were stimulated every 15 s until the potential stabilized. Exogenous A T E in a total volume of 15-20 ~tl, was then added directly to the chamber with a Hamilton syringe. ATP (400 nM) injected into the slice chamber had a dramatic effect on cell excitability. The population spike started to increase 30-60 s following ATP application and reached a maximum size 15-20 min after the addition of ATP (Fig. 1A). The effect

Correspondence: A. Wieraszko, Department of Biology, Boston College, Chestnut Hill, MA 02167, U.S.A. 0006-8993/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)

357 persisted for at least 1.5 h and was not reversed by washing the slice with fresh, ATP-free Ringer solution (Fig. 1A), although elevated potential decreased slightly following perfusion. At present we do not have a reasonable explanation for this perfusion-evoked effect. Shortly after the end of the perfusion, however, the potential stabilized and remained at an elevated level for at least 90 min following ATP application. Results obtained from 20 slices (obtained from 7 B6 mice) showed a 100% increase in the size of the population spike (Fig. 1B). The antidromic response was unchanged or only slightly elevated following ATP application (data not shown). ATP had no influence on the size of the

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Fig. 1. The influence of 400 nM ATP on the size of the population spike recorded from pyramidal neurons. A: representative example of ATP effect. The addition of ATP is marked by an arrow. After 20 min, when the increase in the size of the population spike reached 100%, the slice was washed with ATP-free Ringer solution for 10 min. The time of washing is marked by a horizontal bar above the graph. Note that following washing the potential partially decreased, but did not return to the control level observed before ATP application. Similar results were obtained in 5 other experiments. B: the ATP effect observed in 20 slices obtained from 7 B6 mice. The size of the stabilized potential before ATP addition was taken as 100%. The average size of the potential recorded from 20 slices before ATP application was 1.27 + 0.13 inV. Each point represents the mean increase + S.E.M. obtained from 20 slices.

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potential when applied at concentrations ranging from 1 to 50/~M. At higher ATP concentrations (50-150 ~M), a depression of the population spike occurred. This depression, however, (24.2% + 8.2 at 30 min after ATP application; n = 4) was only temporary, as the potential gradually recovered by itself and eventually reached a level that was higher than that before ATP application (164.6% + 4.4 at 90 min after ATP application; n = 4) (Fig. 2). When the concentration of ATP exceeded 180 /~M, the potential was suppressed irreversibly and did not recover either by itself, or after washing the slices with ATP-free Ringer solution (Fig. 3). The potential could be restored, however, after addition of 0.7 /~M 3,4-diaminopyridine (AP). These results demonstrate that ATP has a dual mode of action on pyramidal neurons. At low concentrations (nanomolar range), ATP induces a long-lasting increase in the size of the population spike, whereas at higher concentrations (uM range), ATP depresses the population spike. We have demonstrated previously3° that high frequency stimulation, which evokes LTP, simultaneously induces the release of ATP. Our present results show that exogenous ATP, applied at a concentration similar to that observed in the release experiments, can induce a long-lasting increase of the population spike. Our findings suggest that ATP, released during high frequency stimulation, may play a role in LTP. We do not yet know, however, if ATP evokes LTP or simply resembles LTP. Considering the biochemical events which accompany LTP, we can speculate on the mechanism by which ATP induces a stable

358 increase in the size of the population spike. Since the ATP effect is prolonged, enzymatic reactions may be involved. One mechanism might involve the phosphorylation of a specific protein (F1) by protein kinase C (ref. 1). Kinase C is activated by diacylglycerol, a product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis 3. Furthermore, exogenous ATP is known to stimulate PIP2 hydrolysis in brain synaptosomes 12. It is therefore possible that ATP, either released during intensive stimulation or added exogenously, can trigger events leading to kinase C activation and F1 phosphorylation. This assumption is attractive since protein phosphorylation is a pivotal biochemical event in the mechanism of LTP 1'2a'23. Because ATP can serve as a substrate for ectokinases 9"m, the phosphorylation of membrane proteins by ectokinases may also be important in modulating nerve cell excitability. ATP may also act by modulating intracellular calcium levels. ATP can facilitate calcium influx into smooth muscle 2 and can increase free calcium level in cultured nerve cells 1°. An increase in intracellular calcium concentration is another crucial step for initiation of LTP 8'15'~8. Moreover, 1,4,5-inositoltriphosphate (IP3), another product of PIP2 hydrolysis, can increase intracellular calcium concentration 3. The concentration of ATP, which influenced PIP2 hydrolysis 12 and increased free calcium level in cultured cells 1° was in the range of 100 ktM. ATP used at this concentration in our system potentiated synaptic response after a brief period of

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Fig. 3. The influence of 250 /~M ATP on the size of the population spike. Note that after ATP application, the potential decreased sharply and remained depressed even after the slice was washed with ATP-free Ringer solution. The time of the wash is marked by horizontal bar above the graph. The potential could be restored, however, after addition of 3,4-diaminopyridine (AP) at a concentration of 0.7/aM.

reversible depression (Fig. 2). Hence, released ATP may initiate the chain of reactions leading to LTP by stimulating PIP2 hydrolysis, activating kinase C, and influencing free calcium level in target neurons. The depressing effect of ATP on hippocampal neurons that we observed was also observed previously 5'v. However, our new findings show that this depression is reversible by itself, as long as the ATP concentration does not exceed 180 #M. Moreover, the potential recovers to a level higher than that observed before ATP application. The depressing effect of ATP may result from a direct action of ATP 5"v or from a product of ATP hydrolysis, e.g. adenosine 19. Adenosine is known to inhibit hippocampal neurons 22. In either case, the concentration of both inhibitory factors (elevated ATP concentration or adenosine) diminishes with the time: ATP being removed through ectoenzyme hydrolysis 9'19 and adenosine being removed through high affinity uptake 21. The overshoot in recovery of the potential suggests that together with depression, ATP can cause a long-lasting change in synaptic efficiency. This change, expressed as a potentiation, is observed after the depressive effect of ATP or adenosine disappears. At higher concentrations of added ATP, the potential does not recover by itself or after washing with fresh Ringer solution. This suggests that higher concentrations of ATP can permanently depress synaptic activity. The ATP-induced depression, however, can be overcome by 3,4-diaminopyridine. Aminopyridines facilitate the calcium influx into the nerve cell27 and it has been demonstrated that also AP, the drug used in our study, facilitates the calcium influx to the hippocampal nerve endings during electrical stimulation 16. Thus, the recovery of the potential suppressed by higher ATP concentrations after subsequent AP addition shows that recovery to the proper calcium level inside the cell was enough to reverse the ATP effect. Further experiments are needed to reveal the molecular mechanism of ATP action, especially as in these experiments AP was used only to demonstrate that cells were not irreversibly impaired by ATP action. Although the mechanism of ATP-induced synaptic depression is not clear, changes in the membrane permeability evoked by higher ATP concentrations 4, could also be responsible for the observed effect. Our results introduce new information on the role

359

of A T P in modulating synaptic activity and demonstrate a multifunctional action of A T P on cell excitability. The net effect of A T P will depend primarily on its concentration in the extracellular space. This concentration can be achieved through a delicate balance between the a m o u n t of A T P released and the rate of A T P hydrolysis by ectoenzymes. We recently outlined a mechanism of

We have shown for the first time that a naturally occurring c o m p o u n d (ATP), released during nerve activation, can p e r m a n e n t l y potentiate synaptic response in a m a n n e r similar to that of electrical stimulation. Further studies are needed to establish the biochemical basis for this ATP-evoked synaptic potentiation.

epilepsy in seizure-prone mice that involves a perturbation of this balance 24.

Supported by NSF G r a n t BNS 8644955 and N I H Grants 24826 and 23355.

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