Progress in Neurobiology Vol. 36, pp. 329 to 341, 1991 Printed in Great Britain.All rights reserved

0301-0082/91/$0.00 + 0.50 © 1991 PergamonPress plc

THE E L E C T R O P H Y S I O L O G Y OF A D E N O S I N E IN THE M A M M A L I A N C E N T R A L N E R V O U S SYSTEM R. W. GREENE*and H. L. HAASt *Harvard Medical School and Brockton Veterans Administration, MA 02401, U.S.A. ?Johannes Gutenburg-Universitiit, Mainz, F.R.G. (Received 11 December 1990)

CONTENTS 1. Introduction 2. Postsynaptic effects 2.1. Steady-state increase in potassium conductance 2.1.1. Ionic selectivity 2.1.2. Voltage sensitivity 2.1.3. Effects of calcium and potassium antagonists 2.2. Modulation of calcium and voltage dependent potassium currents 2.2.1. Enhancement of calcium dependent potassium current 2.2.2. Lack of effect on I A and IQ 2.3. Indirect effects on calcium current 3. Inhibition of evoked synaptic potentials 3.1. Site of action 3.2. Mechanism of action 4. Mediators of electrophysiological effects 4.1. Receptor subtypes 4.1.1. Postsynaptic responses 4.1.2. Presynaptic responses 4.2. Second messengers 5. Electrophysiolosy of endogenous adenosine 5.1. Antagonist evoked excitation 5.2. Antagonism of uptake and increased catabolism 5.3. Effects in low calcium, high magnesium 6. Activity in pathophysiological states 6.1. Epilepsy 6.2. Hypoxia 7. Conclusions Acknowledgements References

1. INTRODUCTION The ubiquitous distribution of adenosine (AD) throughout the mammalian central nervous system (CNS) is partially if not entirely due to the central role of A D and its related compounds in intracellular energy metabolism (Lehninger, 1982). The demonstration of an uneven distribution of high affinity extracellular A D receptors (Bruns et aL, 1980), of high affinity uptake and catabolic enzyme systems for A D (Geiger and Nagy, 1984) and of evoked release of A D (Wojcik and Neff, 1982) suggests an additional extracellular role for AD. With respect to the evoked release of AD, the relationship to metabolic state is marked. Experimental manipulations in the CNS which increase metabolic demand relative to metabolite availability, including hypoxia, hypoglycemia, Address correspondence to: R. W. Greene, Neuroscience lab 151C, V.A.M.C., 940 Belmont Street, Brockton, MA 02401, U.S.A.

329 330 330 330 330 331 332 332 333 333 334 334 334 335 335 335 335 336 337 337 337 337 338 338 338 339 339 339

potassium evoked depolarization, exposure to convulsants and electrically evoked seizure activity (Pull and McIlwain, 1972; Berne et al., 1982; McIlwain and Poll, 1986), increase the extracellular concentration of AD. Extracellular studies from both/n rive and in vitro preparations have shown that exogenously administered A D reduces the excitability of neurons in all major divisions of the CNS which suggests that the extracellular function is at least in part as an inhibitory neuromodulator (Phillis and Wu, 198 l; Dunwiddie, 1985). This effect may also lessen the metabolic demand of nervous tissue. Taken together, the extracellular function and metabolically correlated release of A D are consistent with a homeostatic role for AD as a mediator of a negative feedback interaction between the metabolic and electrophysiological states (Greene and Haas, 1985; Mcllwain and Poll, 1986). Over the last twenty years, our understanding of the electrophysiological role of A D in the mammalian central nervous system has advanced, especially with

329

330

R.W. GREENEand H. L. HAAS

ADENOSINE

respect to the mechanisms of action. The following focuses upon the alterations in electrophysiological membrane properties elicited by exogenous and endogenous AD and their relationship to neuronal excitability.

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2. POSTSYNAPTIC EFFECTS In the mammalian CNS two postsynaptic inhibitory effects elicited by AD have been described. The most efficacious, as regards a reduction in neuronal excitability, is a steady-state hyperpolarization of membrane potential accompanied by a decrease in input resistance. This was first described on CAI rat and guinea pig hippocampal neurons (Okada and Ozawa, 1980; Siggins and Schubert, 1981; Segal, 1982) and more recently in the locus ceruleus (Shefner and Chiu, 1986), and medial pontine reticular formation (Gerber, Haas and Greene, unpublished observations). Similar effects are also observed in the human hippocampus (Haas et al., 1987) and neocortex (McCormick and Williamson, 1989). AD can elicit this hyperpolarization when synaptic activity is blocked either with calcium antagonists including magnesium, cobalt (Segal, 1982; Siggins and Schubert, 198l) and cadmium (Greene and Haas, 1985) or with TTX (Proctor and Dunwiddie, 1983; Greene and Haas, 1985; Trussell and Jackson, 1985). The second postsynaptic AD effect is an enhancement of accommodation and the associated long duration afterhyperpolarization present in cortical neurons (Haas and Greene, 1984). The result of this enhancement is a dynamic modulation of neuronal responsiveness such that long duration but not short duration excitatory input, is selectively inhibited. This action is observed only at relatively low concentrations of AD. At higher concentrations, the former effects predominate (see Section 2.2.1; Greene and Haas, 1985). Described below are the mechanisms of the postsynaptic actions with respect to ionic and voltage sensitivity. 2.1. STEADY-STATE INCREASE 1N POTASSIUM CONDUCTANCE

2.1.1. Ionic selectivity' The steady-state hyperpolarization and decrease in input resistance elicited by AD is mediated by an increase in potassium conductance as demonstrated in cultured striatal neurons (Trussell and Jackson, 1985) and adult hippocampal cells (Fig. 1; Gerber et al., 1989). This was established by the demonstration of a relationship between the AD response and the extracellular potassium concentration, consistent with that predicted by the Nernst equation for a change primarily in potassium permeability. 2.1.2. Voltage sensitivity The AD elicited increase in potassium conductance is inwardly rectifying in cultured central nervous system neurons (Trussell and Jackson, 1985). The demonstration of a shift in the V~/2 (the membrane

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FIG. I. The outward current evoked by AD is due primarily to an increase in permeability to potassium. The upper set of 3 current records were obtained in singleelectrode voltage clamp in 3 different extracellular potassium concentrations. AD (1 mu in puffer) was applied by pressure injection indicated by arrows. The downward deflections result from the current required to shift the membrane potential 20 mV for 300msec duration (120). These step commands were employed to assess the AD evoked change in conductance (AGAD= [120mv(Control)-- 12omv(AD)]/2OmV). The current elicited by AD (AIAo=l(during AD at - 7 0 m V ) /(control at - 7 0 mV)) is equal to the product of AG and the driving force (Vh=-70mV-V~¢ver~l). V~versal=261n Ko/K i where Ko is the extracellular potassium concentration (i.e. 2.5, 5.0 or l0 mM) and K~is 140 mi. By substitution and solving the Ko one obtains: Ko = 9,5exp - (AIAD/26AGAD) which is plotted in the lower portion of the figure. The predicted line for a change in potassium permeability would have a slope of I and pass through the origin. The line illustrated was obtained by linear regression and has a slope of 1.16, a Y intercept of -0.8 and r = 0.99. There are two advantages to employing this relationship instead of the direct observation of the reversal potential. First, both the change in current and conductance are used in this determination and second, it avoids hyperpolarization to potentials associated with high conductance states (and thus a short space constant) required by direct observation of the reversal potential (modified from Gerber et al., 1989). potential that produces 1/2 of the maximal conductance) with extracellular potassium concentration suggests that rectification is a property of the conductance and not a result of an interaction with another membrane current (Yakel et al., 1988). This does not appear to be the case in adult hippocampal neurons (Gerber et al., 1989). In the majority of cells examined, ( > 6 0 % ; Fig. 2A) the slope conductance of the AD current over the voltage range of - 1 0 0 mV to - 4 0 mV remained constant. The apparent inward rectification of the AD response, observed in a minority of cells (Fig. 2C), might be attributed to a shunt by an outwardly rectifying intrinsic current. Support for this idea was obtained by applying tetraethylammonium (TEA; 10 mM) to the perfusate of neurons showing an apparent inwardly rectifying AD response. At this concentration, the primary antagonistic effect of the TEA will probably be on the voltage and calcium dependent potassium current, Ic (Fig.

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FIG. 2. The conductance evoked by AD is voltage insensitive. (A) The current-voltage relationship of the AD steady-state current characteristic of over 60% of the neurons examined. Plots were constructed by digital subtraction of the whole cell I/V plot obtained during control from that in the presence of AD. Ramp voltage commands of 2.5 mV/sec under single electrode voltage clamp control were used. (B) The current blocked by TEA (10 mM). Note the outward rectification beginning at - 6 5 inV. (C) In less than 40% of the neurons examined, AD evoked a current with inward rectification beginning at about - 65 mV. (D) When examined in the presence of TEA and AD evoked current no longer showed inward rectification. Thus, it is possible a shunt by TEA-sensitive current is responsible for the observed inward rectification (modified from Gerber et al., 1989). 2B; Herman and Gorman, 1981). In the presence of TEA, the inward rectification of the A D current was no longer observable. Rather, the AD conductance was voltage insensitive. An alternative explanation for these results is that TEA limits the local extracellular increase in potassium concentration (by antagonism of potassium conductance) as the neuron is depolarized. A depolarization of the reversal potential for potassium (brought about by the increase in extracellular potassium) might then be limited so that the A D potassium current is not as greatly reduced. This effect may occur in addition to a shunt effect. The difference in voltage sensitivity of the A D conductance observed in cultured and adult neurons may result from a difference in the magnitude of expression of two distinct A D responses. In both culture and slice preparations, the response to AD is mediated by a G-protein (see Section 4.2). However, at least four types of G-protein activated potassium channel have been demonstrated in excised patches from cultured hippocampal neurons (Van Dongen,

1988). Both inwardly rectifying and voltage insensitive G-protein activated potassium channels were observed. Studies from the in vitro hippocampai slice preparation suggest that in the adult postsynaptic membrane the AD receptor is associated predominantly with the latter.

2.1.3 Effects of calcium and potassium antagonists The steady-state AD potassium conductance in adult hippocampal neurons is not only voltage insensitive, but also insensitive to calcium. This response is not reduced by blockade of calcium flux across the membrane with cadmium or by buffering of intracellular calcium concentration with intracellular injection of EGTA (Fig. 3; Greene and Haas, 1985). The A D conductance is resistant to blockade by two potassium channel antagonists, tetraethylammonium (TEA) and 4-aminopyridine (4-AP). They were applied in concentrations sufficient to antagonize a significant portion of the delayed outward currents present in CAI neurons for TEA (10 mM) and the

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FIG. 3. Antagonists of calcium and potassium currents do not affect the AD mediated hyperpolarization with the exception of barium. AD (I00 #u) was applied during the time indicated by the horizontal bar. Downward deflections are due to the membrane potential response to intracellularly injected constant current pulses used to test the input resistance. The concentrations of antagonists used were cadmium (Cd2+, 100 # M), tetraethylammonium (TEA, 10 mM),intracellular EGTA (icEGTA, 1 M), 4-aminopyridine (4-AP, 100 #M; however at a 5 mM concentration, the AD response is more that 50% blocked), barium (Ba2+, 2 mM).The traces with TEA, 4-AP and barium were obtained in the presence of TTX. Calibration: 2 min, 10 mV. (Modified from Greene and Haas (1989). In: Hippocampus; New Vistas. Eds. V. Chan-Palay and C. Kohler. Alan R. Liss: New York.) transient outward currents for 4-AP (500/tM; Fig. 3; Greene and Haas, 1985). At higher 4-AP concentrations (5 mM) the AD response is partially blocked. Barium, in addition to its ability to readily pass through calcium channels (Hagiwara and Byerly, 1981) is a potent but less specific antagonist of potassium currents including Ic (Connor, 1979), the M current (Constanti et al., 198 l) and I K (Armstrong et al., 1982). Complete blockade can be obtained by the addition of 2 mM barium to the perfusate (Fig. 3; Trussell and Jackson, 1987; Gerber et al., 1989). The studies described above demonstrate that the conductance evoked by AD shares several characteristics with the conductance responsible for S-current described in invertebrates. Both are ligand gated (S-current is enhanced by FMRFamide) and voltage and calcium insensitive (Brezina et al., 1987; Brezina, 1988). Further both conductances are blocked by barium and high concentrations of 4-AP (Shuster and Sigelbaum, 1987; Brezina, 1988).

2.2. MODULATIONOF CALCIUM AND VOLTAGE DEPENDENT POTASSIUMCURRENTS

2.2.1. Enhancement of calcium dependent potassium current Many cortical neurons accommodate to constant excitatory input, in association with a long duration ( > 1 see) afterhyperpolarization (L-AHP). The current responsible for these phenomena has been characterized in hippocampal pyramidal cells as a calcium dependent and voltage independent potassium current, /Anp (Lancaster and Adams, 1986). AD, at concentrations of less than 50 #M in the perfusate, enhances accommodation, the L-AHP (Haas and Greene, 1984) and /AnP (Fig. 4). At AD concentrations greater than 50/~M, the enhancement of the L-AHP was not observed. In fact, it was often reduced under these circumstances. However, this was always associated with the steady-

ELECTROPHYSlOLOGY OF ADENOSINE

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ADENOSINE 20~M,

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FIG. 4. Adenosine enhances accommodation and the associated long duration afterhyperpolarizationand IAap. (A) Top trace indicates current pulse (400 pA amplitude) injected intracellularly, which evokes some accommodation before and an enhanced accommodation during exposure to AD (20 #M[/sc]). (B) At a slower time scale, the long duration afterhyperpolarization which follows a burst of action potentials (depolarized off scale) is of greater duration and in this case of greater amplitude during AD (20/~M) exposure. (C) Under single electrode voltage clamp control and following a step command to - 2 0 mV, the relaxing outward currents responsible for the afterhyperpolarization may be observed at - 6 0 mV. They are enhanced during AD (5/ZM) exposure. (Modified from Greene and Haas (1989). In: Hippocampus; New Vistas. Eds. V. Chan-Palay and C. Kohler. Alan R. Liss: New York.) state A D evoked increase in potassium conductance. The potassium conductance increase could reduce the calcium current by a shunting effect (i.e. it may hasten the repolarization of the membrane potential during an action or synaptic potential to a range negative to that needed to open calcium channels, see Section 2.3). As a result, the reduction of IAxp in the presence of high concentrations of A D may at least in part be accounted for by the reduction of calcium flux into the neuron. Two possible additional, but not exclusive, mechanisms include a direct shunt of/AHP by the A D conductance and an increase in adenylate cyclase activity due to A2 receptor activation (at high AD concentrations; Section 4.1) which could also decrease I^Hp (Section 4.2). 2.2.2. Lack o f effect on 14 and I o Two of the voltage sensitive potassium currents known to be modulated by neurotransmitters are IA (Aghajanian, 1985; Akins et al., 1990) and I 0 (similar in most respects to In; Bobker and Williams, 1989; Pape and McCormick, 1989). The name, IA, is employed here to refer to the family of subtypes of transient outward current which exhibit voltage semiJPN 36/6--F

tive activation and inactivation (Butler et al. 1989; Solc et al., 1987; Greene et al., 1990). IA activates near the action potential threshold resulting in an initial, but transient, refractory period to depolarizing excitatory input. I^ also inactivates in this membrane potential range which limits its duration of action (i.e. a 'transient' effect). IQ (Halliwell and Adams, 1982) is activated at hyperpolarized levels and does not inactivate. Because the reversal potential of I 0 is depolarized to the potential range in which it is active, this current tends to prevent hyperpolarization of the membrane potential. Neither of these currents was enhanced by AD (Fig. 5, Gerber et al., 1989). Furthermore, the antagonists, 4-AP and CsC1, were ineffective blockers of the A D response at the concentrations effective for antagonism of IA (the 4-AP sensitive subtype called ID in the hippocampus; Storm, 1988) and IQ (Halliwell and Adams, 1982), respectively.

2.3. INDIRECT EFFECTS ON CALCIUM CURRENT

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In the peripheral nervous system, somatic calcium current is directly reduced by A D (Henon and McAfee, 1983; Dolphin et al., 1986; MacDonald et al., 1986). In marked contrast to the CNS, the PNS neurons tested (both superior cervical ganglia and dorsal root ganglia) did not exhibit an A D evoked increase in potassium conductance which resulted in a technically less demanding analysis of calcium currents. A similar situation was observed with acutely isolated neonatal spinal cord neurons of the dorsal horn, namely, that AD (in addition to ATP, serotonin, norepinephrine, somatostatin and dynorphin A) directly antagonized calcium currents with no reported effect on potassium currents (Sah, 1990). In the adult hippocampus, AD reduces the calcium dependent action potential observed in the presence of TTX (Proctor and Dunwiddie, 1983; Haas and Greene, 1984). However, such a reduction might be due to either a decrease in calcium current or an increase in potassium current. In fact, the decrease of inward calcium current elicited by AD had a similar time course of onset and recovery to the AD elicited potassium current. Furthermore, the amplitudes of each effect were positively correlated (Fig. 6). In another study of the adult hippocampal slice under voltage clamp control there was no A D effect on calcium currents observed (Halliwetl and Scholfield, 1984). The holding potential of - 3 2 mV employed in this study was well above threshold for calcium current activation so that the intracellular calcium concentration was probably not within the physiological range. This may explain the observed absence of AD evoked potassium current. It also raises the possibility that the observed absence of A D elicited calcium current depression was due to the same mechanism. Nevertheless, these findings were confirmed by the demonstration that during complete blockade of the A D potassium current with barium (2 mM), A D had no observable effect on the slow inward calcium (and barium) currents (Fig. 6). Upon removal of the barium, inhibition of the slow inward currents by AD was restored (Gerber et al., 1989). Apparently, the primary mechanism for A D evoked

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A

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B 50ms_.i 0.5 -40 -90 FIG. 5. IQ and IA are not affected by AD. (A) Under single electrode voltage clamp control, voltage command steps from -55 mV to -95 mV elicit IQ (a slow inward or downward relaxation). AD does not alter the relaxation, only the instantaneous current. (B) From a holding potential of -90 mV, step commands to -40 mV evoke a transient outward or upward current (especiallynoticeable by comparison of the steps to - 60 mV and - 50 mV). AD does not affect the transient IAcurrent. (Modified from Greene and Haas (1989). In Hippocampus; New Vistas. Eds. V. Chan-Palay and C. Kohler. Alan R. Liss: New York.) reduction of calcium current is by the indirect means of an increase in AD potassium current. This current may act as a shunt to reduce the time a neuron (or dendrites of a neuron) might otherwise spend at the depolarized potential, necessary to allow the inward flux of calcium through the voltage sensitive calcium channel gates. Thus it is possible that under physiological conditions, the calcium flux due to action potential firing or synaptic depolarizations is reduced.

3. INHIBITION OF EVOKED SYNAPTIC POTENTIALS 3.1. SITEOF ACTION AD has been shown to depress extracellularly recorded field potentials which result from the excitatory postsynaptic response to stimulation of an afferent fiber tract in olfactory cortex (Kuroda et al., 1976; Scholfield, 1978), hippocampus (Schubert and Mitzdorf, 1979; Dunwiddie and Hoffer, 1980; Okada and Kuroda, 1980) and the neostriatum (Malenka and Kocsis, 1988). In all these regions the excitatory postsynaptic response is due primarily to an excitatory postsynaptic potential (EPSP; Fig. 7). Evidence for localization of the AD elicited depression of the EPSP to either the pre- or postsynaptic elements is at present circumstantial. In the olfactory cortex there does not appear to be a postsynaptic AD response (Scholfield, 1978). In the hippocampus this is clearly

not the case. Nevertheless, AD was observed to increase paired pulse facilitation to Schaeffer collateral stimulation in CA1 neurons (Dunwiddie and Haas, 1985). This is most consistent with a reduction in transmitter release. Further support comes from biochemical studies in the hippocampus (Dolphin and Archer, 1983; Fastbom and Fredholm, 1985) and other regions which demonstrate an AD evoked suppression of transmitter release. A variety of different transmitter systems are similarly affected including acetylcholine, serotonin, dopamine, GABA, and glutamate (reviewed by Dunwiddie, 1985). However, the modulation of release of transmitter elicited by biochemical methods such as exposure to veratridine or high concentrations of potassium, may be different from the modulation of the release of transmitter by more physiological mechanisms. 3.2. MECHANISMOF ACTION There is as yet no direct evidence from which an assessment of a presynaptic mechanism might be obtained. At least three different mechanisms might be considered as candidates. One is a direct action of AD to suppress presynaptic calcium flux as has been observed on the postsynaptic membrane of peripheral neurons. A second alternative is an indirect suppression of presynaptic calcium flux due to a shunt by an increased potassium conductance, observed postsynaptically in the hippocampus. Finally, at the neuromuscular junction, Silinsky (1986) has proposed that

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FIG. 6. AD does not reduce the slow inward currents during antagonism of the AD evoked potassium current by barium. (A) Single electrode records made with cesium chloride in the recording electrode (2 M) and barium (2 mM) in the perfusate. Voltage step commands to - 1 0 m V (Vho~d=--70mV) evoked slow inward currents, normally carried by calcium through high threshold calcium channels, that were not appreciably altered by exposure to AD. (B) When control perfusate (Ca, 2.5 mM; Mg, 1.4 raM; Ba, 0 mM) was used in the same cell, peak inward current was reduced (upper graph) in proportion and with a similar time course to the AD elicited outward current (lower graph). This is consistent with a reduction of calcium current resulting from a shunt by the potassium outward current (modified from Gerber et aL, 1989). AD evoked presynaptic inhibition impairs calcium dependent release at an intracellular site rather than the flux of calcium across the membrane. It is also noted that these mechanisms are not exclusive of one another.

4. MEDIATORS OF E L E C T R O P H Y S I O L O G I C A L EFFECTS 4. I. RECEPTOR SUBTYPES There are two subtypes of A D receptors described in the mammalian cortex, classified as A~ and A2. The A~ receptor mediates inhibition of adenylate cyclase activity and has a greater affinity for A D than does the A2 receptor which increases adenylate cyclase activity (Van Calker et al., 1979; Londos et al., 1980).

Activation of the outward potassium current in cultured striatal neurons is mediated most likely by an AI receptor (Trussell and Jackson, 1989). The response--concentration plots for the agonists Lphenyl-isopropyl-adenosine (L-PIA), its D-isomer (D-PIA), N-ethylcarboxyadenosine (NECA) and A D revealed a 1/2 maximal effect sequence (from lowest to highest concentration) of L - P I A > D PIA > NECA > AD, characteristic of the A, receptor (Snyder, 1985). Evidence for receptor specificity of the postsynaptic AD effect in the adult hippocampal slice was obtained by examination of the extracellularly recorded population spikes following antidromic activation of CA1 neurons in the presence of low calcium high magnesium (Dunwiddie and Fredholm, 1989). Perfusion of the hippocampal slice with this low calcium medium completely blocks evoked synaptic transmission. Nevertheless, these conditions evoke spontaneous cell firing, spontaneous epileptiform field potentials and epileptiform repetitive population spikes (an antidromic spike followed by afterdischarges) following antidromic stimulation of CA1 axons in the alveolus (Haas and Jefferys, 1984; Haas et al., 1984). The repetitive population spikes are probably a result of electrical field effects and a shift of potassium from the intraceilular to extracellular space. Inhibition of this discharge by exogenous AD (Haas et al., 1984) is most likely to result from direct effects on the CAI neuronal membrane. By measurement of the AD evoked depression of the first afterdischarge and the inhibition of this depression by 8-cyclopentyltheophylline (8-CPT) a Schild plot was constructed which gave an estimated K o for 8-CPT of between 26-78 nM (95% confidence limit). This is consistent with mediation by an A~ receptor and clearly distinguishable from the estimated KD of 1.7/~M for the A2 receptor evoked increase in cAMP formation. 4.1.2. Presynaptie responses

In the olfactory cortex, there is no evidence for a postsynaptic A D response, thus the AD elicited depression of the evoked EPSP is likely to be a predominantly presynaptic effect. A comparison of the order of potency of six agonists gave a response profile most consistent with mediation by the A~ receptor subtype (McCabe and Scholfield, 1985). Unfortunately the situation is less clear using agonists in the hippocampus. This is due in part to the presence of electrophysiologically active AD receptors on both pre- and postsynaptic membranes (Section 3) and in part to the geometry of the CAI dendrites, which makes an accurate appraisal of the relative contribution of pre- and postsynaptic AD effects technically difficult. In particular, synaptic spines possess a shape consistent with relative electrical isolation from the rest of the dendrite and neuronal soma. These spines are located in dendritic regions known to possess the most dense concentration of AD receptors (Lee et al., 1983; Tetzlaff et al., 1987). If the postsynaptic AD effect upon the evoked EPSP is mediated by receptors on the spines, then depression

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FIG. 7. Adenosine depresses the extraceUularly recorded evoked excitatory postsynaptic population response. On the left is a schematic to indicate the location of: (1) the stimulating electrode in the striatum radiatum or lacunae so that axons of passage of CA3 neurons may be stimulated causing an excitatory postsynaptic potential in the CA1 dendrites; (2) the EPSP may be extracellularly recorded from the striatum radiatum as a negative potential (illustrated on the bottom); (3) extracellular recording in the pyramidal layer may include both the positive potential of the EPSP and the larger negative potential of the population action potential which was elicited by the EPSP (illustrated on the top). On the right is a plot of the stimulus strength (ordinate) versus the evoked potential amplitude (abscissa) of the EPSP (triangles) and population spike (circles). Empty symbols reflect data obtained before and filled symbols, during perfusion with AD (20/~M). of the EPSP might occur in the absence of an observable effect in the main body of the dendrite or the soma. Thus, a postsynaptic effect may have the appearance (on an intracellular record) of a presynaptic effect. 4.2. SECONDMESSENGER The best characterized postsynaptic response to AD, consisting of a hyperpolarization associated with a decrease of input resistance is dependent upon a GTP-binding protein. Using whole cell patch clamp on cultured hippocampal neurons, Trussell and Jackson (1987) demonstrated antagonism of the AD response by pretreatment with pertussis toxin that inactivates G i and Go (Gilman, 1984; Sternweis and Robishaw, 1984) and by intraceilular application of the poorly metabolized analog of GDP, GDPflS, which inhibits G-proteins by the antagonism of GTP binding (Eckstein et al., 1979). Further, the addition of GTP to the patch pipette prevented washout of the AD response (a result of intracellular dialysis by the patch pipette) in a dose-dependent manner. These findings have been confirmed in the in vitro hippocampal slice by pretreatment with pertussis toxin two days prior to slice preparation which prevented the AD evoked hyperpolarization (Zgombick et al., 1989). AD is known to both inhibit and increase adenylate cyclase activity in the brain (Londos et al., 1980). The inhibition of cyclase activity and AD evoked hyperpolarization both appear to be mediated by the

A 1 receptor. Furthermore, both are blocked by pertussis toxin (Zgombick et al., 1989). This raises the possibility that the latter effect is a result of the decrease in the cAMP. However, in cultured CNS neurons, the electrophysiological AD effect is unaffected by exposure to either forskolin which greatly stimulates adenylate cyclase activity, or to cAMP analogs resistant to phosphodiesterase (Trussell and Jackson, 1987). The same has been observed in the hippocampal slice (Greene and Haas, unpublished observations). However, another electrophysiological effect of AD, is on IAHe that is modulated by intracellular cAMP levels (Madison and Nicoll, 1986). The amines, histamine (Haas, 1984) and noradrenaline (Madison and Nicoll, 1986) act to decrease /AnP by increasing cAMP levels. AD enhances the long duration A H P (see Section 2.2.1) and this could result from A~ receptor activation of a G-protein which inhibits cAMP formation. A probable presynaptic effect of AD is the suppression of transmitter release elicited by non-specific neuronal stimulation (Section 3.1). In cultured cerebellar neurons this suppression was shown to be blocked by preincubation in pertussis toxin (Dolphin and Prestwich, 1985). A similar finding of pertussis toxin sensitivity of the AD elicited suppression of the end plate potential at the mammalian neuromuscular junction was reported (Silinsky et al., 1989). In this preparation, the site of action is clearly presynaptic (Ginsborg and Hirst, 1972). There is some evidence for the involvement of protein kinase C in the hyperpolarization and in the

ELECTROPHYSIOLOGYOFADENOSINE depression of the evoked EPSP by AD. The addition of phorbol esters, activators of protein kinase C, also block these electrophysiological AD effects on hippocampal neurons in culture (Yakel et al., 1988) or in the slice (Worley et al., 1987).

5. ELECTROPHYSIOLOGY OF ENDOGENOUS ADENOSINE The concentration of adenosine in the extracellular CNS fluid is about 1 #M with little variation throughout the CNS. However, these levels may be increased by as much as 2 orders of magnitude in hyperactive tissues, probably as a consequence of the saturation of the powerful high affinity uptake systems present in CNS tissue (for review see Wu and Phillis, 1984; Dunwiddie, 1985). Thus, it is conceivable that within the micro-environment of the neuronal dendrites and AD receptors, AD concentrations vary to an even greater extent, perhaps under physiological as well as pathophysiological conditions. 5.1. ANTAGONISTEVOKEDEXCITATION The methylxanthines, theophylline, isobutylmethylxanthine and 8-phenyltheophylline (8-CP) have been shown to competitively antagonize the suppression of evoked population EPSPs recorded in vitro in response to the application of exogenous AD, in the olfactory cortex (Scholfield, 1978; Okada and Kuroda, 1980; McCabe and Scholfield, 1985) and hippocampus (Dunwiddie and Hoffer, 1980). It was noted that these antagonists had excitatory effects of their own. In the hippocampus, the methylxanthine, caffeine, was shown to affect all electrophysiological properties affected by AD but with the opposite polarity (Fig. 8; Greene et al., 1985). The effects included a depolarization of the membrane potential, an increase in input resistance, a decrease in the voltage insensitive calcium dependent potassium current and an enhancement of the evoked population EPSP and population spike. 8-CP (l/~i) has also been shown to depolarize the resting membrane potential and increase resting input resistance (Zgombick et al., 1989). These data are consistent with a mechanism of action as an antagonist of endogenous, electrophysiologically active AD. They may also suggest a mechanism for caffeine's well appreciated stimulant effect (Snyder, 1985). 5.2. ANTAGONISMOF UPTAKEAND INCREASED CATABOLISM Iontophoretic application of adenosine uptake inhibitors depressed cell firing in the cerebral cortex, in vivo, even in the absence of exogenous AD (Phillis et al., 1979). Both the blockade of AD reuptake with hexobendine and the enhancement of AD catabolism with adenosine deaminase (ADA) have effects upon evoked synaptic transmission in the hippocampal slice preparation. The blockade of reuptake depressed the EPSP and ADA enhanced it (Dunwiddie and Hoffer, 1980). Similar findings with uptake inhibitors, dipyridamole (Motley and Collins, 1983)

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FIG. 8. Caffeine elicits an enhancement of the evoked EPSP and antagonizes and AD mediated depression of the evoked EPSP. Upper 4 traces show the extracellularly recorded EPSP from the cell layer (upper two traces) and the dendritic layer (lower two traces) before (on the left) and during exposure to caffeine (100 #M). The plot on the bottom is of a long concentration/response curve of the AD elicited suppression of the evoked EPSP before (squares) and in the presence (circles) of caffeine (50 #M). The curves are consistent with a competitive antagonism by caffeine of the electrophysiological response elicited by AD (modified from Greene et al., 1985). and nitrobenzylthioinosine (NBTI; Sanderson and Scholfield, 1986) have been observed in the olfactory cortex. Further evidence that these effects result from action of endogenous AD on electrophysioiogical properties of hippocampal pyramidal cells derives from the observations: (1) NBTI mimicked the AD elicited hyperpolarization and decrease in input resistance of CAI neurons; (2) the efficacy of the AD antagonist, caffeine, to increase the evoked EPSP was significantly enhanced in the presence of NBTI, similar to the effect of adding exogenous AD; (3) in the presence of ADA, caffeine was no longer effective in increasing the evoked EPSP, presumably because the effect of endogenous AD was already reduced due to catabolism by the ADA (Fig. 9; Haas and Greene, 1988). Thus, in the in vitro preparation, AD appears to exert an inhibitory tone during electrophysiological activity under control conditions. 5.3. EFFECTSIN LOW CALCIUM,HIGH MAGNESIUM The source of the electrophysiologically active AD is unknown. However, it is unlikely that calcium dependent synaptic release can be the sole source. In the presence of TTX or of low calcium, high magnesium in the perfusate which blocks calcium dependent synaptic potentials (Haas and Jefferys, 1984), NBT! was shown to mimic the depressant effect of AD. Similarly, in low calcium, high magnesium perfusate, ADA increases neuronal excitability (Fig. 10; Haas and Greene, 1988; Fowler, 1988). It is conceiv-

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FIG. 9. The enhancement of the evoked CA1 EPSP by caffeine is reduced in the presence of adenosine deaminase and increased in the presence of nitrobenzylthioinosine.The amplitude of the evoked EPSP (measured as indicated in the inset, with a set latency of 4 msec to avoid contamination by the positive going population spike) is plotted against time before (open circles) and during (closed circles) exposure to the catabolic enzyme of adenosine, adenosine deaminase (ADA in "A") and the adenosine uptake inhibitor nitrobenzylthioinosine (NTI in "B"). The stimulus strength was readjusted in both cases so that the EPSP amplitude was the same after agent exposure as during control. This required a decrease of stimulus amplitude for ADA and an increase for NTI. Caffeine was then applied for the durations indicated by the horizontal bars. In "A" the effects of ADA is consistent with less endogenous adenosine in the extracellular fluid and in "B" the effect of NTI is consistent with more endogenous adenosine (modified from Haas and Greene, 1988). able that the A D reuptake is a part of an A D bidirectional transport system (and as such, a potential source of AD), sensitive to metabolic state and A D concentrations.

6. ACTIVITY IN P A T H O P H Y S I O L O G I C A L STATES 6.1. EPILEPSY Epileptiform activity can be induced in the hippocampal slice by the addition of convulsants which interfere with inhibitory transmission such as bicuculline or penicillin or in the absence of synaptic transmission by exposure to perfusate with low Ca2+/high Mg :÷ (a reduction of divalent cation concentration, Taylor and Dudek, 1982; Haas and Jefferys, 1984). Adenosine has been demonstrated to antagonize induced epilepsy in vivo (Maitre et al., 1974), convulsant evoked epileptiform activity in vitro (Dunwiddie et al., 1981) and low Ca2+/high Mg 2+ evoked epileptiform activity in vitro (Fig. 1 l; Haas et al., 1984; Lee et al., 1984). Further, in the presence of the selective A~-receptor antagonist, 8-cyclopentyl1,3-dipropylxanthine, CA3 neurons in the slice preparation develop sustained epileptiform activity (Alzheimer et al., 1989).

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FIG. 10. The firing rate of CAI neurons is increased by adenosine deaminase and decreased by nitrobenzylthioinosine in the absence of calcium dependent synaptic activity. The firing rate was determined from extracellular single unit recordings (illustrated in insets) and plotted against time. In both "A" and "B" the perfusate contained 0.2 mM calcium and 4 mM magnesium to block synaptic activity. (A) Adenosine deaminase (ADA) which decreases the endogenous adenosine concentration, increased the firing rate. (B) Nitrobenzylthioinosine (NTI) which increases the endogenous adenosine concentration, mimics the effect of adenosine (AD) to decrease the firing rate (modified from Haas and Greene, 1988). The A D actions in the low Ca:+/high Mg 2÷ epilepsy model are postsynaptic and thus, likely to be mediated by the postsynaptic A D mechanisms discussed above. As the extracellular concentration of AD begins to increase, so might the A D elicited enhancement of the voltage-insensitive calcium dependent potassium conductance. This will selectively inhibit long-duration excitatory input while having little or no effect upon the short duration (0-50 msec) input. As AD levels increase further with increasing epileptiform activity, the voltage and calcium insensitive potassium conductance may be activated and, eventually predominate, resulting in a powerful nonspecific depression of neuronal excitability. In circumstances involving synaptic transmission in the generation of epileptiform activity, AD may have additional mechanisms of anti-epileptic action. Acting at the presynaptic terminals, A D may cause the depression of excitatory transmitter release. Unfortunately, little is understood at present about the nature of this depression to predict any selectivity of its action for different patterns of synaptic release. 6.2. HYPOXIA Ischemic states can be mimicked in vitro by replacement of perfusate saturated with oxygen by that

ELECTROPHYSIOLOGYOF ADENOSINE

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339

is consistent with an electrophysiological inhibitory role for endogenous AD. This inhibition is likely to be mediated by the same mechanism as described for exogenous AD. Further, the endogenous AD can depress excitability in the absence of calcium dependent synaptic release. To the extent that the in vitro preparation mimics the physiological state, endogenous AD appears to exert a physiological inhibitory tone in the hippocampal cortex. Under conditions of metabolic stress when the extracellular AD levels rise, one might expect this tone to increase, thus decreasing the metabolic load due to electrophysiological activity. Acknowledgements--We gratefully acknowledge Drs S.

FIG. I1. Epileptiform activity in the hippocampal slice is antagonized by adenosine. Antidromic stimulation of CA1 neurons causes a population antidromic spike followed by recurrent population spikes in media containing 0.2 mM Ca and 4 mM MR. The first recurrent spike is indicated by the star in the lower left-hand trace (calibration =2mV, 8 msec). The lower right-hand trace shows the absence of recurrent spikes during exposure to adenosine (AD). In the upper record, the amplitude of the first recurrent spike is plotted against time to show the duration of AD evoked suppression of the epileptiform recurrent spike. (Modified from Greene and Haas (1989). In: Hippocampus; New Vistas. Eds. Alan R. Liss: New York.) saturated with nitrogen. One of the earlier ensuing events is a profound depression of neuronal excitability (Hansen, 1985) and a release of AD into the extracellular fluid (Berne et al., 1982). The depression is at least in part mediated by a postsynaptic increase in a potassium conductance which is blocked by intracellular cesium and may be calcium dependent unlike that elicited by AD (Leblond and Krnjevic, 1989; Krnjevic and Leblond, 1989). Further, the AD elicited increase in potassium conductance may be prevented by pretreatment with pertussis toxin (to prevent the G-protein activation). Under these conditions, the hypoxia induced hyperpolarization and decrease in input resistance appeared unaffected (Spuler and Grafe, 1989). It would thus appear that mediators other than AD are responsible for the hypoxia induced depression. However, a role for AD in hypoxia as suggested by the delay of hypoxia induced depression of the population spike in the presence of AD antagonists (Fowler, 1989), cannot be excluded. 7. CONCLUSIONS An inhibitory neuronal tone may be exerted by AD in the mammalian nervous system by means of three mechanisms. First, AD evokes a voltage and calcium insensitive potassium conductance; second, AD enhances a voltage insensitive calcium dependent potassium conductance; finally, AD depresses synaptic potentials by an unknown presynaptic mechanism. Activation of voltage and calcium insensitive potassium conductance is dependent upon a G-protein associated with an Al-receptor. Evidence based upon the actions of antagonists of AD reuptake and the AD catabolic enzyme, in vitro,

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