Molecular and Cellular Action of Caffeine in the Brain
- Actions of Caffeine in the Brain with Special Reference to Factors That Contribute to Its Widespread Use

III. Molecular and Cellular Action of Caffeine in the Brain

A. Fundamental Biochemical Actions

The biochemical mechanism that underlies the actions of caffeine at doses achieved in normal human consumption must be activated at concentrations between the extremes (between barely effective doses and doses that produce toxic effects; see Fig.

B. Adenosine Levels in Brain and Other Tissues

The hypothesis that we consume coffee because it blocks the actions of endogenous adenosine at its receptors is only tenable if adenosine is present in sufficient concentrations to activate the adenosine receptors already under basal conditions. We must therefore critically assess this postulate.

Adenosine is a normal cellular constituent. The intracellular level is regulated by the balance of several enzymes. Adenosine is formed by the action of an AMP-selective 5'-nucleotidase, and the rate of adenosine formation via this pathway is mainly controlled by the amount of AMP. Therefore, the important factor determining the rate of adenosine formation via this pathway is the relative rates of ATP breakdown and synthesis. These are in turn determined by the rate of energy utilization and the availability of metabolizable substrate.

There are two enzymes that constitute the major pathways of adenosine removal: adenosine kinase and adenosine deaminase. The latter enzyme is present mostly intracellularly but is also found in some extracellular compartments. The preferred substrate of the enzyme is not adenosine but 2-deoxyadenosine (Fredholm and Lerner, 1982

Extracellular ATP is very rapidly hydrolyzed to adenosine and other metabolites. Thus, if ATP is released from neuronal (or glial) cells, e.g., as a transmitter or an intercellular signal, it will provide a source of extracellular adenosine. It seems likely that this may be significant in some circumstances and in some locations. However, extracellular ATP is not the major source of adenosine released from brain slices during field stimulation (at least when relatively low frequency stimulation is used) or following hypoxia/hypoglycemia. This is shown by the fact that agents that block extracellular AMP hydrolysis fail to affect the rate of adenosine release significantly (Lloyd et al., 1993). Thus, intracellular adenosine formation is quantitatively most important.

Intra- and extracellular adenosine concentrations are kept in equilibrium by means of equilibrative transporters. These transporters are blocked by several agents such as nitrobenzylthioinosine, propentofylline, dipyridamole, and dilazep. In addition there are sodium-dependent, concentrating transporters that move extracellular adenosine into cells. These latter transporters are not blocked by the above agents, and their precise role in the CNS is unknown. When inhibitors of equilibrative transport are given, the levels of adenosine rise in the CNS despite a decrease in the release of adenosine metabolites such as inosine and hypoxanthine (Andiné et al., 1990; Fredholm et al., 1994b). The reason for this has been discussed elsewhere (see Fredholm et al., 1994b). Adenosine, once released, can secondarily be taken up by cells and metabolized to inosine and hypoxanthine. It should, however, be pointed out that transport inhibitors block the overall release of adenine nucleotide breakdown products (Jonzon and Fredholm, 1985), as expected for a model where equilibrative transporters are critically important. Furthermore, the addition of L-homocysteine in the presence of transport inhibitors leads to a very substantial reduction in the efflux of adenosine. The reason is that an excess of L-homocysteine forces the S-adenosylhomocysteine hydrolase reaction to occur in reverse and intracellular adenosine levels are very much reduced. When intracellular levels are decreased the extracellular levels also go down.

From these facts it can be deduced that adenosine levels in the extracellular fluid should be raised whenever there is a discrepancy between the rate of ATP consumption and ATP synthesis. In addition, it is expected that drugs that interfere with the key enzymes and with the transporters should affect adenosine levels. Extracellular adenosine levels have been measured using microdialysis. In the first paper using this method it was shown that the level of adenosine, while initially high, stabilized at about 1 µM within a few hours of implantation of the dialysis probe (Zetterström et al., 1982). It was also shown that the level of adenosine was raised about 3-fold following a mild hypoxia. The level of adenosine can increase dramatically to 10 µM or more following ischemia (Andiné et al., 1990; Dux et al., 1990). However, a later study showed that it took much longer to reach the true equilibrium level and that consequently the disturbance produced by the microdialysis probe lasted for perhaps 24 h (Ballarin et al., 1991). Our current best estimate of the basal level of adenosine in the brain of awake, unrestrained rats is between 30 and 300 nM. Interestingly, these levels are close to the estimated levels of adenosine in plasma (Reid et al., 1991). There is one report to the effect that caffeine, particularly prolonged administration of caffeine, increases the levels of adenosine in plasma dramatically (Conlay et al., 1997) at least in rats, and that this effect is receptor-mediated. This finding clearly needs to be reproduced---especially in humans.

We turn now to the question of whether there are adenosine receptors that are activated not only by the high adenosine levels seen in ischemia, but also by the low (high nanomolar concentrations) physiological levels.

C. Adenosine Acts on Several Types of G-Protein-Coupled Receptors

1. Receptor Subtypes. At present four distinct adenosine receptors, A1, A2A, A2B, and A3, have been cloned and characterized in several species (Fredholm et al., 1994a

Although A3 and A2B receptors are unlikely to be important, A1 and A2A receptors are activated at the low basal adenosine concentrations measured in resting rat brain. Thus, these receptors are likely to be the major targets for caffeine and theophylline. A1 and A2A receptors are both G-protein-coupled. The A1 receptor is coupled to the pertussis toxin-sensitive G-proteins Gi-1, Gi-2, Gi-3, Go1, and Go2. In agreement with this, activation of A1 receptors can cause inhibition of adenylyl cyclase and of at least some types of voltage-sensitive Ca2+-channels such as the N- and the Q-channels, and activation of several types of K+-channels, phospholipase C and phospholipase D. Consequently, a host of different cellular effects can ensue (see Fredholm et al., 1994a,1995). A2A receptors associate with Gs-proteins; therefore, activation of these receptors causes the activation of adenylyl cyclase and perhaps also activation of some types of voltage-sensitive Ca2+-channels, especially the L-channel. Thus, A1 and A2A receptors have partly opposing actions at the cellular level. This is interesting because the two types of receptor are sometimes coexpressed in the same cell. It is therefore important to consider where these two adenosine receptors are located.


2. Receptor Distribution. A1 and A2A receptors in the brain can be localized by receptor autoradiography with radioactive ligands. In addition, the sites of receptor synthesis can be determined using in situ hybridization. Adenosine A1 receptors are present in almost all brain areas, with the highest levels in hippocampus, cerebral and cerebellar cortex, and certain thalamic nuclei (Goodman and Snyder, 1982

Adenosine A2A receptors are found to be concentrated in the dopamine-rich regions of the brain, irrespective of whether ligand binding or mRNA is used for the localization (Fig. 2). This association was in fact noted a long time ago when it was shown that in cell-free homogenates from these regions, and only from these regions, adenosine stimulated adenylyl cyclase activation (Fredholm, 1977; Premont et al., 1979). In the first direct studies on the localization of A2A receptors, a number of radiolabeled agonists were used. One of the agonists used most frequently was [3H]CGS 21680 (Jarvis and Williams, 1989; Parkinson and Fredholm, 1990). More recently it has become apparent that CGS 21680 is not an optimal ligand. For example, it has been found to have a relatively low affinity for A2A receptors in nonrodent species. At human A2A receptors for example those expressed in Chinese hamster ovary cells, CGS 21680 has an affinity close to 100 nM, whereas its affinity at rat A2A receptors is closer to 10 nM (see Ongini and Fredholm, 1996). A second factor that limits its usefulness as a radioligand is that it is an agonist and that its affinity consequently depends on the association of the receptor with G-proteins. This association is highly variable between preparations and methods used. Finally, it has been found that CGS 21680 binds to sites that are clearly different from A2A receptors (Johansson et al., 1993b; Johansson and Fredholm, 1995). These sites are present in cortex and hippocampus and can be clearly differentiated from the A2A receptors by the use of selective antagonists (Lindström et al., 1996). In fact, in many respects these non-A2A-receptor binding sites for [3H]CGS 21680 show many characteristics of an A1 receptor (Cunha et al., 1996). It has recently been shown that [3H]SCH 58261, a nonxanthine antagonist, can be used successfully to study the distribution of A2A receptors (Ongini and Fredholm, 1996). When this radioligand is used there is little evidence for significant A2A receptor binding outside striatum, nucleus accumbens, and tuberculum olfactorium.

In situ hybridization, using either oligodeoxynucleotide probes or riboprobes, similarly reveals a very selective localization of A2A receptor mRNA to the same dopamine-rich regions of the brain. Very little mRNA is detected in other regions of the brain. This is somewhat surprising given the amount of functional data that clearly suggests the presence of functionally important A2A receptors in hippocampus and cortex.


The in situ hybridization technique makes it possible to determine which cells express A2A receptor mRNA. It was observed that A2A receptor mRNA was colocalized with dopamine D2 receptors in enkephalin-expressing, medium-sized spiny neurons in the dorsal striatum (Schiffmann et al., 1991; Fink et al., 1992; Johansson et al., 1993a). It has later become clear that this colocalization of A2A and D2 receptors extends also to the core and shell regions of the nucleus accumbens and to the tuberculum olfactorium (Svenningsson et al., 1997b) (Fig. 3). On the other hand, the neurons that express dopamine D1 receptors and Substance P do not express adenosine A2A receptor mRNA. Furthermore, none of the above reports detected any significant expression of A2A receptor mRNA in the large aspiny cholinergic neurons. One group did report A2A receptor mRNA in cholinergic neurons using in situ hybridization (Dixon et al., 1996) as well as functionally important A2A-like receptors regulating acetylcholine release from cholinergic synaptosomes (Kirk and Richardson, 1994; Kurokawa et al., 1994). As shown in Fig. 3, this finding could not be replicated in studies using riboprobes for both the A2A receptor and choline acetyl transferase despite the fact that these probes show a much higher sensitivity and specificity (Svenningsson et al., 1997b). The reason for this discrepancy between the results of different studies is unclear.

D. Caffeine Affects Transmitter Release and Neuronal Firing Rates via Actions on Adenosine A1 Receptors

The inhibitory effect of adenosine on transmitter release was first noted in the peripheral nervous system, but similar effects in the CNS were soon demonstrated (see Fredholm and Hedqvist, 1980

As discussed previously (Fredholm and Dunwiddie, 1988), adenosine appears to use several mechanisms in order to produce inhibition of transmitter release. The electrically evoked release, but not the spontaneous release of neurotransmitter, is strongly dependent on the concentration of calcium in the extracellular environment (see Fredholm and Hu, 1993). On the other hand, the electrically evoked release is poorly affected by buffers of intracellular calcium, whereas the spontaneous transmitter release is strongly affected. This supports the contention that calcium entering via voltage-dependent calcium channels and acting on docked vesicles in the neighborhood of the channel is important. It is interesting to note that adenosine acting on A1 receptors has been shown to decrease calcium entry via N-type channels in hippocampal CA1 and CA3 neurons (Scholz and Miller, 1992; Mogul et al., 1993). However, in some types of neurons the effect of adenosine is only moderately or not at all affected by omega-conotoxin, a selective inhibitor of N-type channels (Fredholm, 1993). This could mean that adenosine acts either on some other type of calcium channel (Takahashi and Momiyama, 1993), perhaps the putative Q-type channel (Sather et al., 1993; Takahashi and Momiyama, 1993). Another possibility is that adenosine affects, directly, a calcium-sensitive member of the release machinery, a contention for which there is some support (Silinsky, 1984; Scholz and Miller, 1992; Thompson et al., 1992). However, when it has been possible to actually measure Ca2+ influx, the reduction has been found to adequately account for the decrease in transmitter release (Yawo and Chuhma, 1993; Wu and Saggau, 1994). There is also some evidence that increases in cyclic AMP in nerve endings are associated with an increase in transmitter release (Chavez-Noriega and Stevens, 1994). Because activation of adenosine A1 receptors is known to cause a decrease in cAMP formation, it is conceivable that this may also be a mechanism of decreased transmitter release---at least under some circumstances.

There is also considerable evidence that adenosine acts to decrease the rate of firing of central neurons (Phillis and Edstrom, 1976). This effect appears to be quite general and is due to an activation of potassium channels via adenosine A1 receptors (Dunwiddie, 1985). When the effect of endogenous adenosine at these receptors on glutamatergic neurons is blocked by caffeine, it leads to epileptiform activity in vitro (Dunwiddie, 1980; Dunwiddie et al., 1981), and this could be the mechanism by which methylxanthines produce seizures in vivo.

It is also known that caffeine increases the turnover of several monoamine neurotransmitters, including 5hydroxytryptamine (5-HT) dopamine, and noradrenaline (Fernström and Fernström, 1984; Bickford et al., 1985; Fredholm and Jonzon, 1988; Hadfield and Milio, 1989). There is evidence that methylxanthines increase the rate of firing of noradrenergic neurons in the locus ceruleus (Grant and Redmond, 1982). The increase in noradrenaline turnover is probably the explanation for the fact that methylxanthines also reduce the number of beta-adrenoceptors in rat brain (Fredholm et al., 1984; Shi et al., 1993a). It has also been shown that the mesocortical cholinergic neurons are tonically inhibited by adenosine and that caffeine consequently increases their firing rate (Rainnie et al., 1994). It was postulated that this effect is of importance in the electroencephalogram (EEG) arousal following caffeine ingestion. Because dopamine and noradrenaline neurons also are involved in arousal, there is ample neuropharmacological basis for assuming that central stimulatory effect of caffeine could be related to inhibition of adenosine A1 receptors. Also there are increases in 5-hydroxytryptamine receptors, muscarinic receptors, and delta-opioid receptors following higher doses of caffeine (Shi et al., 1993a, 1994). The functional relevance, if any, of these changes remains to be elucidated.

There is considerable evidence for a link between adenosine A1 receptors and dopamine D1 receptors (see Ferré et al., 1997). Thus, blockade of adenosine A1 receptors enhances motor effects of D1 receptor agonists. Infusion of an adenosine A1 receptor agonist into the caudate-putamen does not per se modify the levels of GABA in the entopeduncular nucleus, the output structure of the dopamine D1 receptor-expressing, medium-sized GABAergic neurons, but blocks the stimulatory effect of a D1 receptor agonist (Ferré et al., 1996). There are several possible mechanisms that could underlie these behavioral and neurochemical effects. It has been shown that activation of adenosine A1 receptors influence the binding of dopamine D1 agonists (Ferré et al., 1994, 1996, 1998). There are also more indirect interactions, and an involvement of N-methyl-D-aspartate (NMDA) receptors has been implicated. It is interesting to note that a recent study (Harvey and Lacey, 1997) presented strong evidence that combined dopamine D1 and NMDA receptor stimulation increases the release of adenosine, which then acts at adenosine A1 receptors to decrease the release of the excitatory neurotransmitter. Some of these interactions between A1, D1, and NMDA receptors are schematically represented in Fig. 4. In addition, D1 receptors in the ventral tegmental area (VTA) interact with adenosine A1 receptor effects (Bonci and Williams, 1996).

E. Caffeine Effects on Dopaminergic Transmission Are Exerted Mainly via Actions on Adenosine A2A Receptors

As noted above A2A receptors are located preferentially in the subpopulation of the medium sized spiny GABAergic neurons that project to globus pallidus, a subpopulation in which they are colocalized with dopamine D2 receptor mRNA. These colocalized receptors have been shown to interact functionally. Thus, activation of A2A receptors has been shown to decrease the affinity of dopamine binding to D2 receptors (Ferré et al., 1991

There is evidence that these interactions between adenosine A2A and dopamine D2 receptors observed in vitro have functional correlates in intact striatum. Thus, it is interesting that dopamine administered in the striatum has been shown to block the release of GABA in the globus pallidus (Ferré et al., 1993) and that this effect is reduced by endogenous adenosine. Furthermore, activation of adenosine A2A receptors increases GABA release from pallidal slices (Mayfield et al., 1993). The effects of adenosine on striatal GABA release are much more complex, possibly indicating a complex interaction between different neuronal populations. In slices of striatum, adenosine A2A receptor agonists do not directly influence the release of dopamine or acetylcholine (Jin and Fredholm, 1997), but adenosine A2A receptor stimulation has been shown to block the inhibitory effect of a dopamine D2 receptor agonist on acetylcholine release from striatal slices (Jin et al., 1993). This could indicate that part of the dopamine D2 receptor-mediated control of acetylcholine release from striatal slices is indirect and mediated via actions exerted at GABAergic neurons.

F. Identifying the Neuronal Substrates For Caffeine by Examining Changes in Immediate Early Genes---High Dose Effects

An increased neuronal activity is often accompanied by an expression of so-called immediate early gene (IEGs) such as c-fos, c-jun, junB, junD, NGFI-A (also called zif/268), and NGFI-B. Thus it is possible to determine which neuronal pathways are activated by caffeine by examining the effect of caffeine on immediate early gene expression. Caffeine causes a concentration-dependent increase in c-fos expression, which is confined to the striatum (Johansson et al., 1994

Because amphetamine and cocaine are known to act by releasing dopamine and because caffeine is presumed to act in part by increasing dopaminergic transmission, it is of interest to compare the effects of these three agents. A recent study revealed a gross morphological difference in the pattern of c-fos induction: cocaine and amphetamine increase the c-fos mRNA expression throughout the striatum, not least in the nucleus accumbens; caffeine, on the other hand, increases c-fos mRNA expression primarily in dorsolateral straitum (Johansson et al., 1994). Furthermore there is a marked difference at the cellular level. Amphetamine and cocaine primarily increase c-fos in the cells that express dopamine D1 receptors and Substance P, but not in those that express D2 receptors and met-enkephalin. By contrast, caffeine increases c-fos expression in both types of cells (Johansson et al., 1994). These data point to differences between the two types of agents, differences that could have some bearing on the question of whether caffeine is an addictive drug much like cocaine and amphetamine (Griffiths and Woodson, 1988a-c), but, as noted, the doses used to elicit IEG expression are high and not necessarily relevant in discussing behavioral stimulation.

The effect of an increase in the release of dopamine on IEG expression in the nucleus accumbens was directly studied by electrical activation of the medial forebrain bundle (Chergui et al., 1996). Burst activation of these neurons causes a marked increase in the evoked release of dopamine in the nucleus accumbens as assessed by voltametry. It is also associated with a marked increase in the expression of several IEGs, including c-fos, jun-B, NGFI-A, and NGFI-B. This increase can be blocked by dopamine D1 receptor antagonists and is confined to the striatonigral neurons that express D1 receptors. This shows that increased dopamine release---whether brought about by nerve activation or by pharmacological means---causes a D1 receptor-mediated increase in IEG expression. Because high doses of caffeine increase c-fos both in D1- and D2-expressing neurons, the mechanism underlying its actions cannot be explained solely by an increase in dopamine.

As noted above, there are adenosine A1 receptors on virtually all types of neurons that have the ability to decrease transmitter release. They are certainly present on the dopaminergic neurons (see Jin et al., 1993; Jin and Fredholm, 1997). However, they are also present on glutamatergic neurons. The striatum receives a strong glutamatergic input from both cortex and thalamus (see Gerfen, 1992), and part of the caffeine-induced increase in c-fos could be due to elevated release of glutamate. Indeed, at least part of the elevation in c-fos could be blocked by NMDA receptor antagonists (Svenningsson et al., 1996). Because the blockade was not complete, it is clear that additional mechanisms are also operative, but it may be relevant that the largest effect of NMDA receptor antagonism was observed in nucleus accumbens.

Caffeine injections lead to an increased expression not only of c-fos but also of other members of the same family of IEGs, notably c-jun and jun-B. Furthermore, there is an increased expression of the AP-1 transcription factor (Svenningsson et al., 1995b). Moreover, there are later changes in the expression of neuropeptides that are known to have AP-1-sensitive regulatory elements, notably preproenkephalin. These results suggest that even a single, albeit high, dose of caffeine can induce changes in gene expression that could lead to adaptive changes in the brain. The mRNA for four different neuropeptides, dynorphin, enkephalin, neurotensin/neuromedin, and Substance P, is elevated in the striatum by high doses of caffeine (Svenningsson et al., 1997a). Two of these, neurotensin/neuromedin and Substance P, are dependent on a rise in c-Fos as evidenced by the effect of a specific antisense oligonucleotide. By contrast, mRNA for dynorphin and enkephalin, were unaffected by blocking c-Fos increases, suggesting that other transcription factors are more important. Cyclic AMP response element-binding protein (CREB) is a likely candidate.

G. Low Doses of Caffeine Selectively Decrease the Activity of Striatopallidal Neurons in the Striatum and Their Counterparts in the Nucleus Accumbens

The high doses of caffeine used in these previous studies lead to a behavioral depression in experimental animals (see Daly, 1993

Two recent studies examined the expression of mRNA for NGFI-A and NGFI-B in an attempt to reveal effects of low, behaviorally relevant doses of caffeine (Svenningsson et al., 1995a, 1997c). They showed that lower doses of caffeine (7.5-25 mg/kg) decrease the expression of mRNA for NGFI-A and NGFI-B in striatum (see Fig. 5). Indeed, the effect seen at the lowest dose was almost 75% of that maximally observed, suggesting that the threshold effect may be on the order of a few milligrams per kilogram. This may be the first evidence for direct neurochemical changes induced by such low, clearly stimulant doses of caffeine. As noted above, the only known biochemical action of caffeine, in the concentrations reached following administration of doses similar to those attained during normal human caffeine consumption, is blockade of adenosine receptors. Because the effect was most clear-cut in the striatum, where A2A receptors are abundant, the data suggest that antagonism at adenosine A2A receptors plays an important role in mediating the effects of caffeine. This is further supported by the finding that the caffeine-induced changes are located specifically to the striatopallidal neurons, which express A2A receptors in high abundance. It has also been shown that the effect of a low dose of caffeine can be mimicked by the selective adenosine A2A receptor antagonist SCH 58261, but not by the selective adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; Fig. 5) (Svenningsson et al., 1997c).

There is a parallelism between caffeine (or SCH 58261)-induced increase in locomotion and a decrease in the expression of the mRNA for some IEGs in the striatum (Svenningsson et al., 1995a, 1997c). The parallelism does not necessarily imply a direct causal relationship. Clearly the fall in mRNA cannot be the cause of the altered motor behavior since the latter occurred very rapidly. Conversely, the alteration in locomotor behavior is unlikely to cause the change in mRNA expression, because other drugs such as amphetamine cause an increase in locomotor behavior and an increase in the expression of mRNA for NGFI-A (Svenningsson et al., 1995a). The possibility exists, however, that the parallelism may be due to the fact that a single mechanism is the cause of a change both in mRNA and in locomotion after caffeine.

There is reason to believe that a reduction of intracellular levels of cyclic AMP is important for the observed decrease in the expression of the IEGs, because both NGFI-A and NGFI-B have CRE-like binding sites in their 5' flanking sequence (Watson and Milbrandt, 1989; Sheng and Greenberg, 1990). Adenosine A2A receptors are coupled to G-proteins that activate adenylyl cyclase. By antagonizing the actions of adenosine at these receptors, caffeine would decrease intracellular cyclic AMP levels. Dopamine D2 receptors are coupled to Gi-proteins, and decrease the levels of cyclic AMP. It should be pointed out that a D2 agonist will be able to depress cyclic AMP formation only if there is a high basal rate of cyclic AMP generation. Adenosine acting on A2A receptors is a probable mediator of such basal cyclic AMP generation (see Fig. 4).

These considerations focus the attention on the cyclic AMP system in the basal ganglia. Indeed, there is evidence that cAMP-dependent protein kinase is very important in the acquisition of cocaine self-administration and also in relapse into cocaine-seeking behavior (Self et al., 1998). As illustrated in Fig. 4, cAMP formation is stimulated via D1 receptors in the GABAergic neurons of the direct pathway, whereas adenosine A2A receptors mediate the important cAMP-raising signal in the neurons of the indirect pathway. Additional support for this scheme has recently been provided by the observation that D1 agonists and A2A agonists cause additive effects on striatal cAMP and on cAMP-dependent phosphorylation of DARPP-32 (Svenningsson et al., 1998a).

Bidirectional changes in gene expression following low and high doses of caffeine were also found for jun-B. The basal expression of jun-B is known to be relatively high in striatum (Mellstrom et al., 1991), and it has been reported that the expression of this IEG increases markedly following administration of a high dose of caffeine (Svenningsson et al., 1995b). Interestingly, it has been reported that cyclic AMP can regulate also the expression of jun-B (de Groot et al., 1991).

A working hypothesis is illustrated in Fig. 4. It is assumed that the level of cyclic AMP is important to determine the expression of mRNA for NGFI-A, NGFI-B, and Jun B in striatopallidal neurons. It is further assumed that the rate of cyclic AMP production is importantly controlled by adenosine, acting on A2A receptors to stimulate adenylyl cyclase, and by dopamine, acting on D2 receptors to inhibit the enzyme. In agreement with this basic hypothesis, the D2 receptor agonist quinpirole was found to induce a marked reduction of the expression of mRNA for NGFI-A and NGFI-B. Quinpirole does not alter the expression of c-fos (Paul et al., 1992) unless c-fos expression is enhanced, e.g., by reserpine treatment (Cole and Di Figlia, 1994). Caffeine (7.5-25 mg/kg) had an effect of equal magnitude, and its effect was not clearly additive to that of quinpirole. Because the effect of caffeine was confined to the striatopallidal neurons, the data suggest that these neurons are the target also for quinpirole.

It is known that neuroleptic drugs with D2 antagonistic properties cause a rapid and transient increase in IEG expression; this effect has been attributed to a removal of an inhibitory D2 receptor tone (Robertson et al., 1992; Merchant and Dorsa, 1993). In one study (Svenningsson et al., 1998b), haloperidol was given with or without caffeine and the animals were sacrificed 30 min later. Under these circumstances the expected increase in IEGs was observed after the D2 antagonist. The effect of the D2 antagonist was reduced by caffeine in the dorsomedial striatum and nucleus accumbens, but it was increased in the caudal part of striatum (Svenningsson et al., 1998b). This suggests that caffeine not only acts on the basal ganglia neurons but also affects the striatal inputs. In another study (Svenningsson et al., 1995a) the D2 receptor antagonist raclopride was given 4 h before sacrifice, and then there was no significant effect of the antagonist per se, probably because IEG levels had returned to control by this time. Furthermore, raclopride did not inhibit the depressant effect of caffeine given simultaneously with raclopride (Svenningsson et al., 1995a). These findings can be explained if adenosine and dopamine are both tonically active at their respective receptors. Thus, when dopamine receptors are blocked with raclopride, the stimulatory effect of adenosine is unhampered, leading to a transient increase in the IEG expression; when the adenosine receptors are also blocked, gene expression is brought down to essentially normal levels. The finding is less easy to explain if it is assumed that the major effect of adenosine A2A receptor stimulation is to regulate signaling via the D2 receptors. Thus, we have to assume that adenosine plays an important role in regulating gene expression in striatopallidal neurons that is independent of its established ability to influence the affinity of dopamine as an agonist at D2 receptors (Ferré et al., 1992). The scheme in Fig. 4 also indicates that GABA release in the pallidum may be regulated by adenosine and dopamine in opposite directions, and as noted above, studies of GABA release support this proposal.

Given that adenosine acting on A2A receptors is expected to increase the release of GABA in globus pallidus, caffeine is expected to decrease it. As a consequence of the decreased release of the inhibitory transmitter, caffeine is then also expected to increase activity in this brain area. This contention has been borne out in studies examining the expression of IEGs in globus pallidus following caffeine or selective adenosine A2A receptor antagonists (Le Moine et al., 1997; Svenningsson and Fredholm, 1997). Furthermore, there are important synergistic effects of adenosine A2A receptor antagonism and stimulation of dopamine D1 receptors (Pinna et al., 1996; Le Moine et al., 1997). It is also of potential relevance that the human adenosine A2A receptor gene has been linked to a potential schizophrenia locus on chromosome 22 (Deckert et al., 1997). If this tentative identification holds up, the link between adenosine and dopamine-related functions would be strengthened.

The link between adenosine A2A receptors and dopamine-related effects in the striatum is further supported by the finding that a selective adenosine A2A receptor agonist, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N- ethylcarboxamido adenosine (APEC), can antagonize the motor stimulant effects of amphetamine. The A2A agonist also reduced the effects of amphetamine on c-Fos in nucleus accumbens core and shell (Turgeon et al., 1996). These authors also demonstrated effects of an A1 receptor agonist and thus supported several previous reports that not only A2A but also A1 receptors are important in the regulation of striatal function (Ferré et al., 1997). Indeed, blockade of adenosine A1 receptors has been shown to potentiate the motor stimulation afforded by a dopamine D1 receptor agonist (Popoli et al., 1996b). Conversely, stimulation of A1 receptors blocks the EEG arousal afforded by D1 receptor stimulation (Popoli et al., 1996a).

). Therefore the induction of IEG expression might reflect behavioral depression rather than the behavioral stimulation that is the basis for the widespread human use of caffeine. It is known that the basal expression of mRNA for NGFI-A (Milbrandt, 1987) and NGFI-B (Milbrandt, 1988) is relatively high in striatum (Watson and Milbrandt, 1990; Schlingensiepen et al., 1991; Worley et al., 1991; Bhat et al., 1992). Several studies have shown that the striatal levels of NGFI-A mRNA can be regulated via dopaminergic transmission and an increase in the expression of the gene is seen following treatment with D1 agonists, D2 antagonists, and indirect dopamine agonists like cocaine and amphetamine (Cole et al., 1992; Moratalla et al., 1992; Nguyen et al., 1992). In addition, it has been reported that a significant reduction of NGFI-A mRNA occurs following chronic treatment with cocaine (Bhat et al., 1992).). However, the increase does not become apparent until caffeine doses exceed 50 mg/kg, i.e., doses clearly higher than those required to elicit behavioral stimulation (see below). This could mean that the caffeine-induced increase in immediate early genes is related to the second phase of caffeine action, which involves a behavioral depression. Alternatively, the dose-response relationship could indicate that substantially higher concentrations are required to observe a generalized c-fos increase than are needed to activate a sufficient number of neurons to produce a behavioral stimulation. Some support for the latter contention is provided by the finding that other central stimulants, including amphetamine and cocaine, have to be given in very much higher concentrations to induce c-fos than to cause behavioral stimulation.), but not antagonist binding affinity. This type of change mimics that observed following the addition of sodium ions. However, the effect of the A2A receptor agonist was at least as large in the presence as in the absence of sodium ions. The interaction could not be observed when high levels of dopamine D2 and adenosine A2A receptors were transiently expressed in Cos-7 cells (Snaprud et al., 1994). In these cells the receptors were not functionally coupled to an effector response. This suggests that the interaction may not occur between the receptor molecules only but that some interactions with other membrane components are also necessary. Conversely, in fibroblasts stably transfected with both A2A and D2 receptors there was a clear-cut interaction at the binding level despite the fact that the A2A receptors in these cells were very poorly coupled to adenylyl cyclase (Dasgupta et al., 1996). This indicates that the interaction at the level of binding does not require the full effector response. The latter contention is also supported by the fact that the binding studies were conducted on broken cell preparations and under conditions when adenylyl cyclase activity and protein phosphorylation can be expected to proceed at negligible rates. More recently this binding interaction was observed in Chinese hamster ovary cells cotransfected with A2A and D2 receptors, and in this cell type there were also very clear-cut interactions at the second messenger level and beyond.; Fredholm and Dunwiddie, 1988). There is some evidence that the release of excitatory transmitters is more strongly inhibited by adenosine than that of inhibitory neurotransmitters (Fredholm and Dunwiddie, 1988). This would be in keeping with a proposed role of adenosine as a homeostatic regulatory factor that serves to match the rate of energy consumption to the rate of substrate supply. The receptors involved are similar to adenosine A1 receptors.; Fastbom et al., 1987). Only moderate levels are found in caudate-putamen and nucleus accumbens. The corresponding mRNA shows a somewhat different distribution (Mahan et al., 1991; Reppert et al., 1991), indicating that some of these receptors are located on nerve terminals rather than cell bodies (Johansson et al., 1993a). Indeed, the presence of presynaptic adenosine A1 receptors mediating inhibition of transmitter release has been demonstrated on virtually all types of neurons [for review see Fredholm and Dunwiddie (1988)]. In the caudate-putamen, adenosine A1 receptor mRNA was found to be present, albeit in low abundance, on all the major types of neurons (Ferré et al., 1996).; Table 3). Of these subtypes, the rat A3 receptor was originally shown to be but little affected by many methylxanthines, including caffeine. In humans, the A3 receptor is blocked by caffeine with a KD of close to 80 µM. Therefore, this receptor is not the best target for caffeine actions in humans. The A2B receptor has been shown to require higher concentrations of adenosine for activation than those found in resting animal tissues. Thus, inhibition of adenosine actions at this receptor is similarly unlikely to provide an explanation for the actions of caffeine under physiological conditions. Under pathophysiological conditions, however, A2B receptors are likely to be activated by endogenous adenosine and caffeine may then very well act also on these receptors. ). The Km for adenosine is well above 5 µM and adenosine deaminase is therefore of particular importance when adenosine levels are high (Arch and Newsholme, 1978). Adenosine kinase, by contrast, has a Km level in the range of physiological intracellular adenosine concentrations. Indeed, blockade of adenosine kinase has a much larger effect on the rate of adenosine release than does blockade of adenosine deaminase (Lloyd and Fredholm, 1995). Another enzyme of importance is S-adenosylhomocysteine hydrolase. This enzyme sets the equilibrium between S-adenosylhomocysteine and adenosine + L-homocysteine. When the level of the amino acid is low, this enzyme serves to generate adenosine. On the other hand, when the level of L-homocysteine is raised, it can trap adenosine formed via AMP breakdown as S-adenosylhomocysteine inside the cell. This reaction has been used to demonstrate that the bulk of the adenosine formed by energy deprivation or electrical field stimulation in hippocampal slices is formed intra- rather than extracellularly (Lloyd et al., 1993).1). This tends to rule out the direct release of intracellular calcium [probably via an action on ryanodine receptors (McPherson et al., 1991)], which occurs only at millimolar concentrations. Also the inhibition of cyclic nucleotide phosphodiesterases (Smellie et al., 1979; see Fredholm, 1980; Nehlig and Debry, 1994) occurs at rather higher concentrations than those attained during human caffeine consumption. Xanthines can influence 5'-nucleotidase and alkaline phosphatase, but these actions are also exerted only at millimolar concentrations (Fredholm et al., 1978; Fredholm and Lindgren, 1983). In fact, the only known mechanism that is significantly affected by the relevant doses of caffeine is binding to adenosine receptors and antagonism of the actions of agonists at these receptors (see Fredholm, 1980, 1995). Thus, in the remainder of this section, adenosine receptor antagonism is taken to be the mechanism of action of caffeine even though there are data, especially from behavioral experiments, that could be interpreted as evidence for some other, as yet unidentified mechanism of action (see, e.g., Garrett and Holtzman, 1995).

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