Chronic theophylline treatment in vivo increases high affinity adenosine A1 receptor binding and sensitivity to exogenous adenosine in the in vitro hippocampal slice

Brain Research, 542 (1991) 55-62 Elsevier

55

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Chronic theophylline treatment in vivo increases high affinity adenosine A1 receptor binding and sensitivity to exogenous adenosine in the in vitro hippocampal slice Carl R. Lupica 1, Michael F. Jarvis 2'* and Robert F. Berman 1 1Wayne State University, Departmentof Psychology, Biopsychology Laboratory, Detroit, MI 48202 (U.S.A.)and 2ResearchDepartment, Pharmaceuticals Division, C1BA-GEIGY Corporation, Summit, NJ 07901 (U.S.A.) (Accepted 4 September 1990) Key words: Electrophysiology; Adenosine A 1 receptor; Up-regulation; Theophylline; Hippocampus; Cyclohexyladenosine

The present investigation examined the effects of chronic treatment with the adenosine receptor antagonist theophylline in vivo, on in vitro hippocampal electrophysiology and adenosine A1 receptor binding in the same animals. Adult rats were injected once daily (i.p.) with theophylline for 1 week at 75 mg/kg, followed by an additional week at 100 mg/kg, or with saline for the same 2-week period. Two days following the last injection, hippocampal slices were prepared and population spikes recorded from the pyramidal cell layer of area CAI were elicited by Schaffer collateral-commissural fiber stimulation. The degree of inhibition caused by superfused adenosine was compared between hippocampal slices from theophylline- and saline-treated rats. Tissue from the contralateral hippocampus was used in [3H]cyclohexyladenosine ([3H]CHA) receptor binding. Hippocampi from theophylline-treated animals showed a significantly greater number of [3H]CHA binding sites (apparent Bmax; 125% of control, P < 0.05), without a significant change in binding affinity, and were more sensitive than controls to the inhibitory effects of adenosine on the population spike response. These results suggest that chronic adenosine receptor antagonism results in the up-regulation of adenosine A1 receptors which are functional and physiologically relevant in the in vitro hippocampus, and further supports the hypothesis that methylxanthine tolerance is mediated, at least in part, by an increase in adenosine receptor density.

INTRODUCTION

receptors in the hippocampal formation are located in the stratum radiatum and stratum oriens of CA1 and CA322"

Adenosine has been proposed as a neuromodulator due to its potent effects at inhibiting synaptic transmission in the central nervous system 25. This proposal is also supported by the observation that adenosine exerts a tonic inhibitory influence upon CNS activity which can be uncovered in the presence of competitive antagonists (e.g. methylxanthines) and catabolic enzymes (e.g. adenosine deaminase) 12'15. A t least two subtypes of extracellular adenosine receptors exist, as defined by radioligand binding and actions on cyclic adenosine 3"-5"-monophosphate (cAMP) formation. Adenosine acts at the A1 receptor subtype to inhibit the formation of c A M P at nanomolar concentrations 1°,32,53, while the A2 receptor mediates the accumulation of c A M P at micromolar concentrations of adenosine. Receptor binding studies reveal that [3H]cyclohexyladenosine ([3H]CHA) specifically labels A1 receptors throughout the brain 23'27'31, with the hippocampal formation containing one of the highest densities of A1 binding sites 23. Most of the A1

23,27

Adenosine inhibits field excitatory postsynaptic potentials (EPSPs) and population spikes in the in vitro hippocampus 14'41"49, and potency comparisons among adenosine agonists are consistent with adenosine interacting with A1 receptors in this structure 13'34'43. Methylxanthines (e.g. caffeine and theophylline) are competitive antagonists at A1 and A2 adenosine receptors 46'53, inhibiting adenosine-mediated actions on c A M P accumulation and radioligand binding. Consonant with adenosine receptor antagonism, methylxanthines have been hypothesized to enhance neural activity resulting in behavioral stimulation. This hypothesis is supported by studies showing a high degree of correspondence among the abilities of methylxanthines to enhance locomotor activity 51, block the effects of behaviorally depressant doses of adenosine agonists 4, and compete for [3H]CHA binding sites 2s. Chronic treatment with caffeine or theophylline in-

* Present address: Rorer Central Research, King of Prussia, PA 19406, U.S.A. Correspondence: C.R. Lupica. Present address: University of Colorado Health Sciences Center, Department of Pharmacology C236, 4200 E. 9th Ave., Denver, CO 80262, U.S.A.

56 c r e a s e s t h e n u m b e r of b r a i n a d e n o s i n e A1 b i n d i n g sites (i.e. 1 0 - 3 5 % i n c r e a s e s in a p p a r e n t B . . . . ) in m a n y n e u r a l regions 6"9"19'24"35"37"39"42"52.H o w e v e r , m e t h y i x a n t h i n e - i n d u c e d u p - r e g u l a t i o n o f A 1 b i n d i n g d o e s n o t o c c u r in all brain areas, possibly reflecting heterogeneous sensitivity of t h e b r a i n to t h e e f f e c t s o f m e t h y l x a n t h i n e s . T h i s c o u l d b e d u e to d i f f e r e n t i a l access o f t h e s e c o m p o u n d s to b r a i n regions,

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MATERIALS AND METHODS

Subjects Male Long Evans rats, weighing 125-300 g, purchased from Harlan Laboratories (Indianapolis, IN) were used in these experiments. These animals were housed in a vivarium in which a 12-h light/dark cycle was maintained (lights on at 07.00 h), and had ad libitum access to food and water through the drug administration procedure.

Chronic drug administration protocol Twenty-six animals received theophylline (1,3-dimethylxanthine; Sigma, St. Louis, MO) dissolved in 0.9% NaCI. Due to the relative insolubility of high concentrations of theophylline at room temperature, the solution was warmed to 37 °C prior to each i.p. administration. The drug was administered in one daily dose (given between 12.00 and 14.00 h) at 75 mg/kg for 1 week followed by 100 mg/kg/day for an additional week. Twenty-three control subjects received the same volume of 37 °C saline for the same period of time as the drug injected animals.

Receptor binding Tissue homogenate preparation. Hippoeampal tissue from the hemisphere opposite that used in electrophysiological recordings and any tissue remaining after slice preparation (see below) were analyzed for adenosine receptor density and affinity using Scatchard-Rosenthal plots 44'47. The hippocampal tissue was homogenized in 20 vols. of ice-cold 50 mM Tris HC1 buffer, pH 7.4 at 25 °C. The tissue was then centrifuged at 48,000 g for 10 min at 4 °C. The supernatant was discarded and the pellet resuspended in buffer to give a concentration of approximately 20 mg/ml. To this suspension 2 IU/ml adenosine deaminase was added and the homogenate incubated at 37 °C for 30 rain. This homogenate was then centrifuged at 48,000 g for 10 min at 4 °C and the resulting pellet resuspended in buffer to a final tissue concentration of approximately 4 mg/ml. [3H]CHA binding. Saturation studies were conducted to determine the binding affinity (Kd) and density (Bmax) of [3H]CHA (spec. act. 27-35 Ci/mmol, Dupont-NEN, Boston, MA) binding sites in hippocampal membranes from individual animals (n =49). Membranes were incubated with 5 concentrations of [3H]CHA ranging from 0.1 to 30 nM. Non-specific binding was determined in the

presence of 2(I/~M 2-chloroadenosine (2-CADO). Binding reactions were conducted in 500 itl final volumes, initiated by the addition of 250 ,ul membrane suspension (150-200 ,ug protein/ml), and incubated at 25 °C for 2 h. The reaction was terminated by vacuum filtration through Whatman GF/B glass fibers under reduced pressure in a Brandell cell harvester (Gathersburg, MD). The filters were washed 2 x 5 ml with ice-cold buffer, placed in scintillation vials, cocktail added and bound radioactivity determined by standard liquid scintillation spectrometry. Protein concentrations were determined by the method of Bradford 7 using bovine serum albumin (BSA) as the standard.

Electrophysiology Hippocampal slice preparation and recording. A modified T.C. Earle's balanced salt solution (Sigma, St. Louis, MO) was used as artificial cerebrospinal fluid (ACSF) throughout the physiological portion of these experiments. ACSF was prepared daily and contained the following (in mM): dextrose 10, NaC1 120, KCI 5.4, CaCI 2 2.5, MgSO 4 0.83, NaH2PO 4, 0.9, NaHCO 3 21; at pH 7.4. Stock solutions of adenosine (Sigma) were prepared approximately once per week in Earle's solution at 100x the final desired concentration. Two days following the last injection subjects were decapitated, the brains removed using chilled surgical instruments and rinsed with ice-cold ACSK that had been perfused with a 95% Oz-5% CO2 mixture. Transverse bippocampal sections (400/~m nominal thickness) were prepared using a tissue Chopper (Stoelting, Chicago, IL), and transferred within 5 min to a recording chamber maintained at 33 °C using a DC, proportional temperature regulation unit (Frederick Haer, New Brunswick, ME). Slices were taken only from dorsal hippocampus due to the differential distribution of adenosine receptors and excitability in dorsal versus ventral hippocampus 2~'29. Hippocampal tissue from the opposite hemisphere and any remaining after slice preparation was dissected, frozen on dry ice and stored at -70 °C until the receptor binding assays were performed (up to 3 months). Hippocampal slices from 14 subjects (7 theophylline-treated and 7 control) were recorded at a liquid medium-gas interface to determine the effects of chronic theophylline treatment on paired pulse inhibition. The remaining slices were not perfused until they were to be tested, and were thus maintained at a liquid medium-gas interface. When testing procedures were begun these slices were submerged and a constant flow of fresh oxygenated (95% 02-5% CO2) medium was initiated at a rate of 3 ml/min. Electrophysiological recordings were made with 2-3 Mg2 glass microelectrodes filled with 3 M NaCI, placed in stratum pyramidale of the CA1 region under visual guidance. Sehaffer collateralcommissural fibers were electrically stimulated using a concentric, bipolar, tungsten microelectrode (75 /~m o.d.) placed in stratum radiatum of area CA3. Monophasic 0.1 ms pulses of 6-30 V were delivered to the synaptic pathway at 90 s intervals and the voltage adjusted to evoke a population spike of 1-2 mV amplitude. The tips of recording and stimulating electrodes were initially placed on the surface of the slice, which was then evaluated for the presence of paired-pulse inhibition (at a 20 ms interpulse interval) and a stable response over a 15-min period; these two criteria were used for the selection of viable slices and electrode positions. Once an acceptable response was found, the tip of the recording electrode was advanced in 5 /~m increments, with a hydraulic microdrive (David Kopf Instruments, Tejunga, CA), to a depth (between 65 and 210 #m) at which a maximal response could be observed at a given stimulus intensity. Electrophysiological signals were amplified 1000x and filtered at 10 and 10,000 Hz using a differential amplifier (DAM-50, World Precision Instruments, New Haven, CT). Signals were monitored, stored on floppy disk and analyzed with a Nicolet (Madison, WI) model 4094C digital oscilloscope. Fig. 2.1 shows a representative evoked response, and graphically demonstrates how each potential was measured. After the stimulus intensity was set to evoke a stable response slices were evaluated for paired pulse inhibition (PPI) by stimulating

57 twice in rapid succession through a single stimulating electrode located in stratum radiatum. Interpulse intervals (IPI) were 10, 20, 30, 50, 100 and 400 ms in duration, and paired-pulses were delivered every 90 s until the paired-pulse sequence was completed. Five responses at each interpulse interval were averaged to construct paired-pulse curves prior to and during adenosine superfusion. After the paired-pulse sequence a stable baseline was recorded and adenosine superfusion initiated by switching to a media bottle that contained the drug at 5, 11.2 or 25/~M. After establishment of a stable response to adenosine ( - 5 min), the stimulus intensity was reset to provide a population spike that was similar in amplitude to the control response, and PPI evaluated in the presence of adenosine. Once the paired-pulse sequence was completed in adenosine, washout was initiated by switching to an adenosine-free media bottle. Any slice not showing /> 85% recovery from adenosine was not used in any of the subsequent analyses. Data analysis. T h e degree of inhibition during adenosine superfusion at successive 90-s intervals, determined as a proportion of I> 5 min of averaged control responses prior to adenosine superfusion, was compared across chronic treatment groups using a repeated measures analysis of covariance (ANCOVA, BMDP-2V), with the baseline response used as a covariate. The effects of adenosine on PPI were determined by dividing the second evoked response (P2) by the first (P1), at each interstimulus interval before and during adenosine perfusion. These proportion scores were analyzed using a repeated measures ANOVA (BMDP-2V). Data from the binding experiments were analyzed and plotted using the iterative curve fitting program LIGAND 36.

similar ( - 80%) reduction of the population spikes in both experimental and control slices (Figs. 2.4 and 3). Thus, although the inhibitory action of adenosine per se upon population spikes was found to be significant at all concentrations, chronic theophylline treatment did not result in any additional sensitivity to adenosine at 25/~M. Paired-pulse inhibition refers to the observation that the second population spike evoked by a pair of closely timed afferent pulses (< 50 ms) is typically smaller than

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Electrophysiology Slices from theophylline-treated subjects were more sensitive than control slices to the inhibitory effects of adenosine on population spikes at the 5 (n = 5) and 11.2 /~M (n = 8) concentrations (F(L32) = 11.1, P < 0.01), as shown in Fig. 2.2 and 2.3. At 5/~M adenosine caused a maximal 40% decrease in population spike amplitude in slices obtained from theophylline-treated animals and a 27% reduction in population spike size in control slices (Fig. 3). Similarly, 11.2/~M adenosine resulted in a 78% maximal response decrease in slices from theophyllinetreated animals while responses from control slices exhibited a 52% suppression of this response at the same concentration (Fig. 3). At 25 /~M adenosine caused a

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[3H]CHA binding Saturation experiments revealed that chronic theophylline treatment resulted in a significantly greater density of [3H]CHA binding sites in hippocampal membranes as compared to controls (Bma x _ 1 S.E.M. = 485 + 26 fmol/mg protein and 387 + 35 fmol/mg protein, respectively; P < 0.05), with no change in binding affinity (K d + 1 S.E.M. = 1.0 + 0.1 and 1.1 + 0.2 nM, respectively). Representative Scatchard-Rosenthal plots demonstrating a greater number of [3H]CHA binding sites in the theophylline-treated animals as compared to the control animals are shown in Fig. la, while the average change in Bm~~ values is shown in Fig. lb.

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Fig. 1. Effects of 2 weeks treatment with theophylline on hippocampal [3H]CHA binding, a: representative Scatchard analyses from a theophylline-(Theo) treated animal and a control animal treated with saline. Although Bin,x values are higher than the average in both controls and theophylline-treated hippocampi, this figure shows that this treatment caused an increase in apparent B,,ax without altering K d over a 0.1-30 nM range of [3H]CHA concentrations. Specific binding was determined in the presence of 20 gM 2-chloroadenosine. b: average [3H]CHA Bma~ values in hippocampal homogenates taken from animals treated with theophylline (n = 26) or saline (n = 23). Chronic theophylline treatment caused a significant 25% increase in the amount of specifically bound [3H]CHA (P < 0.05, t-test) without a change in binding affinity (mean K u = 1.0 + 0.1 and 1.1 + 0.2 nM, respectively).

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Fig. 2.1: a representative population spike recorded from stratum pyramidale of the CA1 layer illustrating how these potentials were measured. Line A delineates the maxima of the evoked extracellular EPSP following the negatively deflecting population spike, line B delineates the maxima of the EPSP prior to the population spike. The difference between A and B was halved (marked by the broken line), and the absolute value between this and the maximum negative deflection of the population spike (marked by line C) taken as the population spike amplitude. The longer arrow indicates the amplitude measured by this method, the smaller arrow indicates the stimulus artifact. 2, 3 and 4: averaged chronological records showing the effect of superfused adenosine on population spike amplitude in hippocampal slices taken from chronically theophyiline-(Theo) and saline-treated animals. Each point represents a mean percent (+ S.E.M.) change from baseline. The solid bars represent the periods during which adenosine was superfused at the indicated concentration. Slices taken from theophylline-treated animals were significantly more sensitive to the inhibitory effects of adenosine at 5/~M (n = 5 theophylline-treated and 5 controls, respectively), and 11.2/~M (n = 8 and 11). but not 25/~M (n = 7 and 9; P < 0.01, repeated measures ANCOVA).

the response evoked by the first pulse. This is thought to reflect the activation of recurrent inhibitory circuitry, and the strength of this inhibitory circuitry 2'3. In slices maintained in the interface condition, inhibition of the second population spike was seen at 10, 20 and 30 ms IPIs (77, 70 and 36% inhibition, respectively), while facilitation of the second response was seen beginning at the 50-ms IPI (Fig. 4a). Analysis of chronic treatment effects on PPI in the interface condition revealed a highly significant effect of IPI on the degree of inhibition of P2 (Fig. 4a) (F(5,6o) = 45.8, P < 0.001). However, no significant difference between chronic treatment groups

on this measure was detected. Based upon the observation of Lee and Schubert 3° that bath application of adenosine reduces the degree to which the second response is suppressed, we hypothesized that hippocampal slices taken from both chronic theophylline- and saline-exposed animals would exhibit less inhibition of P2 during adenosine superfusion. We also hypothesized that slices taken from theophylline-treated subjects would exhibit less inhibition of P2 during adenosine superfusion than controls. No significant differences between slices from chronically saline- or theophylline-treated animals were detected on this measure, though adenosine did

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significantly diminish the degree of inhibition at 25 and 11.2/~M, but not 5/~M (Fig. 4b; ( F ( 1 , 7 ) . = 9.7, P < 0.01; F(1.zz) = 11.1, P < 0.01, respectively).

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The present study d e m o n s t r a t e s that chronic treatment with the adenosine r e c e p t o r antagonist theophylline in vivo can result in a significant increase in [3H]CHA binding and a greater sensitivity of h i p p o c a m p a l pyramidal cells to exogenous adenosine in vitro. These results thus support the hypothesis that chronic methylxanthineinduced up-regulation of adenosine A1 receptors confers greater sensitivity to adenosine and suggests that these additional r e c e p t o r sites are functional and physiologically significant. This differential sensitivity to adenosine was o b s e r v e d at 5 and 11.2/~M but not at 25/~M. This m a y reflect the presence of spare receptors, such that a maximal physiological response is observed with less than maximal r e c e p t o r occupancy, or the limited capacity of r e c e p t o r coupling to the intracellular signal transducer. Potency comparisons among agonists implicate the A1 adenosine r e c e p t o r subtype in the depression of synaptic transmission in the hippocampus, and as the site which is labeled by [3H]CHA13'43,ss. The observed increased sensitivity to adenosine in h i p p o c a m p a l slices taken from t h e o p h y l l i n e - t r e a t e d subjects in the present study is also consistent with adenosine's action at the A1 receptor. Chronic effects of theophylline upon c A M P levels and cyclic nucleotide p h o s p h o d i e s t e r a s e can be discounted as alternative hypotheses for the o b s e r v e d increase in adenosine sensitivity, since c A M P alterations are not correlated with electrophysiological responses to aden-

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INTERPULSE INTERVAL (msec) Fig. 4. a: chronic theophylline effects on paired-pulse responses, as a function of interpulse interval, recorded in stratum pyramidale of area CA1. Slices from controls (Saline; n = 7) and theophyllinetreated (Theo; n = 7) animals were recorded from while at the interface of artificial cerebrospinal fluid and a warmed oxygenated atmosphere. Each symbol represents the mean proportion of the second population spike (P2) to the first (P1) in a paired pulse sequence. There were no significant differences between slices from theophyiline- and saline-treated animals in this condition, b: comparison of paired-pulse curves prior to (Pre-adenosine), and during superfusion with 5, 11.2 and 25 juM adenosine. Since there was no significant shift in paired pulse curves as a result of chronic theophylline treatment, the data were collapsed across chronic theophylline and saline treatment groups. Statistical analysis showed that there was a significant shift in these paired-pulse curves at the 11.2 and 25/~M concentrations, but not at the 5/~M concentration of adenosine (**, P < 0.001; *, P < 0.01; 11.2 and 25/~M adenosine concentrations, respectively, vs Pre-adenosine baseline; repeated measures ANOVA). The 200- and 400-ms interpulse were omitted in b for clarity.

osine 13'5°. F u r t h e r m o r e , since chronic methylxanthine t r e a t m e n t does not alter binding o f the adenosine uptake site label [3H]nitrobenzylthioinosine35, theophylline-induced downregulation of this site is not a tenable

60 explanation for the observed effect. Thus, we favor the hypothesis that the observed increase in [3H]CHA binding found in this study represents an increase in AI receptor density in hippocampus that underlies the enhanced sensitivity to adenosine. Closely paired suprathreshold activation of afferent fibers in the CA1 region of the hippocampal slice can lead to a marked reduction in the size of the secondary response which is thought to reflect the strength of recurrent inhibition 2'3'~'~6. Thus, an increase in the size of the second response as a result of drug action can be interpreted as reflecting a decrease in recurrent inhibition. No differences in PPI were detected between slices taken from chronic theophylline animals or controls tested in the interface condition, suggesting that chronic theophyiline treatment did not merely result in some more general physiological deficit. A significant, concentration-dependent, decrease in inhibition of the second population spike in a paired pulse sequence was seen in the presence of adenosine, which agrees with the finding of Lee and Schubert 3° in hippocampus, and Scholfield and Steep s in olfactory cortex. However, there was no differential effect of adenosine on the reduction of inhibition between slices from chronic theophylline animals or controls. The absence of a chronic theophylline treatment effect on PPI, and the observation that this treatment resulted in a greater sensitivity of unpaired responses to adenosine, suggests that while recurrent inhibition may be reduced by adenosine, the adenosine receptors which mediate this response (presumably located on interneurons) may be less sensitive to the chronic effects of theophylline than those responsible for unpaired adenosine-mediated inhibition. The localization of [3H]CHA binding sites to interneurons 38 and pyramidal cells in the hippocampal formation, suggests that adenosine receptors involved in these separate responses may indeed be associated with different neural elements. Furthermore, adenosine is known to decrease potassiumstimulated release of preloaded [3H]GABA in cortical and striatal slices 25'26, and can decrease G A B A turnover in hippocampus 56, although this effect has been contested in dentate gyrus lj. Tolerance and cross-tolerance following chronic methylxanthine treatment has been reported in a variety of biological systems. An increase in hypotension to enREFERENCES 1 Ahlijanian, M.K. and Takemori, A.E., Cross tolerance studies between caffeine and N-6-L-phenylisopropyladenosine (PIA) in mice, Life Sci., 38 (1986) 577-588. 2 Andersen, P., Eccles, J.C. and Loyning, Y., Pathway of postsynaptic inhibition in the hippocampus,J. Neurophysiol., 27 (1964) 608-619. 3 Andersen, P., Gross, G.N., Lomo, T. and Sveen, O,, Partici-

dogenous adenosine can be seen following extended caffeine treatment 54, and prolonged administration of caffeine can diminish the potentiation of locomotor behavior seen with acute treatment is, as well as its ability to attenuate adenosine agonist induced analgesia 1. Also, acute intravenous administration of caffeine can elevate the spontaneous firing rates of midbrain reticular neurons in vivo, which is eliminated with chronic exposure9; an effect that is correlated with an increase in [3H]CHA binding. Studies examining the biochemical effects of chronic adenosine receptor antagonism have shown that R-PIA induced inhibition of adenylate cyclase was increased by 35% 24, and Fredholm et al. 2° have reported that chronic theophylline treatment leads to a potentiated ability of adenosine to inhibit noradrenaline release. More recent studies have shown that methylxanthineinduced adenosine receptor up-regulation is positively correlated with a shift in the sensitivity to chemoconvulsants in rats 4°'52, and alters postictal behavior following electrically induced tonic-clonic seizures 5'33. In addition, chronic caffeine treatment can protect against hippocampal damage produced by ischemia, which parallels a 17% up-regulation of [3H]CHA binding 45. It should be noted that these neuroprotective and anticonvulsant effects of chronic methylxanthine administration mimic the effects of acute adenosine agonist administration. The combined results of these studies and the present investigation suggest that chronic methylxanthine exposure can lead to significant biochemical and behavioral changes which seem to be mediated by an increase in the number of inhibitory adenosine receptors, and that adenosine A1 receptor upregulation may therefore be intimately involved in tolerance and cross-tolerance observed in several biological systems. Also, this observed association between increased adenosine A1 receptor binding and sensitivity to adenosine suggests that receptor increases may represent one homeostatic mechanism utilized by neural systems to maintain inhibitory tone in the presence of receptor antagonists that may be relevant to other neurotransmitter systems as well.

Acknowledgements. This work was supported by a fellowship awarded to C.R.L. by the Wayne State University Interdisciplinary Neuroscience Program and by NIH Grant RR-08167.

pation of inhibitory and excitatory interneurons in the control of hippocampal cortical output. In M. Brazier (Ed.), The Interneuron, University of California Press, Los Angeles, 1969, pp. 415-465. 4 Barraco, R.A., Coffin, V.L., Altman, H.J. and Phillis, J.W., Central effects of adenosine analogues on locomotor activity in mice and antagonism by caffeine, Brain Research, 272 (1983) 392-395. 5 Berman, R.F., Jarvis, M.F. and Lupica, C.R., Adenosine

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involvement in kindled seizures. In J.A. Wada, (Ed.), Kindling 4, Plenum, New York, in press. Boulenger, J.P., Patel, R.M., Parma, A.M. and Marangos, P.J., Chronic caffeine consumption increases the number of brain adenosine receptors, Life Sci., 32 (1983) 1135-1142. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. Buzsaki, G., Feed-forward inhibition in the hippocampal formation, Prog. Neurobiol., 22 (1984) 131-153. Chou, D.T., Khan, S., Forde, J. and Hirsh, K.R., Caffeine tolerance: behavioral, electrophysiological and neurochemical evidence, Life Sci., 36 (1985) 2347-2358. Daly, J.W., Adenosine receptors: target sites for drugs, J. Med. Chem., 25 (1982) 197-201. Dolphin, A.C. and Archer, E.R., An adenosine agonist inhibits and a cyclic AMP analogue enhances the release of glutamate but not G A B A from slices of rat dentate gyrus, Neurosci. Lett., 43 (1983) 49-54. Dunwiddie, T.V., Endogenously released adenosine regulates excitability in the in vitro hippocampus, Epilepsia, 21 (1980) 541-548. Dunwiddie, T.V. and Fredholm, B.B., Adenosine receptors mediating inhibitory electrophysiological responses in rat hippocampus are different from receptors mediating cyclic AMP accumulation, Naunyn-Schmiedeberg's Arch. Pharmacol., 326 (1984) 294-301. Dunwiddie, T.V. and Hoffer, B.J., Adenine nucleotides and synaptic transmission in the in vitro rat hippocampus, Br. J. Pharmacol., 69 (1980) 59-68. Dunwiddie, T.V., Hoffer, B.J. and Fredholm, B.B., Aikylxanthines elevate hippocampus excitability: evidence for a role for endogenous adenosine, Naunyn-Schmiedeberg's Arch. Pharmacol., 316 (1981) 326-330. Dunwiddie, T.V., Mueiler, A., Palmer, M., Stewart, J. and Hoffer, B., Electrophysiological interaction of enkephalin with neuronal circuitry in hippocampus. I. Effects on pyramidal cell activity, Brain Research, 184 (1980) 311-330. Fastbom, J. and Fredholm, B.B., Effects of long-term theophylline treatment on adenosine Al-receptors in rat brain: autoradiographic evidence for increased receptor number and altered coupling to G-proteins, Brain Research, 507 (1990) 195-199. Finn, I.B. and Holtzman. S.G., Tolerance to caffeine-induced stimulation of motor activity in rats, J. Pharmacol. Exp. Ther., 238 (1986) 542-546. Fredholm, B.B., Adenosine actions and adenosine receptors after one week treatment with caffeine, Acta Physiol. Scand., 115 (1982) 283-286. Fredholm, B.B., Jonzon, B. and Lindgren, E., Changes in noradrenaline release and beta receptor number in rat hippocampus following long-term treatment with theophylline or L-phenylisopropyladenosine, Acta Physiol. Scand., 122 (1984) 55-59. Gilbert, M., Racine, R.J. and Smith, G.K., Epileptiform burst responses in ventral vs dorsal hippocampal slices, Brain Research, 361 (1985) 389-391. Goodman, R.R., Kuhar, M.J., Hester, L. and Snyder, S.H., Adenosine receptors: autoradiographic evidence for their location on axon terminals of excitatory neurons Science, 220 (1983) 967-969. Goodman, R.R. and Snyder, S.H., Autoradiographic localization of adenosine receptors in rat brain using [3H]cyclohexyladenosine, J. Neurosci., 2 (1982) 1230-1241. Green, R.M. and Stiles, G.L., Chronic caffeine ingestion sensitizes the A-1 adenosine receptor-adenylate cyclase system in rat cerebral cortex, J. Clin. Invest., 77 (1986) 222-227. Harms, H.H., Wardeh, G. and Mulder, A.H., Effects of adenosine on depolarization-induced release of various radiolabelled neurotransmitters from slices of rat corpus striatum, Neuropharmacology, 18 (1978) 577-580.

26 Hollins, C. and Stone, T.W., Adenosine inhibition of gammaaminobutyric acid release from slices of rat cerebral cortex, Br. J. Pharmacol., 69 (1980) 107-112. 27 Jarvis, M.E, Autoradiographic localization and characterization of brain adenosine receptor subtypes. In E Leslie and C.A. Altar (Eds.), Receptor Localization: Ligand Autoradiography, Liss, New York, 1988, pp. 95-112. 28 Katims, J.J,, Annau, Z. and Snyder, S.H., Interactions in the behavioral effects of methylxanthines and adenosine derivatives, J. Pharmacol. Exp. Ther., 227 (1983) 167-173. 29 Lee, K.S., Reddington, M., Schubert, P. and Kreutzberg, G.W., Regulation of the strength of adenosine modulation in the hippocampus by a differential distribution of A-1 receptors, Brain Research, 260 (1982) 156-159. 30 Lee, K. and Schubert, P., Modulation of an inhibitory circuit by adenosine and AMP in the hippocampus, Brain Research, 246 (1982) 311-314. 31 Lee, K.S., Schubert, P., Reddington, M. and Kreutzberg, G.W., The distribution of adenosine A-1 receptors and 5"-nucleotides in the hippocampal formation of several mammalian species, J. Comp. Neurol., 246 (1986) 427-434. 32 Londos, C. and Wolff, T., Two distinct adenosine-sensitive sites on adenylate cyclase, Proc, Natl. Acad. Sci. U.S.A., 74 (1977) 582-586. 33 Lupica, C.R. and Berman, R.E, Chronic caffeine modulation of postictal phenomena in amygdala kindled rats, Soc. Neurosci. Abstr., 14 (1988) 1147. 34 Lupica, C.R., Cass, W.A., Zahniser, N.R. and Dunwiddie, T.V., Effects of the selective adenosine A2 receptor agonist CGS 21680 on in vitro electrophysiology, cAMP formation and dopamine release in rat hippocampus and striatum, J. Pharmacol. Exp. Ther., 252 (1990) 1134-1141. 35 Marangos, P.J., Boulenger, J. and Patei, J., Effects of chronic caffeine on brain adenosine receptors: regional and ontogenetic studies, Life Sci., 34 (1984) 899-907. 36 Munson, P.J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem., 107 (1980) 220-239. 37 Murray, T.E, Up-regulation of rat cortical adenosine receptors following chronic administration of theophylline, Eur. J. Pharmacol., 82 (1982) 113-114. 38 Murray, T.E and Cheney, D.L., Neuronal location of N6cyclohexyl-3H-adenosine binding sites in rat and guinea pig brain, Neuropharmacology, 21 (1982) 575-580. 39 Murray, T.E and Sanders, R.C., Chronic theophylline exposure increases agonist and antagonist binding to A1 adenosine receptors in rat brain, Neuropharmacology, 27 (1988) 757-760. 40 Murray, T.E and Sanders, R.C., Temporal relationship between A1 adenosine receptor upregulation and alterations in bicuculline seizure susceptibility in rats, Neurosci. Lett., 101 (1989) 325-330. 41 Okada, Y. and Ozawa, S., Inhibitory action of adenosine on synaptic transmission in the hippocampus of the guinea pig in vitro, Eur. J. Pharmacol., 68 (1980) 483-492. 42 Ramkumar, V., Bumgarner, J.R., Jacobson, K.J. and Stiles G.L., Multiple components of the A1 adenosine receptoradenylate cyclase system are regulated in rat cerebral cortex by chronic caffeine ingestion, J. Clin. Invest., 82 (1988) 242-247. 43 Reddington, M., Lee, K.S. and Schubert, P., An A-1 adenosine receptor characterized by [3H]CHA binding mediates the depression of evoked potentials in the rat hippocampal slice preparation, Neurosci. Lett., 28 (1982) 275-279. 44 Rosenthal, H.E., A graphical method for the determination and presentation of binding parameters in a complex system, Anal. Biochem., 2 (1967) 525-532. 45 Rudolphi, K.A., Keil, M., Fastbom, J. and Fredholm, B.B., Ischaemic damage in gerbil hippocampus is reduced following upregulation of adenosine AI receptors by caffeine treatment, Neurosci. Lett., 103 (1989) 275-280. 46 Sattin, A., and Rail, T.W., The effect of adenosine and adenine

62

47 48

49

50

51

nucleotides on the cyclic adenosine 3"-5"-mono-phosphate content of guinea pig cerebral cortex slices, Mol. Pharmacol., 6 (1970) 12-23. Scatchard, G., The attraction of small proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660-672. Scholfield, C.N. and Steel, L., Presynaptic K+-channel blockade counteracts the depressant effect of adenosine in olfactory cortex, Neuroscience, 24 (1988) 81-91. Schubert, P. and Mitzdorf, V., Analysis and quantitative evaluation of the depressive effects of adenosine on evoked potentials in hippocampal slices, Brain Research, 172 (1979) 186-190. Smellie, F.W., Daly, J.W., Dunwiddie, T.V. and Hoffer, B.J., The dextro- and levorotatory isomers of N-phenylisopropyladenosine: stereospecific effects on cyclic AMP formation and evoked synaptic responses in brain slices, Life Sci., 25 (1979) 1739-1748. Snyder, S.H., Katims, J.J., Annau, Z., Bruns, R.F. and Daly, J.W., Adenosine receptors and behavioral actions of methylxan-

thines, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 3260-3264. 52 Szot, P., Sanders, R.C. and Murray, T.E, Theophylline-induced upregulation of Al-adenosine receptors associated with reduced sensitivity to convulsants, Neuropharmacology, 26 (1987) 11731180. 53 Van Calker, D,, Muller, M. and Hamprecht, B., Adenosine regulates via two different types of receptors the accumulation of cyclic AMP in cultured brain cells, J. Neurochem., 33 (1979) 999-1005. 54 Von Borstel, R.W., Wurtman, R.J. and Conlay, L.A., Chronic caffeine consumption potentiates the hypotensive action of circulating adenosine, Life Sci., 32 (1983) 1151-1158. 55 Williams, M., Purine receptors in mammalian tissues: pharmacology and functional significance, Annu. Rev. Pharmacol. Toxicol., 27 (1987) 315-345. 56 Zambotti, F., Zonta, N., Ferrario, P., Zecca, L. and Mantegazza, P., Effects of 2-chloroadenosine on hippocampal GABA content and turnover, J. Neurotransm., 65 (1986) 167-175.