Brain Research, 567 (1991) 181-187 Elsevier

BRES 17251

181

Research Reports

Functional activity of the adenosine binding enhancer, PD 81,723, in the in vitro hippocampal slice Cynthia A. Janusz 1, Robert F. Bruns 3 and Robert F. Berman 2 Departments of 1Physiology, School of Medicine and 2psychology, Wayne State University, Detroit, M1 48201 (U.S.A.) and 3Biochemical Pharmacology Research, Eli Lilly and Co., Indianapolis, IN 46285 (U.S.A.)

(Accepted 30 July 1991) Key words: Adenosine receptor; Theophylline; Receptor modulator; Hippocampal slice; Electrophysiology; Binding enhancer

The adenosine receptor binding enhancer, PD 81,723, enhances the inhibitory effects of exogenously applied adenosine in a dose-dependent manner in hippocampal brain slices. Extracellular recordings were obtained from the CA1 cell layer while electrically stimulating the stratum radiatum. Application of 1, 10 or 32/~M PD 81,723 in the presence of adenosine resulted in a dose-dependent reduction in the amplitude of the population spike which could be partially reversed by theophylline. In addition, hippocampal slices exposed to adenosine showed greater paired-pulse facilitation compared to control and this facilitation was significantlyenhanced by the presence of PD 81,723. PD 81,723 had no effect when administered alone, but required the presence of adenosine. These results demonstrate that in addition to enhancing adenosine receptor binding, PD 81,723 also enhances the functional activity of adenosine in the hippocampal slice. INTRODUCTION

Adenosine is an important neuromodulator in the central nervous system (CNS) with anticonvulsant, hypnotic, anxiolytic and centrally mediated hypotensive actions. It has powerful neuromodulatory influences on synaptic transmission, with its effects being predominantly inhibitory 8. Endogenous adenosine has been shown to depress spontaneous neuronal firing in mammalian CNS neurons 27 and inhibit evoked synaptic activity in vitro in olfactory tg, hippocampus 9'24 and cortical 3~ slices. The inhibition by adenosine may occur at both presynaptic and postsynaptic sites of action. PresynapticaUy, adenosine inhibits transmitter release s'11, possibly via a reduction in calcium entry into presynaptic nerve terminals. Postsynaptically, adenosine inhibits neuronal cells through an increase in potassium conductance 12'37. Both mechanisms are important to the inhibitory action of adenosine, but the presynaptic effects may be primary 8. Adenosine is released from brain slices26 and synaptosomes 18. Several sources for released adenosine have been suggested 2'8'4°. Adenosine can be produced intracellularly and exported by facilitated diffusion 32'33, or can be produced from hydrolysis of extraceUular ATP 2"23. The effects of adenosine are mediated through cell surface receptors, A1 and A23s, with opposite effects on adenylate cyclase activity. The A~ subtype inhibits cy-

clase activity, while the A 2 subtype stimulates cyclase activity. Receptor subtype-selective agonists and antagonists have been reported 2'6. Pathophysiological conditions that increase metabolic demand (i.e. hypoxia, seizures) increase extracellular adenosine concentrations in nervous tissue 41 through the hydrolysis of cellular AMP 23. The protective role of adenosine in ischemia suggests that agents that potentiate the actions of adenosine could be valuable tools in the treatment of ischemic disease resulting from myocardial infarction or stroke 2L39. Unfortunately, adenosine agonists acting directly at the receptor have been shown to have many clinically undesirable side effects7, thus limiting the use of this strategy ~4. These side effects occur as a result of widespread activation of adenosine receptors, rather than activation of adenosine receptors localized to the tissue experiencing the oxygen deficit. Therefore, compounds exhibiting a selective modulation of adenosine receptors might prove to be more useful in ischemia, since augmentation of endogenous adenosine would occur in ischemic areas without an enhancement of adenosinergic actions in non-ischemic areas. Recently, a new class of compounds has been shown to enhance adenosine receptor binding 4. These compounds originated from a series of 2-amino-3-benzoylthiophenes that were intermediates in the synthesis of benzodiazepine-like compounds 36. Several compounds

Correspondence: R.F. Berman, Mott Center for Human Growth and Development, Wayne State University, 275 E. Hancock, Detroit, MI 48201, U.S.A.

182 from this series, including P D 81,723, were f o u n d to e n h a n c e b i n d i n g of a d e n o s i n e A1 agonists 4. T h e e n h a n c e m e n t a p p e a r e d to be specific to the A 1 a d e n o s i n e receptor as agonist b i n d i n g to A 2 receptors a n d o t h e r G p r o t e i n - l i n k e d receptors was n o t e n h a n c e d . I n the p r e s e n t study, we d e m o n s t r a t e that P D 81,723 e n h a n c e s the i n h i b i t o r y actions of e x o g e n o u s l y applied a d e n o s i n e o n C A 1 h i p p o c a m p a l electrophysiology in the rat, using the b r a i n slice p r e p a r a t i o n . P D 81,723 was n o t effective w h e n a d m i n i s t e r e d alone, b u t r e q u i r e d the presence of a d e n o s i n e . MATERIALS AND METHODS In addition to the electrophysiological studies, binding studies were carded out to confirm the binding enhancer's effect in the hippoeampus. Hippocampi were dissected from frozen rat brains (Pel-Freez, Rogers, AR), and membranes were prepared as previously describeda. Binding of 0.2 nM [3H]CHA (Du Pont/NEN, Boston, MA, 34.4 Ci/mmol) to 0.4 mg original wet weight of rat hippocampal membranes was carded out for 4 h at 25 °C in 50 mM Tris-HCl pH 7.7 with 0.1 U/ml adenosine deaminase (BoehringerMannheim no. 102-105) and 1 mM EDTA. Other details of the method were as described4. For the electrophysiological experiments, male Long-Evans rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing between 125 and 200 g were used. Animals were housed in groups of two under a 12 h light-dark cycle, with food and water available ad libitum. Slices of the hippocampus were prepared as follows. Animals were decapitated and the brains quickly removed. The brain was placed on its dorsal surface, and the hippocampus was then rapidly dissected on ice (4 °C) using a ventral approach as previously described35. The hippocampus was then placed onto a tissue slicer (Stoelting Co., Wood Dale, IL), and 400o/~mslices cut at a 70° angle to the septotemporal axis. Slices were immediately placed into ice-cold (4 °C), oxygenated (95% 02/5% CO2) artificial cerebrospinal fluid (ACSF) consisting of (in mM): CaCI2 2.4, MgSO4 1.3, KC1 5, NaC1 120, NaH2PO 4 1.24, Na/-ICO3 26 and dextrose 10. Approximately 6-8 slices were taken from a single hippocampus. All slices were transferred to ACSF within 7 rain from the time of sacrifice to ensure viability of the tissue. Slices were then gently transferred to the recording chamber. The recording chamber was constructed of transparent Plexiglas, and contained deionized water maintained at 34 °C via a DC proportional te~nperature regulation unit (Frederick Haer, Brunswick, ME). In addition, the chamber was perfused continuously with 95% 02/5% CO 2. A slice well, containing 2 ml of ACSF, was placed in the chamber, upon which nylon netting was stretched to act as a support for the brain slices. Under these conditions, stable electrophysiological activity can be maintained in brain slices for 8-12 h. Slices were allowed to equilibrate to the chamber for 1 h before electrophysiological recording began. Extracellular recordings were made with glass micropipettes filled with 2 M NaCI, and with impedances between 1 and 4 MG. The tip of the recording electrode was placed in the pyramidal cell layer at a depth of 70-100 /~m using a hydraulic micropositioner (Model 650, David Kopf Instruments). A concentric bipolar, tungsten microstimulating electrode 5, insulated with teflon and sputter-coated with gold, was used to stimulate the Schaffer collateral fibers in the stratum radiatum of the CA1 hippocampal subfield. Evoked responses were recorded within the stratum pyramidal of the CA1 subfield. ExtraceUular field potentials were amplified 1000-fold (World Precision Instruments DAM-50), visualized on a Tektronix 4094C digital storage oscilloscope, and stored on disk for later analysis of peak lateneies and amplitudes. Electrical stimulation was delivered via a Grass S-48 stimulator and PSIU-6 stimulus isolation

unit every 60 s as monophasic square-wave pulses at 0.1 ms pulse duration. Stimulation levels were adjusted to yield a 1.5 mV population spike. This level was typically below 20 V. Slices requiring greater than 30 V to evoke a 1.5 mV population spike were not used. Hippocampal slices were illuminated from below and visualized microscopically at 40x magnification. Only slices with clear and well-defined cellular layers were used for recording. Any slices showing evidence of epileptiform activity or multiple evoked population spikes were not used. In the paired-pulse procedure, two pulses (P1 and P2) separated by 10-i00 ms were delivered to the Schaffer collateral pathway. At short interpulse intervals (e.g. 10--30 ms) in normal hippocampal slices, the population spike evoked by the second pulse (P2) is typically smaller in amplitude than that evoked by the first (P1). This paired-pulse phenomenon has been attributed to feedforward and recurrent inhibition of the CA1 pyramidal cells 1'1°. At longer interpulse intervals (i.e. >50 ms) the response to P2 is facilitated (i.e. paired-pulse facilitation). All dose response-curves were determined using the static bath procedure 35. In this procedure, responses were recorded from slices maintained at the gas-liquid interface while suspended on nylon netting on the 2 ml slice well. After a stable baseline had been established, 1-12.5/~1 of a concentrated drug solution (80x desired final concentration) were added to the slice-well using a calibrated microliter syringe. Fifteen minutes were allowed for the drug to diffuse and reach final concentration in the well at which time the evoked potential became stabilized and responses recorded. All reagents and drugs used were purchased from Sigma Chemicals (St. Louis, MO), except for the benzodiazepine receptor antagonist, Ro 15-1788, which was a gift from Dr. E.I. Teitz, Department of Pharmacology, Medical College of Ohio, and the binding enhancer, PD 81,723, which was synthesized at Eli Lilly and Co., Indianapolis, IN, by the method of Tinney et al. 36. All drugs were dissolved in vehicle in concentrations 80x greater than final desired concentrations. Adenosine and theophylline were dissolved in 0.9% buffered saline. Ro 15-1788 and PD 81,723 were dissolved in 100% dimethyl suifoxide, (DMSO). Population spike amplitudes were measured before and after drug administration. These data were statistically analyzed by analysis of variance (ANOVA) followed by appropriate post hoc individual comparisons (BMDP Statistical Software, Los Angeles, CA). Paired-pulse data were expressed as percent change (i.e. P2/P1 x 100%) over the interpulse intervals used (10-100 ms), and analyzed similarly.

RESULTS P D 81,723 s t i m u l a t e d the b i n d i n g of the A 1 agonist ligand [3H]N6-cyclohexyladenosine ( [ 3 H ] C H A ) by up to 160% (Fig. 1), confirming that the e n h a n c e m e n t s h o w n in rat f o r e b r a i n m e m b r a n e s also applies to rat h i p p o c a m pus. M a x i m u m e n h a n c e m e n t o c c u r r e d at 10 /~M P D 81,723, a n d a precipitous d r o p in [ a H ] C H A b i n d i n g was seen at P D 81,723 c o n c e n t r a t i o n s above 20/~M (data n o t shown), as previously o b s e r v e d 4. T h e greater e n h a n c e m e n t in the p r e s e n t e x p e r i m e n t (160% c o m p a r e d to 45% in ref. 4) is d u e to several modifications in b i n d i n g conditions (in particular, addition of E D T A ) designed to optimize the degree of e n h a n c e m e n t by this agent. R e p r e s e n t a t i v e e v o k e d field potentials are s h o w n in Fig. 2 A , a l o n g with a d i a g r a m of a n in vitro h i p p o c a m p a l slice d e m o n s t r a t i n g the p l a c e m e n t s of the stimulating a n d r e c o r d i n g electrodes (Fig. 2B). A s s h o w n in the Figure,

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Functional activity of the adenosine binding enhancer, PD 81,723, in the in vitro hippocampal slice.

The adenosine receptor binding enhancer, PD 81,723, enhances the inhibitory effects of exogenously applied adenosine in a dose-dependent manner in hip...
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