Arachidonic acid metabolites do not mediate modulation of neurotransmitter release by adenosine in rat hippocampus or striatum

76

Brain Research. 527 (1990) 76-80 Elsevier

BRES 15822

Arachidonic acid metabolites do not mediate modulation of neurotransmitter release by adenosine in rat hippocampus or striatum Thomas V.

Dunwiddie 1'2, Marianne Taylor 1, Wayne A. Cass 1, Frank A. Fitzpatrick 1 and Nancy R. Zahniser 1

t Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80206 (U.S.A.) and 2VeteransAdministration Medical Center, Denver, CO 80262 (U.S.A.)

(Accepted 6 March 1990) Key words: Arachidonic acid; Lipoxygenase inhibitor; Cyclooxygenaseinhibitor; Phospholipase A2 inhibitor; Electrophysiology; Dopamine; Striatum; Hippocampus; Synaptic modulation

The possible involvement of arachidonic acid metabolites as mediators of the modulation of neurotransmitter release by adenosine, acetylcholine, and GABA was examined in brain slices of rat hippocampus and striatum. The synaptic modulatory effects of these 3 agents on excitatory transmission in the CA1 region of hippocampus were completely unaffected by a phospholipase inhibitor (p-bromophenacyl bromide, BPB; 10-50/tM), a tipoxygenase inhibitor (nordihydroguaiaretic acid; 5-50/~M), the cyclooxygenase inhibitor indomethacin (10-20 /~M), and a cyclooxygenase/lipoxygenaseinhibitor (U53059; 5-10/~M). BPB was also found to be ineffective in altering the modulation of transmission by adenosine in the perforant path, and the adenosine inhibition of electrically stimulated release of endogenous dopamine from striatal slices. Arachidonic acid itself also had no effect on synaptic transmission. While these experiments do not rule out such a role for arachidonic acid or its metabolites in mammalian brain, they suggest that in a number of systems the inhibition of transmitter release must occur through an entirely independent mechanism.

INTRODUCTION Adenosine inhibits synaptic transmission at many synapses and appears to do so by inhibiting the release of transmitter from the presynaptic terminal 5. Within the hippocampal formation, we have shown that adenosine modulates the efficacy of synaptic transmission at the excitatory Schaffer collateral and commissural synapses onto the CA1 pyramidal neurons via an action at a presynaptic A1 adenosine receptor 6'7. In the rat striatum, adenosine also inhibits dopamine release 11'18'21, and we have recently demonstrated that this also appears to be due to actions at an A1 receptor 1°. Despite the fact that adenosine's inhibitory actions on release have been characterized in a variety of systems, the mechanism by which it inhibits transmitter release remains obscure. Previous work has suggested that adenosine may inhibit release by hyperpolarizing the presynaptic nerve terminal via activation of a potassium conductance 25, that it may act by directly inhibiting the voltage-dependent calcium conductances that mediate calcium entry into the presynaptic nerve terminal 17'27, or that it may affect the

calcium sensitivity of processes in the nerve terminal that mediate release 29. Regardless of the ionic mechanism(s) involved, it is also possible that any of these processes could be affected indirectly via the activation of second messenger systems coupled to adenosine receptors. Arachidonic acid metabolites formed by a lipoxygenase pathway have been proposed as mediators of the inhibition of synaptic transmission at sensory neuron synapses in Aplysia 23"28. In order to determine whether mammalian brain adenosine receptors might utilize an arachidonic acid metabolite(s) as a second messenger to mediate the inhibition of transmitter release, we characterized the elects of pharmacological manipulation of this system on the inhibition of excitatory transmission in slices of the rat hippocampus, and on the inhibition of release of endogenous dopamine from slices of rat striatum. More specifically, we have characterized the effects of inhibitors of phospholipase A2, which is an enzyme that can liberate arachidonate from membranes and which could be the first step in the formation of putative mediators. Furthermore, we have also examined the effects of inhibitors of the cyclooxygenase and

Correspondence: T. Dunwiddie, Department of Pharmacology, C-236, University of Colorado Health and Sciences Center, 4200 E. 9th Ave., Denver, CO 80262, U.S.A.

77 lipoxygenase enzymes that w o u l d lead to the f o r m a t i o n of a r a c h i d o n i c acid m e t a b o l i t e s such as prostaglandins, t h r o m b o x a n e s and leukotrienes.

MATERIALS AND METHODS

Animals Male Sprague-Dawley rats (Sasco Animal Laboratories, Omaha, NE) weighing 150-250 g were used for all experiments. They were housed in groups of 4-6 under a 12-h light-dark cycle with food and water available ad libitum. At the beginning of the experiments, subjects were decapitated, and the hippocampus or striatum was dissected free of surrounding tissue.

Electrophysiology Transverse sections taken from the middle portion of the hippoeampi were prepared, as described previously9'16'19. Slices were cut at 400/~m on a Sorvall tissue chopper and placed in medium consisting of (in mM): NaCI, 124; KCI, 4.9; KH2PO 4, 1.2; MgSO4, 2.4; CaCI2, 2.5; NaHCO 3, 25.6; and glucose, 10 (pH 7.5), that had been saturated with 95% 02/5% CO 2 and transferred to a recording chamber maintained at 33 + 1 °C. During recording slices were continuously superfused with medium over both surfaces. Electrophysiological recordings were made with 2-3 MI2 glass microelectrodes, and synaptic responses were elicited by monophasic 0.1 ms pulses of 6-30 V delivered via twisted nichrome wire stimulators. After a stable baseline was obtained, slices were either superfused with 50 /~M adenosine, or pretreated with various inhibitors for 10--30 min and then superfused with 50/~M adenosine. This concentration of adenosine was chosen because it elicits a robust, but not maximal, inhibition of the synaptic response. All drugs solutions were made up at 100-1000x the desired final concentration in water, or at 600-3000x the desired concentration in 100% DMSO. Arachidonic acid was made up as a 60 mM solution in DMSO, then added directly to the buffer to achieve the final concentration. Although 30 /~M arachidonic acid remained in solution, 60/tM clearly did not, indicating that saturation occurred somewhere between these two concentrations. Control experiments in which DMSO alone was added to the perfusion fluid in equivalent amounts did not show any alteration in adenosine sensitivity.

RESULTS A s has b e e n previously o b s e r v e d 8~s, superfusion of h i p p o c a m p a l brain slices with a d e n o s i n e inhibited the e v o k e d field excitatory postsynaptic p o t e n t i a l (EPSP) response (Fig. 1), which r e c o v e r e d r a p i d l y u p o n termination of the a d e n o s i n e superfusion. P r e t r e a t m e n t before the adenosine superfusion with p - b r o m o p h e n a c y l bromide (BPB, 1 0 / ~ M ) , which inhibits the liberation of arachidonic acid from m e m b r a n e s by p h o s p h o l i p a s e A 2 as well as by the phosphoinositol-specific p h o s p h o l i p a s e C ( P I - P L C ) 13, had no effect u p o n s u b s e q u e n t responses to adenosine (Fig. 1). A similar lack of effect o f BPB was o b s e r v e d in o t h e r slices tested with concentrations of BPB ranging from 10 to 50/~M (Fig. 2). Because the liberation of arachidonic acid by phospholipase A 2 represents the first step in the synthesis of various m e t a b o l i t e s , this s e e m e d the best initial choice for disrupting this system. H o w e v e r , since t h e r e are alternative pathways such as the P I - P L C p a t h w a y for the formation of arachidonic acid and since it has been

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Endogenous dopamine release Striatal slices (400/zm) were incubated in a metabolic shaker at 34 °C for 60 min in Krebs' buffer: (in mM) NaC1, 118; KCI, 4.7; glucose, 11.1; NaHCO3, 25; MgCI2, 1.2; NaH2PO4, 1.0; CaCI2, 1.3; ascorbic acid, 0.11 and Na2EDTA , 0.004; saturated with 95% 02/5% CO2(see ref. 16 for details). The slices were then transferred to glass superfusion chambers and superfused (1 ml/min) at 34 °C for 30 min, followed by 20 min of superfusion with Krebs' buffer containing 10 /~M GBR 12909 to inhibit the synaptic dopamine transporter and to obtain detectable levels of dopamine. Overflow of dopamine was evoked by electrical stimulation (1 Hz/1 min). Ten rain after the first stimulation, superfusion was begun with 50/zM adenosine and/or 10/~M p-bromophenacyl bromide (BPB). After 30 min of drug superfusion, a second release was elicited identical to the first. BPB was dissolved in DMSO, and an equivalent amount of DMSO (0.05% final concentration) was added to control slices. Dopamine levels were determined using high-performance liquid chromatography (HPLC) coupled with electrochemical detection (EC) as described in detail previously12. Overflow of dopamine was expressed as pg/ml/mg wet weight of tissue.

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Fig. 1. Co-perfusion with BPB does not alter responses to adenosine in hippocampal slices. In A, a representative slice was superfused with 50 ~M adenosine alone, 10 ~M BPB alone, or BPB and adenosine together as indicated by the bars along the bottom. BPB by itself had no effect upon the magnitude of the field EPSP, and did not alter the response to adenosine. B illustrates the averaged responses from the periods indicated by the numbered line segments at the top of A. In each case, adenosine markedly reduced the field EPSP response, and the response recovered completely upon washing.

78 suggested that PI-PLC is not inhibited by BPB in brain 22, we also tested a number of agents that inhibit subsequent steps in the metabolic conversion of arachidonic acid to various mediators. Nordihydroguaiaretic acid (NDGA) inhibits lipoxygenases as well as possibly phospholipase A22, indomethacin (INDO) inhibits the cyclooxygenase pathway 24, and U53059 inhibits both cyclooxygenase and lipoxygenase pathways 2°. However, none of these inhibitors had any significant effect upon the depressant effect of adenosine on hippocampal field EPSP responses at the concentrations tested (Fig. 2). We also examined the effect of 10-30 MM U73122, another inhibitor of phospholipase A23 in 4 slices, but it was without effect as well. Although these data suggest that adenosine may not act via arachidonic acid metabolites, other inhibitors of synaptic transmission might utilize these compounds as second messengers. To test this possibility, we conducted experiments similar to those with adenosine, but substituted the GABA B receptor agonist baclofen (5 MM) or the cholinergic receptor agonist carbachol (5 #M) for adenosine. Again, pretreatment of the slices with 10 MM BPB had no significant effect upon the inhibitory responses produced by these compounds (Fig. 3). We also examined the direct effects of arachidonic acid on excitatory transmission; if adenosine were acting via an arachidonic acid metabolite, then arachidonic acid itself would be expected to mimic the effects of adenosine. However, superfusion of slices with nominal concentrations of 15, 30 and 60 MM arachidonic acid had no

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significant effects on the synaptic responses. Even at the highest concentration, where the formation of precipitate indicated that the medium was saturated with arachidonic acid, superfusion resulted in a non-significant -2.9 _+ 2.8% change in the field EPSP amplitude after 30 min (n = 4 slices, 2 animals). Yet another possibility was that metabolites of arachidonic acid might modulate transmitter release in some systems, but not in the excitatory Schaffer collateral and commisural inputs to the CA1 region. To examine this possibility we first characterized the effects of BPB on adenosine responses in the perforant path input to the dentate gyrus. BPB at a concentration of 10 MM had no effect upon the inhibition by 50 MM adenosine of the field

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Fig. 4. The effect of BPB on adenosine inhibition of evoked dopamine overflow from striatal slices. These chronological records indicate the mean -+ S.E.M. efflux of endogenous dopamine over 1 rain intervals from striatal slices (n = 5 rats for each group). Stimulation was at 1 Hz for 1 rain (3-4 rain, 41-42 rain), and slices were pretrated with 50 ~tM adenosine (B) or adenosine + 10 MM BPB (C) for 30 min prior to the second period of stimulation as indicated. GBR 12909 (10 MM) was included to inhibit dopamine uptake.

79 EPSP evoked by perforant path stimulation. Depression was 56 + 3% in controls and 60 + 5% in BPB pretreated slices (n = 4). We also characterized the actions of BPB on the inhibitory effects of adenosine on evoked dopamine overflow from striatal slices. In these experiments, dopamine release was evoked initially by electrical field stimulation under control conditions; slices were then pretreated with drugs and a second release was initiated. The mean responses to this protocol are illustrated in Fig. 4. When slices were treated with vehicle only, the response to the second period of stimulation was almost identical to the first (mean response was 98% of the first). In slices pretreated with 50/~M adenosine, the second response was decreased by 30 + 5% (P < 0.05 compared to control and BPB alone), whereas slices pretreated with both 10 # M BPB and adenosine showed a 29 + 5% decrease (P > 0.05). BPB alone induced a 5.4 + 2.1 increase in the second response. DISCUSSION Although arachidonic acid metabolites are apparently involved in the modulation of synaptic transmission in Aplysia 23"28, the present studies clearly suggest that they do not play a corresponding role in the modulation of transmitter release in several neurotransmitter systems in the mammalian brain. One synapse that has been studied in great detail insofar as such modulation is concerned is the excitatory amino acid (probably glutamatergic) synapse onto the dendrites of the CA1 pyramidal cells of the hippocampus. Adenosine, acetylcholine, GABA, and neuropeptide Y all diminish transmission at this synapse through an apparently presynaptic (and perhaps common) mechanism 1,4,s,14,~5,3°. However, this type of modulatory action does not appear to be affected by the phospholipase A2, lipoxygenase, or cyclooxygenase inhibitors that we tested. Even the lowest concentrations of REFERENCES 1 Ault, B. and Nadler, J.V., Baclofen selectively inhibits transmission at synapses made by axons of CA3 pyramidal ceils in the hippocampal slice, J. Pharmacol. Exp. Ther., 223 (1982) 291-297. 2 Billah, M.M., Bryant, R.W. and Siegel, M.I., Lipoxygenase products of archidonic acid modulate boisynthesis of plataletactivating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2, Z Biol. Chem., 260 (1985) 6899-6906. 3 Blaesdale, J., Bundy, G., Bunting, S., Fitzpatrick, E, Huff, R., Sun, E and Pike, J.E., Inhibition of phospholipase dependent processes by U 73122, Adv. Prostaglandin Thromboxane Leukotrine Res., 19 (1989) 590-593. 4 Colmers, W.E, Lukowiak, K. and Pittman, Q.J., Neuropeptide Y action in the rat hippoeampal slice: site and mechanism of presynaptic inhibition, J. Neurosci., 8 (1988) 3827-3837. 5 Dunwiddie, T.V., The physiological role of adenosine in the

these drugs tested (10 # M BPB, 5 # M N D G A ) , which completely abolished responses to FMRFamide in Aplysia 23, did not alter hippocampal synaptic responses. We have also observed that at these concentrations both BPB and N D G A block dopamine uptake into striatal slices (Cass et al., in preparation), so it is clear that accessibility of the drug to the tissue is not a problem. Thus, it does not seem likely that adenosine, acetylcholine, or G A B A modulate transmitter release in the hippocampus via an arachidonic acid metabolite-mediated system. Furthermore, the lack of any effect of arachidonic acid itself suggests that it is unlikely that there are other, perhaps unknown modulators that do use an arachidonic acid metabolite as a mediator. Finally, the adenosine modulation of dopamine release in the striatum, which we have shown to be mediated via an Al-like receptor similar to the receptor that modulates transmission in the hippocampus, also is unaffected by BPB. It seems quite likely that metabolites of arachidonic acid may play important roles in intercellular communication in the brain, as they do in so many peripheral systems. If arachidonic acid metabolites serve as second messengers in the hippocampus, it seems more likely that they mediate long-term modifications in synaptic responses rather than more transient forms of modulation. Long-term potentiation of synaptic responses in the hippocampus has been reported to be sensitive to disruption by these agents at concentrations similar to those used in the present study 22'31. Unlike the Aplysia, the actions of inhibitory synaptic modulators do not appear to be mediated by arachidonic acid metabolites in any of the systems that we have tested. Acknowledgements. This research was supported by Grants NS 26851 and DA 02702, Training Grant A A 07464, and the Veterans Administration Medical Research Service. We would like to thank Tom Worth for technical assistance with these experiments. central nervous system, Int. Rev. Neurobiol., 27 (1985) 63-139. 6 Dunwiddie, T.V. and Fredholm, B.B., Adenosine receptors mediating inhibitory electrophysiological responses in rat hippocampus are different from receptors mediating cyclic AMP formation, Naunyn-Schmiedeberg's Arch. Pharmacol., 326 (1984) 294-301. 7 Dunwiddie, T.V. and Fredholm, B.B., Adenosine A1 receptors inhibit adenylate cyclase activity and neurotransmitter release and hyperpolarize pyramidal neurons in rat hippocampus, J. Pharmacol. Exp. Ther., 249 (1989) 31-37. 8 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. 9 Dunwiddie, T.V. and Lynch, G.S., Long-term protentiation and depression of synaptic responses in the rat hippocampus: localization and frequency dependency, J. Physiol., 276 (1978) 353-367. 10 Dunwiddie, T.V., Worth, T., Lupica, C. and Taylor, M., Mechanisms underlying synaptie modulation by adenosine in rat

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