Alcohol, Vol. 9, pp. 541-546, 1992

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Actions of Ethanol and Acetate on Rat Cortical Neurons: Ethanol/Adenosine Interactions J. W . P H I L L I S , l M. H. O ' R E G A N A N D L. M. P E R K I N S

Department o f Physiology, Wayne State University School o f Medicine, Detroit, M I Received 3 M a r c h 1992; Accepted 14 M a y 1992 PHILLIS, J. W., M. H. O'REGAN AND L. M. PERKINS. Actions of ethanol and acetate on rat cortical neurons: Ethanol~adenosine interactions. ALCOHOL 9(6) 541-546, 1992.-Recent studies have suggested that ethanol may exert some of its central depressant actions by increasing the extracellular levels of adenosine in the brain. Ethanol can inhibit the cellular uptake of adenosine, thus increasing its extracellular concentration. After ethanol metabolism by the liver, blood acetate levels are elevated and acetate metabolism in the brain could also lead to the production of adenosine. Rat cerebral cortical cup release experiments failed to reveal any elevation in the extracellular levelsof either adenosine or inosine following the intrapcritoneal (IP) administration of ethanol (1.5 g/kg) or acetate (2 g/kg). IP-administered ethanol (0.5 and 1.0 g/kg) enhanced the magnitude and duration of the inhibition by iontophoretically applied adenosine of the spontaneous firing of rat cerebrocortical neurons; an action which would be consistent with the block of adenosine uptake. Acetate, applied iontophoretically, depressed the spontaneous firing of 63eta of the cerebrocortical neurons tested. 8-p-Sulphophenyltheophylline, an adenosine antagonist, was ineffective at blocking these inhibitions, indicating that adenosine generation is unlikely to have played a major role in the acetate-evoked depression of cerebral cortical neurons. Adenosine

Ethanol

Alcohol

Acetate

Uptake

RECENT evidence suggests that ethanol-induced changes in central nervous system (CNS) excitability may involve the neuromodulator adenosine and acetate, a metabolic product of ethanol metabolism in the liver. Dar et al. (8) initially proposed a possible role of adenosine in some of the CNS effects of ethanol on the basis of their observation that pretreatment with theophylline, an adenosine antagonist, prior to acute ethanol administration, markedly reduced the duration of ethanol-induced sleep and similarly decreased the duration and intensity of motor incoordination. In contrast, dipyridamole, an adenosine uptake blocker, potentiated the motor incoordination and prolonged the hypnosis elicited by ethanol. Further evidence for an involvement of brain adenosine in ethanolinduced motor incoordination was obtained in experiments in whi I chronic administration of the adenosine antagonists, caffeine or isobutylmethylxanthine, resulted in enhanced ethanol-induced incoordination and increased whole brain adenosine binding (9). The observation that acute ethanol administration enhances the number of adenosine A~-binding sites in the rat brain (3) furnishes a potential explanation for the involvement of adenosine in ethanors effects. Ethanol also increases extracellular levels of adenosine in $49 lymphoma cells by inhibiting adenosine uptake via the nucleoside trans-

Neurons

In vivo

porter without affecting its transporter mediated efflux (15). Ethanol also inhibits adenosine transport by rat cerebral and cerebellar cortical synaptosomes in pharmacologically relevant concentrations (4,21), suggesting that its depressant actions on the CNS may be a result of its potentiation of the local extracellular concentration, and thus the effects, of endogenously released adenosine. Indeed ethanol has been demonstrated to cause a dose-dependent increase in basal and stimulated adenosine release from rat cerebellar synaptosomes (5). Acetate, resulting from ethanol metabolism in the liver, is released into the circulation and utilized in a number of tissues, including the brain (1). Acetate readily crosses the blood-brain barrier (11,16) and brain acetate levels are elevated following ethanol administration (1). Acetate has behavioral effects on rats comparable to those of ethanol (1), which may involve an adenosine-mediated mechanism (12,13,22,24). It is thought that the transformation of acetate into acetyl CoA, in an ATP utilizing reaction, results in AMP formation. AMP is subsequently converted to adenosine by the enzyme 5 '-nucleotidase. The purpose of the present study was to explore the actions of ethanol and acetate on rat cerebral cortical neurons and to

Requests for reprints should he addressed to Dr. John W. Phillis, Department of Physiology, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, MI 48201. 541

542

PHILLIS, O'REGAN AND PERKINS

evaluate the potential involvement of adenosine in their effects. The experiments described in this report include both electrophysiological and adenosine release studies. METHOD

For all experiments, male Sprague-Dawley rats (350-450 g; Charles River) were anesthetized with haiothane. After insertion of a tracheal cannula, anesthesia was sustained with methoxyflurane in air (cortical release experiments) or methoxyflurane in a mixture of nitrous oxide and oxygen [70/30 v/v (electrophysiological experiments)]. Body temperature was maintained at 37°C with a heating pad controlled by a rectal probe. For the electrophysiological experiments, the rats were mounted in a stereotaxic frame. A small hole was drilled through the parietal bone 2 mm lateral to the sagittal suture and 1.5 mm posterior to the coronal suture line. A slit was made in the dura to expose the sensorimotor cortex. All exposed tissues were covered with a thin layer of 2% agar in saline to prevent drying. Recording of neuronal activity and iontophoresis of drugs was accomplished with multibarrelled micropipettes prepared from fiber-fill capillaries. The central recording barrel and one side barrel were filled with 2 M NaC1. This side barrel was used for current neutralization. The remaining side barrels were filled with various combinations of adenosine (0,1 M, pH 4.0), sodium acetate (0.1 M, pH 7.8), acetylcholine chloride (0.1 M, p H 5.2) and 8-p-sulphophenyltheophylline (0.05 M, pH 5.7). Drug effects were evaluated by observing changes in the rate of firing of deep (900-1400 #) spontaneously active cortical neurons. Adenosine was applied repetitively at fixed intervals from the micropipette and the duration of the depression of spontaneous firing it elicited was measured. The mean duration of at least three adenosinergic inhibitions recorded prior to ethanol administration was compared with that of three or more adenosine applications following ethanol administration. Acetylcholine was used to facilitate the firing of cholinoceptive cortical neurons; many of which can be identified as corticospinal neurons (17). Acetate and 8-p-sulphophenyltheophylline were applied by iontophoretic ejection from the multibarrelled micropipettes. Ethanol, diluted in saline, was administered intraperitoneally (IP) at doses of 0.5 and I g/kg. Only one or two injections of ethanol, separated by at least 3 hours, were given to each animal to avoid residual drug effects. Ten rats (5 ethanol, 5 acetate) were prepared for cerebral cortical release experiments. The right femoral artery was cannulated for measurement of arterial blood pressure and withdrawai of arterial blood samples for blood gas and p H analysis. Following placement of the animal's head in a Narashige SH-8 nontraumatic head holder, a continuous midline incision was made along the top of the skull. The dorsal and dorsolaterai surfaces of both cerebral hemispheres were exposed by removal of the overlying frontal and parietal bones, with a thin strip of bone left intact along the midline to protect the dorsal sagittal venous sinus. The dura mater-arachnoid complex overlying both hemispheres was reflected, and oval cortical cups suspended in flexible mounting brackets were lowered onto both hemispheres. Each cup was filled with an artificial CSF solution to ensure that there was no leakage of fluid from the cups. The dorsal surface of the head was then covered with 4% agar in CSF to protect the exposed tissue surfaces and stabilize the cups. Arterial blood pressure was recorded on a Grass polygraph. After a 30-rain equilibration period, the cups were emptied

and refilled with 250 #1 of warmed sterile artificial CSF (19), which had been bubbled with a gas mixture of 5% carbon dioxide in 95% nitrogen. Cup fluid was maintained at 37°C with a heat lamp. The adenosine deaminase inhibitor deoxycoformycin (19) (500/~g/kg) was administered intravascularly 20 min prior to the onset of collection of cup perfusate contents. After two basal 10-rain collections of cortical perfusate, either alcohol (1.5 g/kg) or acetate (2 gm/kg) was administered intraperitoneally and a further eight successive 10-min samples were then collected. The collected cortical perfusates were ejected into chilled microvials, centrifuged at 120(O and then analyzed for their adenosine and inosine contents by HPLC using previously published techniques (19,20). Statistical analysis for changes in perfusate purine levels following ethanol or acetate administration was by ANOVA. RESULTS

Systemically Administered Ethanol Potentiates the Inhibitory Effects of Adenosine on Cerebral Cortical Neurons Systemic administration of ethanol was found to prolong the inhibitory effects of iontophoretically applied adenosine on the spontaneous firing o f cerebral cortical neurons. Potentiation of adenosine's depressant actions (Fig. 1A) was apparent within 3-5 min of ethanol administration and reached a maximum at 10-12 min (Fig. IB). The magnitude of the inhibition of firing was also augmented. Partial recovery following ethanol administration had occurred by 20-min postethanol administration (Fig. 1C). Recovery was complete 30-40 min later. Ethanol (0.5 gm/kg, IP) was tested on six neurons. It depressed the spontaneous firing of three neurons and prolonged the inhibitory effects of adenosine in all of the neurons. When the duration of inhibition was measured from the onset of the application o f adenosine to recovery of the basal spontaneous firing rate, ethanol increased the mean duration o f inhibition from 71.2 _+ 5.79 s (SEM) to 92.66 _+ 7.2 s (t9 < 0.05). Ethanol (1.0 g/kg), tested on nine neurons, depressed the spontaneous firing of seven of these and potentiated adenosine's inhibitory effects on all of them. The mean duration of adenosine-evoked inhibition of firing was increased from 59.1 +_ 6.14 s to 90.0 _+ 5.4 s (p < 0.01). Ethanol (0.1 g/kg) failed to alter the duration of the inhibitory action of adenosine on the one neuron tested.

Locally Administered Acetate Inhibits the Spontaneous Firing o f Cerebral Cortical Neurons Acetate was applied iontophoreticaUy with currents of 20120 nA on 54 neurons in five rats. The spontaneous firing of 34 (63%) of these neurons was inhibited as a result of the acetate application. Examples of the inhibitory actions of acetate on two responsive neurons are presented in Fig. 2. Acetate (25 nA for 75 s) depressed the firing of the neuron illustrated in Fig. 2A. Recovery of spontaneous firing had occurred 120 s after the application was terminated. A similar result was obtained on the neuron presented in Fig. 2B. Inhibition of spontaneous firing was evident in both of these neurons prior to the cessation of the application current. On many other neurons, the inhibitory effect of acetate became clearly apparent only after termination of the acetate application. On 12 of these neurons, acetate actually induced a weak, rapid onset, increase in firing. The excitation of six of these neurons reversed to a depression once the application had ceased. Acetate may therefore have a rapid onset, short duration, excitant

ETHANOL, ACETATE, AND ADENOSINE

543 of adenosine-evoked inhibitions was 44.6 + 3.9 s; as compared to 47.2 + 5.0 s during acetate applications.

AD

A

Effects o f Ethanol and Acetate Administration on Purine Release From the Cerebral Cortex

401-

Arterial blood gas and pH values recorded during the first and last CSF collection periods, together with the mean arterial blood pressure (MABP) at these time points, are presented in Table 1. Ethanol (1.5 g/kg, IP) had no effect on arterial pH, PaCO2 or PaO2, but did significantly reduce MABP from 117.8 + 6.6 to 87.4 + 8.9 mmHg (/7 < 0.05). Acetate (2 g / kg, IP) administration resulted in an increase in the pH of femoral arterial blood from a control value of 7.39 ± 0.015 to 7.51 + 0.018 (/7 < 0.01). This was accompanied by a nonsignificant reduction in MABP. PaO2 and PaCO2 were unaltered. The effects of ethanol (1.5 g/kg) and acetate (2 g/kg) on adenosine and inosine release into cerebral cortical perfusates are illustrated in Fig. 3. Neither agent had a significant effect on adenosine or inosine concentrations in the perfusates.

B4

DISCUSSION C

~

The major findings of this study are that ethanol is capable of potentiating the inhibitory action of adenosine on cerebral cortical neurons, and that locally applied acetate inhibits the spontaneous firing of these neurons. The effect of alcohol on neuronal membranes is complex and has been postulated to involve membrane components such as the NMDA receptor (14), the GABA A receptor (23), membrane transport systems, and second messenger signals

~

40

(10). 0

~ 1 lmin

FIG. 1. Rate meter recording of the firing of a spontaneously active rat cerebral cortical neuron. Adenosine (AD) was applied by a current of 42 nA during the periods indicated by the horizontal bars. (A) Control responses to adenosine. (B) Responses recorded 10-17.5 rain after IP administration of ethanol (0.5 g/kg). (C) Responses recorded 40 min after ethanol administration showing partial recovery.

action on some neurons, which tends to mask the appearance of its depressant action during the acetate application. The duration of the depressant action of acetate was variable, with some neurons recovering slowly over periods of 5-12 min when it was applied with large currents for 2 min or more. Twenty neurons were unaffected by acetate applications with currents of up to 120 nA. The adenosine antagonist, 8-p-sulphophenyltheophylline, was tested for its ability to reverse acetate-induced inhibitions o f nine neurons. On one neuron, 8-p-sulphophenyltheophylline (20 nA) apparently reversed the inhibition, as the spontaneous firing frequency abruptly returned to control levels after application o f the adenosine antagonist. On four neurons, 8-p-sulpho,~henyltheophylline (20-25 nA) caused a small increase in ~.w firing frequency during its application, but did not reverse the acetate effect. 8-p-sulphophenyltheophylline application was without effect on the firing o f the remaining neurons tested. Potential interactions between acetate and adenosine were tested on six neurons. Acetate (10-60 nA) failed to potentiate the inhibitory effects of adenosine. The mean control duration

Dar et al. (3,6-9) have presented evidence that adenosine may be a participating factor in the ethanol-induced modulation of central excitability. Clark and Dar (5) demonstrated that ethanol (25-200 mM) significantly increased basal release of adenosine from cerebellar synaptosomes in a dose-dependent manner. At pharmacologically relevant concentrations of 2.5-100 raM, ethanol inhibited the uptake of adenosine by rat cerebellar synaptosomes (4), an action that may account for the adenosine-releasing action of ethanol, especially since Nagy et al. (15) have shown that adenosine can increase extracellular adenosine in $49 lymphoma cell cultures by inhibiting its uptake via the nucleoside transporter. Of considerable significance was the observation by this group that ethanol, unlike dipyridamole, did not inhibit transporter mediated purine efflux. Inhibition of the adenosine uptake system in rat cerebral cortical neurons would account for the ethanol-induced potentiation of the magnitude and duration of adenosine-evoked inhibitions observed in the present study. The failure of ethanol to enhance endogenous adenosine or inosine release from the rat cerebral cortex observed in the present study, confirms earlier observations on the failure of ethanol to increase the release of 3H-labeled adenosine derivatives from the rat cerebral cortex (18) or to elevate adenosine levels in the cerebral cortex of rats sacrificed by focused microwave irradiation (2). This result is not necessarily inconsistent with the hypothesis that adenosine is involved in ethanol's neuromodulatory actions, in that administration o f potent transport inhibitors (dipyridamole, soluflazine) (20) or an adenosine deaminase inhibitor (deoxycoformycin) (19) also failed to elevate basal adenosine levels in cortical superfusates, even though such agents can alter CNS excitability (21). It is likely that, in anesthetized animals, adenosine release from activated nerve ter-

544

PHILLIS, O'REGAN AND PERKINS

A

Acetate

B

Acetate

25 20

20 20

-

|

|

lmin FIG. 2. Recordings of responses of two spontaneously firing cerebral cortical neurons to iontophoretically applied acetate, (A) 25 nA and (B) 20 hA. Acetate application resulted in the depression of spontaneous firing.

minais or cell bodies makes a minor contribution to overall basal extracellular adenosine levels. Elevations in extracellular adenosine concentrations at active synapses may be obscured by the overall release from multiple cortical areas. Therefore, it remains a possibility that inhibition of adenosine uptake could underlie some of the central effects of ethanol. Carmichael et ai. (1) have postulated that acetate, resulting from ethanol metabolism in the liver, mediates some of the CNS effects of ethanol. Data presented by this group show that, at the 1.5 g/kg dose of ethanol administered in the present study, serum acetate levels would have risen from control values of 0.2 :t: 0.07 mM to a plateau of 1.81 ± 0.09 mM 30 rain after the IP ethanol injection. The 2 g/kg acetate dose used in the present experiments should have elevated serum acetate to over 7 raM. No increase in adenosine or inosine release into the cortical perfusates was observed, even after such large elevations in acetate levels. In support of their proposal, Carmichael et al. (1) were able to demonstrate that sodium acetate, in doses resulting in blood concentrations comparable to those attained after the

administration of I to 2 g/kg of ethanol, had significant CNS effects. Both ethanol and acetate produced a dose-dependent impairment of motor coordination in rats, and this effect of acetate was fully blocked by the adenosine receptor antagonist 8-phenyltheophylline. Locomotor activity in mice was reduced by acetate in a dose-dependent fashion, an effect that was also fully blocked by 8-phenyltheophylline. Carmichael et al. (1) hypothesize that the conversion of acetate into acetyl CoA by central tissues is associated with the generation of adenosine which, after efflux from the cells, depresses synaptic activity by activating extracellular adenosine receptors. However, our failure to observe any increase in adenosine efflux into cortical perfusates after large doses of sodium acetate would be inconsistent with the ethanol/acetate/adenosine hypothesis. Acetate, applied iontophoretically, depressed the spontaneous firing of 63% of the rat cerebral conical neurons tested. This effect was frequently weak, and inconsistent when acetate was applied repeatedly to the same neuron. An initial mild excitant effect of acetate was observed in 12 of the 54 neurons tested, and this effect may account for our observa-

TABLE

1

MEAN ARTERIAL BLOOD PRESSURE, pH AND BLOOD GASES RECORDED DURING PURINE RELEASE EXPERIMENTS Ethanol

Beginning pH PaCO2(mmHg) PaO2(mmHg) MABP(mmHg)

7.39 32.1 94.9 117.8

+ ± + +

0.06 1.5 6.8 6.6

Acetate

End 7.38 30.2 94.9 87.4

+ + ± ±

0.02 0.23 3.42 8.9*

Beginning 7.39 32.7 85.7 128.8

+ ± ± ±

0.01 0.9 3.6 5.4

End 7.51 33.4 85.8 111.4

± 0.02i" + 1.5 + 2.7 + 10.6

Blood pressure was recorded continuously throughout each experiment. The values presented here, together with those for the blood gases and pH, were recorded during the first and last cortical perfusate collection periods. Ethanol or acetate were administered IP after the second collection period. *p < 0.05; tP < 0.01 by paired t-test.

ETHANOL, ACETATE, AND ADENOSINE

A

200

545

B

0 ACETATE

•

200 [

L'TOH

150

150 L~ Z (/) 0 Z

100

0

I

2

•

I

I

I

4

6

8

100

ACL'TA~

g1"O~

l

5O

0

!

1o

n

•

I

2

*

I

I

I

I

4

S

8

1o

COLLECTION PERIOD

COLLECTION PERIOD

FIG. 3. Lack of effect of IP administration of ethanol (EtOH) and acetate on (A) adenosine and (B) inosine release from the rat cerebral cortex. Five rats (ten cortices) were used in both series of experiments. Samples were collected at 10 min intervals and analyzed by HPLC. Ethanol and acetate were administered at the start of the third collection period. The increases in inosine release in sample 3 associated with ethanol administration may have resulted from a transient fall in arterial blood pressure occurring immediately after the injection.

tion that the inhibition o f spontaneous firing was most pronounced after the application o f acetate had been terminated. The adenosine antagonist, 8-p-sulphophenyltheophylline (17), failed to reverse acetate-evoked inhibitions o f spontaneous firing o f eight o f the nine neurons on which it was tested, although its application did result in an increased rate o f firing o f four o f these neurons. The firing o f one neuron did, however, recover immediately after an application o f the adenosine antagonist, suggesting that adenosine generation may have contributed significantly to the depressant action o f acetate on this neuron. The lack o f a pronounced effect o f 8-psulphophenyltheophylline on the other eight neurons suggests

that involvement o f nonadenosinergic mechanisms play an important role in the acetate-evoked depression o f spontaneous firing. Our results, therefore, suggest that adenosine generation is probably not a m a j o r factor in the depressant actions o f acetate on rat cerebral cortical neurons, but do not exclude the possibility that adenosine release makes a minor contribution to the inhibition o f neuronal excitability. Furthermore, the peripheral administration o f ethanol resulted in the potentiation o f adenosine-evoked depression o f spontaneous neuronal activity. This action o f ethanol is unlikely to be mediated by its metabolite acetate.

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13. 14. 15.

16. 17.

methylxanthines on ethanol-induced motor incoordination in mice. Life Sci. 39:1429-1437; 1986. Dietrich, R. A.; Dunwiddie, T. V.; Harris, R. A.; Erwin, V. G. Mechanisms of ethanol: Initial central nervous system actions. Pharmacol. Rev. 41:489-537; 1989. Dhopeshwarkar, G. A.; Subramanian, C.; Mead, J. F. Rapid uptake of [14C] acetate by the adult rat brain 15 seconds after carotid injection. Biochim. Biophys. Acta 248:41--47; 1971. Kiviluoma, K. T.; Peuhkurinen, K. K.; Hassinen, I. E. Adenosine nucleotide transport and adenosine production in isolated rat mitochondria during acetate metabolism. Biochem. Biophys. Acta 974:274-281; 1989. Liang, C. S.; Lowenstein, J. M. Metabolic control of the circulation. Effects of acetate and pyruvate. J. Clin. Invest. 62:10291038; 1978. Lovinger, D. M.; White, G.; Weight, F. F. Ethanol inhibits NMDA-activated ion currents in hippocampal neurons. Science 243: 1721-1724; 1989. Nagy, L. E.; Diamond, I.; Casso, D. J.; Franklin, C.; Gordon, A. S. Ethanol increases extraceUular adenosine by inhibiting adenosine uptake via the nucleotide transporter. J. Biol. Chem. 265:1946-1951; 1990. Oldendorf, W. H. Carrier-mediated blood-brain barrier transport of short chain monocarboxylic organic acids. Am. J. Physiol. 224:1450-1453; 1973. Phillis, J. W.; Edstrom, J. P.; Kostopoulos, G. K.; Kirkpatrick,

546 J. R. Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex. Can. J. Physiol. Pharmacol. 57:1289-1312; 1979. 18. Phillis, J. W.; Jiang, Z. G.; Chelack, B. J. Effects of ethanol on acetylcholine and adenosine efflux from the in vivo rat cerebral cortex. J. Pharm. Pharmacol. 32:871-872; 1980. 19. Phillis, J. W.; O'Regan, M. H.; Walter, G. A. Effects of deoxycoformycin on adenosine, inosine, hypoxanthine, xanthine and uric acid release from the hypoxemic rat cerebral cortex. J. Cereb. Metab. Blood Flow 8:733-741; 1988. 20. Phillis, J. W.; O'Regan, M. H.; Walter, G. A. Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex. Brain Res. 481:309316; 1989.

PHILLIS, O'REGAN AND PERKINS 21. Phillis, J. W.; Wu, P. H. The role of adenosine and its nucleotides in central synaptic transmission. Progr. Neurobiol. 16:187239; 1981. 22. Puig, J. G.; Fox, I. H. Ethanol-induced activation of adenosine nucteotide turnover. Evidence for a role of acetate. J. Clin. Invest. 74:936-941; 1984. 23. Simson, P. E.; Criswell, H. E.; Breese, G. R. Ethanol potentiates ~,-aminobutyric acid-mediated inhibition in the inferior collicuhis: evidence for local ethanol/'y-aminobutyric acid interactions. J. Pharmacol. Exp. Ther. 259:1288-1293; 1991. 24. Steffen, R. P.; McKenzie, J. E.; Bockman, E. L.; Haddy, F. J. Changes in dog gracilis muscle adenosine during exercise and acetate infusion. Am. J. Physiol. 244:387-395; 1983.