Brab~ Research, 588 (1992) 49-57 © 1992 Elsevier Science Publishers B,V, All rights reserved 0005-8993/92/$05.{l{t

49

BRES 17973

Electrophysiological actions of acetate, a metabolite of ethanol, on hippocampal dentate granule neurons: interactions with adenosine N o r a Cullen and P e t e r L. C a r l e n Department of Physiology, Addiction Research Foundation and Pia)fair Neuroscience Unit, Unicersity of Toronto. Toronto, Ont. (Canada) (Accepted 17 March 1992)

Key words: Acetate: Ethanol: Adenosine; Hippocampus; Electrophysiology

Acetate is the primary breakdown product of ethanol metabolism in the liver and has been found in the brain following ethanol ingestion in rats. Systemically administered acetate has been shown to cause motor impairment, an effect which is blocked by the adenosine receptor blocker, 8-phenyltheophylline (8-PT), The effects of sodium acetate were investigated in this study using intracellular recording techniques in rat hippocampal dentate granule cells, and were compared to the actions of ethanol and adenosine individually and in conjunction with 8-PT. Acetate hyperpolarized the membrane at 0.4-0.8 raM. The amplitude and duration of the postspike train aflerhyperpolarization (AHP) were increased by acetate when Ihe cell was repolarized to the control resting membrane potential. Comparable results were seen in voltage clamp. Acetate also decreased spike frequency adaptation, The effects of acetate were mimicked by adenosine (50 p,M) and ethanol (20 raM). The ethanol effects occluded those produced by acetate. All of the effects of acetate, adenosine and ethanol could be inhibited with prior perfusion of 8-PT (I-10 pM). These data suggest that the actions of the major metabolite of ethanol, acetate, may be mediated by adenosine receptor activation,

INTRODUCTION

Acetate is a short-chain fatty acid formed in the liver and released into the general circulation following ethanol ingestion. It is further metabolized in various tissues to CO,, and H20 or used in the synthesis of fatty acids, steroids and ketone bodies ~s'¢'°. Since acetate is an endogenous metabolite that is considered non-toxic, little attention has been given to the possibility that it may contribute to the intoxicating effects of alcohol. Recently, it has been shown that this seemingly innocuous molecule has behavioural effects on rats similar to those of intoxicating doses of ethanol 7. Furthermore, these behavioural effects were blocked by the adenosine receptor blocker 8.phenyltheophyiline (8-PT), suggesting that acetate may affect the central nervous system (CNS) following ethanol consumption via an adenosine mediated mechanism. Acetate has previously been shown to increase adenosine levels in extrahepatic tissues '~7''~°.

Acetate readily crosses the blood=brain barrier ~'~'4~ and is actively metabolized in the brain ~,,7.~,~,3:~,~.~,s~. Endogenous levels of acet.'~te in rat brain are ~tbout 0.22 mM and reach 0.38 + 0.01 mM following low dose ethanol administration (13 raM) 7. Interestingly, acetate has been demonstrated to abate the tremulous component of the ethanol withdrawal syndrome in rats TM. Several investigators have postulated that acetate acts via an adenosine-mediated mechanism "~7''~°''~'p,it is thought that this occurs through the conversion of acetate to acetyl Coenzyme A (acetyl CoA) by acetyl CoA synthetase, known to be present in sheep and rat brain 34. Furthermore, alcohol, acetaldehyde or acetate can produce an increase in acetyl CoA levels s~'. This reaction utilizes adenosine triphosphate (ATP) yielding adenosine 3,5.monophosphate (AMP). Adenosine is produced from AMP by 5'.nucleotidase which has been found in various brain regions including the dentate gyrus :''~'e~ and in association with glial cells "~'~.Several studies have suggested that central adenosine is in-

Correspondence: P.L. Carlen, Playfair Neuroscience Unit, Toronto Western Hospital, 399 Bathurst St., Room 12-413. Toronto, Ont. MST 2S8, Canada. Fax: (1) (416) 369-5745.

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volved in the CNS effects of ethanol "''~'t"and a block of adenosine uptake during ethanol administration has been shown 4". We hypothesize that acetate, a product of ethanol metabolism, contributes to the CNS depres~nt effects of ethanol via adenosine mediated actions. MATERIALS AND METHODS

Hippocampal slices (4(}O pm thick) were prepared from the brains of young adult male Wistar rats (160-22(} g) using previously de.~ribed meth¢~lss and maintained at 35°C in artificial ccrebrospinal fluid (ACSF) which contained (in raM): NaCI 123, KCI 3. KII2PO.= 125. MgSO.t 2. CaCI. 2. NaHCO.~ 26. dextm.~¢ I(}. Intracellular recordings were obtained from a total of 69 neurons using glass micrL~:leclrodes filled with 3 M KCI. 3 M KAc or 3 M KCI and IO mM HEPES with resistances of 60-12l) MH. Acetate and adenosine were dissolved in ACSF to their final concentrations. 8-PT was dissolved in NaOIt. absolute alcohol ( < I mM ethanol for the stock mlution) and A C S F to produce concentr:dions of I-It) ~ M of 8-PT with minuscule amounts of ethanol ( < 1 nM). Sodium acetate, adenosine and 8-PT were obtained from Sigma Corp. Recordings were taken from upper bhtde dentate gyrus granule cells. The intracellular electrodewas used to pass current to artificially depolarize or hyperpolarize tht: cell. a.~ well as to record the cellular voltage responses. The postspike train afterhyperpolarization (AHP) was evoked by I(IO ms depolarizing pulses (0,1 to 1,0 hA), Only the trains with the same number of spikes were compared in control ~)lution (ACSF) and in the drug. Spike frequency adaptation was tested using a tdX) ms depolarizing pulse, (0.1-1,0 nA) and the response was measured as the number of spikes evoked during the same amount of current injection. Bipolar tungsten electrodes stimulated the perfmant path elicitintt an ¢xcit,ttory postsynaptic potential IEPSP) followed by an inhibitory postsynapti¢ pott~ntial (IPSP), Single electrode voltaire clamp recordings were obt,ined via ,an Axoclamp II amplifier (sampling frequency 3=5 klh), A dcpolarizin~ vollctge step of bile70 mV, lasting between 1-20 ms, evoked art out wttrd lail current (/,,h.) which has ~reviously hc~n ,~hown to have the sitm¢ chttracteristic~ as the AIIP*, A cell was considered .~uit. .hie for analysis if it had an input re;slst,nce ~treater than ,|0 MH, spike height greater thtm Oil mV, resting membrane potential more nee.live than = ~.~ mV, and tt stable membrane potential I'or Ill=l~ rain in control solution, Slow events including AIIP's, changes in membrane I~tential, voltage deflections caused by i~ected intracel. lular current and I,,hr,'s were measured from the chart recorder record~ and analyzed visually, Video tape records of fast events (EPSP's, IPSP's and adaptation) were analyzed by a locally devel. aped computer program, Statistical comparisons were made using a Student's one-tailed t.test or an analysis of the vari~mc¢.

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recorder volta~ trac~ shows the resting membrane potential (dashed llne) which was st,hle l'or > I(t mitt before acetate application. A hyperpolariz,tion of 3 mV ensued after approximately 4 rain of 0,4 mM itcct,te p~rfu,~lun, Downward deflections are the: voltage response,~ of the cell to 0,2 nA current pulses used to monitor the input resistance, it: a dos~ =°response curve of the hyperpolarization seen in 31 cells using ll,2-1,tl mM sodium acetate (** P < (I.0(ll; * P < (t.05), C: in voltage clump mode, the holding current is increased, reflecting a hyperpolarizatkm of the membrane potential, In this cell, the holding current was recorded every minute. The 0.4 mM acetate perfuskm was begun at time 0 ( > Ill rain stability), after which the holding current gradually increased gradually. This effect partially washed out,

RESULTS In current clamp mode using KCI electrodes (3 M), the primary effect of sodium acetate was a hyperpolarization after 5-10 rain of drug perfusion (Fig, IA), A dose-response curve of the membrane hyperpolarization (Fig. IB) shows a peak response at (I,4-0,6 mM, This effect partially reversed after I0 min of wash out, Often, even with greater than 30 min washout, there was no reversibility, The degree of hyperpolarization from 0.4 mM acetate was not correlated with the pre-drug resting membrane potential (r = - 0,147; P = 0.6845: n = I0), In voltage clamp mode, the hypcrpolarization was reflected by an increase in the positive

holding current (Fig, IC), This had a similar dose-response relationship as the hyperpolarization data (i.e. peak effect at 0,4-0,8 raM) as seen in Fig. IB. The amplitude and duration of the AHP was reduced by the acetate-induced hyperpolarization and enhanced when the cell was artificially repolarized to the pre-drug resting membrane potential using direct current (DC) injection (Fig. 2A), Acetate did not produce obvious changes in the pattern of spike generation during brief depolarizations (Fig. 2B). The depression of the AHP occurred at concentrations of acetate between 0,1 and 1,0 mM as seen in Fig. 2C (solid line), while the enhancement of the AHP, observed after the

51 acetate-induced hyperpolarization was reversed by DC injection, occurred at 0.4-0.8 mM acetate (Fig. 2C, dashed line). There was no significant change in the half duration of the AHP in current clamp mode, reflecting the short lasting component of the current. The total duration was enhanced with repolarization (125 + 8%,P < 0.0:5, n -- 9 at 0.4 raM), revealing an effect on the long lasting component of the AHP. In voltage clamp mode (Fig. 3), the amplitude and duration of the aflerhyperpolarizing current (l,,hp) was increased. The duration of the /ahp at 0.4 mM was

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Acetate concentration (raM) Fig. 3. A: the l,ihp was evoked following a 10 ms, 60 mV depolarizing voltage step (upper trace). The current is seen as a rapid upward deflection and a slowly decaying downward deflection (outward current), in this cell, 0.2 and 0.4 mM sodium acetate produced an increase in the amplitude and duration of the I,ihp which was reversible. B: the dose-response relationship of amplitude of the /,,hp expressed as a percentage of the control values. The numbers represent the number of cells examined. Bars = S,E.M.; * P < 0.05, • * P < 0.001.

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Fig. 2. Sodium acetate enhances the AHP. A: the AHP is seen as a downward deflection following e train of action potentials (upward deflections attenuated by the chert recorder). After 10 min in 0.4 mM sodium acetate, the cell was hyperpolarized Lind the AHP was markedly reduced. When repolarized to the original membrane potential using DC current injection, the AHP was enhanced compared to control. This effect washed out. B: the pattern of spike generation and the spike amplitude were unaltered during the perfusion of acetate. C: acetate concentration effects on the AHP amplitude. The amplitude of the AHP tended to be depressed in sodium acetate solution with the accompanying hyperpolarization (open cir. cles), and was enhanced at 0.4 mM when the neurons were repolarized by DC current injection (closed circles). Bars = S.E.M.; ** P < 0.001; * P