Acute and chronic actions of ethanol on CA1 hippocampal responses to serotonin

BRAIN RESEARCH ELSEVIER

Brain Research 731 (1996) 12-20

Research report

Acute and chronic actions of ethanol on CA1 hippocampal responses to serotonin Alex H.L. Lau, Gerald D. Frye Department of Medical Pharmacology & Toxicology, Texas A & M University College of Medicine, College Station, IX 77843-1114; USA

Accepted 9 April 1996

Abstract

The effects of acute or chronic ethanol on serotonin (5-HT)-induced membrane hyperpolarization and inhibition of the slow Ca2+-dependent after hyperpolarization (sAHP) were recorded in rat CA1 pyramidal neurons in hippocampal slices using sharp intracellular electrodes. 5-HT (1-100 txM) caused concentration-dependent hyperpolarization of the membrane that was not altered by simultaneous 30 mM ethanol treatment, but blunted by 10 ixM buspirone, a weak 5-HT1A agonist. 5-HT (1-30 I-~M) also partially inhibited ( ~ 40%) the sAHP following a burst of five or more action potentials. Initially ethanol (30 raM) alone did not alter the sAHP, but over a period of 38 min, a slow increase in amplitude ( ~ 40%) was observed. 5-HT-mediated inhibition of the sAHP was significantly greater with ethanol present, regardless of the length of exposure. Pyramidal neurons in hippocampal slices prepared from ethanol-dependent animals showed no obvious signs of withdrawal related hyperexcitability and neither concentration-dependent membrane hyperpolarization nor sAHP inhibition caused by 5-HT were significantly changed from responses in controls. These results suggest that hyperpolarizing responses to 5-HT in hippocampal CA1 pyramidal neurons are functionally resistant to acute or chronic ethanol treatment. 5-HT-mediated inhibition of the sAHP is enhanced by ethanol acutely, but does not show an adaptive change as a result of ethanol dependence. Keywords: Ethanol; Tolerance; Dependence; Serotonin; Afterhyperpolarization;5-HTIAreceptor; 5-HT4 receptor

1. I n t r o d u c t i o n Serotonin (5-HT)-containing neurons originate predominately in the brainstem raphe nuclei, project to many brain areas including limbic structures like the hippocampus and play important roles in a wide range of behavioral phenomena and psychiatric disorders such as alcohol abuse and alcoholism [38]. A complex array of 5-HT receptors including: 5-HT1A, 5-HTIB, 5-HTlc, 5-HT1D, 5-HTIE, 5HT1F , 5-HT2A , 5-I-IT2B, 5-HT2c, 5-HT3, 5-HT4 have been identified as transducers of 5-HT signaling in the central and peripheral nervous systems. Activation of most 5-HT receptors involves G-protein interactions with adenylate cyclase, phospholipase C or ion channels, with the exception of the 5-HT 3 receptor which incorporates a ligandgated cation channel (see for review [19]). At a cellular level 5-HT receptors exert a wide range of actions such as

* Corresponding author. E-mail: [email protected]

postsynaptic 5-HT1A-mediated inhibition or presynaptic 5HT3-mediated excitation [6]. 5-HT-containing neurons and receptors appear to play significant roles in a wide range of ethanol's acute and chronic actions. Ethanol preference and consumption in animal and clinical studies are consistently reduced by increasing net 5-HT release or activating 5-HT t receptors, while subjective effects are reduced by 5-HT 3 antagonists (see for review [38]). In addition, cerebral cortex and hippocampus of alcohol-preferring (P) rats have higher densities of 5-HT1A receptors than non-preferring (NP) animals, while the reverse is true for the raphe nuclei [26]. Ethanol stimulated motor activity is reduced by 5-HT1A agonists [5], while functional tolerance to ethanol and tolerance maintenance by arginineS-vasopressin both require intact 5-HT innervation or activation of 5-HT z and 5-HT 3 receptors [45]. 5-HT receptors also may play a role in the development of physical dependence on ethanol and in withdrawal syndrome related behaviors. For example, withdrawal-related anxiety is prevented by 5-HTIA a g o nists [22], an action that may be due to down-regulation of

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S0006-8993(96)00459-3

A.H.L. Lau, G.D. Frye/Brain Research 731 (1996) 12-20

5-HT1A receptors in cerebral cortex and hippocampus [13,29,41]. Ethanol withdrawal seizures are also exacerbated by antagonizing 5-HT 3 receptors [17]. It is increasingly clear that ethanol directly influences the activity of some 5-HT receptors and may induce neuroadaptive changes in the number or affinity of other 5-HT receptors with chronic treatment. For example, 5-HT 3 receptor-mediated current in cultured neuroblastoma cells and freshly isolated nodose ganglion neurons is enhanced by ethanol [23]. Ethanol also acutely inhibits 5-HTlc stimulation of Ca2+-activated CI- currents in oocytes [37]. Chronic ethanol treatment diminishes the functional capacity of 5-HT receptors measured as a decrease in 5-HTstimulated phosphoinositide turnover in cerebral cortex of ethanol-withdrawn rats [30]. These data suggested an ethanol-induced functional down-regulation of 5-HT2 receptors, even though numbers of 5-HT 2 binding sites remained unchanged [30]. To date relatively few studies have systematically evaluated the acute or chronic effects of ethanol on mammalian CNS 5-HT receptors at the level of individual neurons using electrophysiological means. Such studies would seem important in helping to characterize the functional consequences of changes in receptor number or affinity identified by binding studies. The present study was designed to further characterize the effects of ethanol on the function of CNS 5-HT receptors at the cellular level. Intracellular sharp-electrode recordings were used to evaluate the impact of in vitro or chronic in vivo ethanol exposure on 5-HT-induced membrane hyperpolarization or suppression of the slow Ca 2+dependent AHP in hippocampal CA1 neurons. Recent studies suggest that these actions are the result of two distinct 5-HT receptor subtypes, 5-HT1A and 5-HT4, respectively [1]. As indicated above, 5-HTiA receptors play a particularly important role in the pharmacology of ethanol, but relatively little is known about the role of more recently identified 5-HT4 receptors. The present studies show that acute ethanol enhances the activity of a buspirone resistant 5-HT receptor-mediating inhibition of the sAHP in CA1 pyramidal neurons without evoking an adaptive change in the ability of the receptor to transduce 5-HT signals after exposure leading to physical dependence.

2. Materials and methods 2.1. Animals

Male Sprague-Dawley rats (125-150 g Timco-Harlan Industries, Houston, TX, USA) were initially housed in pairs (22-25°C; lights 0700-1900 h) and given continuous access to standard laboratory rat chow and water. Some animals were used to prepare brain slices without prior ethanol treatment. The remaining animals were used for physical dependence studies. To induce physical depen-

13

dence on ethanol the previously characterized liquid diet method of Frye et al. [15,47] was used. Each rat was housed individually and the first day received rat chow, water and 35 ml of a nutritionally complete liquid diet prepared from purified materials. On the second day, rat chow was removed but an additional 35 ml of liquid diet and water were again available. As of the third day, one half of the animals received water and a liquid diet in which ethanol (0.06 g / m l ) displaced dextrose, isocalorically for an additional 12 consecutive days (ethanol-dependent group). Ethanol concentrations were increased from 0.07 to 0.08 g/ml, after the first 6 days to compensate for the development of metabolic tolerance to ethanol. We have previously shown that this regimen induces daily ethanol consumption of 12 to 16 g / k g / 2 4 h, maintains large amounts of ethanol in the blood stream (up to 2 m g / m l ) and allows continued weight gain (1-2 g/day) [15]. Following withdrawal of ethanol we have routinely observed marked withdrawal signs including susceptibility to andiogenic seizures and forelimb tremor within 3-5 h in animals treated with this protocol indicating the presence of physical dependence on ethanol [15]. For these experiments, animals were decapitated while still intoxicated immediately after removing them from ethanol diets, so signs of withdrawal were rarely observed. The remaining animals (Diet Control) were fed 35 ml of liquid diet without ethanol throughout the 14 day treatment period, an amount previously found to be calorically and nutritionally equivalent to average dally consumption of liquid diet by ethanol-treated animals. 2.2. Hippocampal slice preparation, recording methods and drug application

Standard techniques were used to prepare brain slices and complete intracellular recordings as previously described [16]. Following decapitation, the brain was rapidly removed, cooled and transverse hippocampal slices, 400500 txm thick, were cut on a McIlwain Tissue Chopper (Mickle Lab. Engineering Co. Ltd., Surrey, UK). Slices were held in oxygenated (95% 02, 5% CO 2) physiological solution (raM; NaC1 120, KC1 3, CaC12 2.5, MgC12 1.2, NaH2PO 4 1.2, NaHCO 3 22.6, glucose 11.1) at ~ 30°C for at least 1 h prior to use. Single slices were transferred to a Plexiglas organ bath (2 ml), completely submerged and perfused (4 ml/min, 34°C) with the physiological solution. Intracellular recordings were made from CA1 pyramidal cells using sharp glass electrodes (1.5 mm o.d.; filled with 4 M KAc or 2 M KMeSO4; tip resistances of 40-100 MI~) connected to an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) under bridge or switch-current clamp mode. Voltage and current were displayed on an ink chart recorder (Gould Electronics, Cleveland, OH) or digitized for later analysis. Postsynaptic effects of serotonin were studied by examining either drug-induced hyperpolarization of resting membrane potential or inhibition of the

14

A.H.L. Lau, G.D. Frye~Brain Research 731 (1996) 12-20

slow Ca2+-dependent afterhyperpolarization (sAHP) which immediately followed a series of action potentials activated by depolarizing current pulses (0.1-0.7 nA for 300 ms). Before generating a sAHP, membrane potential was adjusted to - 6 4 mV with depolarizing current. Since the number of action potentials generated during a depolarizing current step influence the amplitude of the sAHP, the effects of 5-HT were quantitated by determining the mean potential change in peak sAHP amplitude for depolarizing current steps which were shown to trigger at least five action potentials under initial control conditions. Absolute ethanol was purchased from the WarnerGraham Co. (Cockeysville, MD), buspirone and 5-HT from Sigma Chem. Co. (St. Louis, MO), and cisapride was a gift from Janssen Research Foundation (Beerse, Belgium). 5-HT and the other agents were prepared fresh on the day of the experiment and applied in the physiological solution superfusing the tissue slices for periods of 5-8 min which allowed sufficient time for an apparent steady state response to be achieved. Cumulative concentration response curves were generated for individual neurons by switching directly from lower to progressively higher concentrations of 5-HT once a stable response was recorded for 2 min. Reversibility of 5-HT effects was determined following each cumulative concentration response measurement by rinsing with drug-free physiological solution until responses returned to baseline levels (10-20 min). No ethanol was added to the superfusion solution in experiments where tissues from ethanol-dependent animals were tested so withdrawal from ethanol began shortly after slices were prepared. Slices were used within 7 h of preparation. Sample means were compared by calculating an independent or a paired-t statistic, as appropriate, using 'Abstat release 6' (Anderson-Bell, Parker Co.). Significance in all statistical tests was assumed when the twotailed P for the 't' statistic was equal to, or less than 0.05, unless otherwise stated (see Fig. 6).

3. Results

3.1. Serotonin and ethanol effects on membrane potential

As previously reported [3], the resting membrane potential of CA1 pyramidal neurons in hippocampal slices prepared from ethanol naive rats was consistently hyperpolarized by 5-HT. As shown for a Control neuron in Fig. 1A, the hyperpolarizing action of 5-HT (1-100 txM) applied cumulatively was concentration-dependent, reached a maximum around 10 ixM and was reversed relatively rapidly when 5-HT superfusion was terminated. The mean maximum hyperpolarization for 15 Control cells was 11.07 _+ 0.53 mV from the resting potential. 5-HT-induced hyperpolarization had an estimated ECs0 of 1.64 _+ 0.41 txM. A small reduction in membrane resistance was apparent, when hyperpolarization was offset with depolarizing cur-

rent, for 5-HT concentrations of 30 txM and above (data not shown). Hyperpolarizing effects of 5-HT are mediated by postsynaptic 5-HT1A receptors via a G-protein linked K + channel [9]. Marked inhibition of the hyperpolarizing actions of 5-HT (1-10 txM; Fig. 1B) by buspirone (10 txM), a weak partial agonist at postsynaptic 5-HT1A receptors [4,14], is consistent with this mechanism. Buspirone alone did not significantly alter membrane hyperpolarization (RMP; before - 5 3 . 4 + 0.4 mV or after 10 txM buspirone - 5 3 . 8 + 1.0 mV; n = 6; P > 0.05). Although swift desensitization occurring in seconds could not be ruled out, slower adaptive changes occurring over 10 or more minutes were unlikely since hyperpolarizing responses to 100 t~M 5-HT applied alone were comparable to those applied ~ 30 min after continuous application of lower 5-HT concentrations (data not shown). To evaluate the effects of acute ethanol treatment, 30 mM ethanol was applied to hippocampal slices by superfusion. This concentration was used since similar blood ethanol concentrations increase punished responding and motor impairment in intact Sprague-Dawley rats [15]. Initial exposure to ethanol caused small changes in membrane potential of some cells, as shown in Fig. 1C, where a slight hyperpolarization was initially observed. However, these effects were not sustained (see Fig. 1C; where initial potential at 0 and 1 txM 5-HT are similar) and no net hyperpolarizing or depolarizing effect of ethanol on membrane potential was present when 1 ~M 5-HT was first applied 10 min after ethanol (mean initial RMP = - 6 2 . 0 _ 1.9 mV; membrane potential in 30 mM ethanol = -63.1 _ 2.1 mV, n = 8; P > 0.05). Also, consistent with our previous findings [16], ethanol did not change resting input resistance (data not shown). In several cells that were initially quiescent, washing out ethanol a n d / o r 5-HT resuited in spontaneous firing as shown on the right in Fig. 1C, however, this observation was not specific to the ethanol treatment protocol and was not examined further. The hyperpolarizing action of 5-HT (1-100 txM) was not dramatically changed by continuous application of 30 mM ethanol (Fig. 1C). 5-HT-induced hyperpolarization (10 t~M) also appeared to be unaffected by higher concentrations of ethanol (100 mM) when tested in a few cells (data not shown). Fig. 2A shows mean results for cells exposed to 1-100 txM 5-HT either alone or in combination with buspirone or ethanol. Buspirone (10 ~M) significantly inhibited 5-HT-induced hyperpolarization at all concentrations of 5-HT above 1 txM, while 30 mM ethanol was without effect at any concentration of 5-HT tested. Hyperpolarizing responses to 5-HT also were studied in hippocampal slices prepared from ethanol-dependent rats or diet controls to determine whether this inhibitory action of 5-HT was diminished by physical dependence on ethanol. In agreement with our previous study in CA1 pyramidal neurons [16], there were no differences in RMP or resting input resistance between the groups (data not shown). Also no consistent increases in neuronal excitabil-

15

A.H.L. Lau, G.D. Frye~Brain Research 731 (1996) 12-20

ity were observed in ethanol-dependent cells during the 7-h period when recordings were made, a time when severe signs of hyperexcitability are present in intact animals undergoing ethanol withdrawal [15]. In general, hyperpolarizing responses to 1-100 IxM 5-HT in cells from Diet Controls (Fig. 2B) were similar to those observed in slices from ethanol naive controls (Fig. 2A). Mean Diet Control responses were concentration-dependent, exhibited maximum increases in membrane polarity of 12.18 + 0.77 mV with 10-30 IxM 5-HT and had an ECs0 of 1.41 _+ 0.40 txM (n = 7). Hyperpolarizing responses to 5-HT in cells from ethanol-dependent rats, tested without ethanol in vitro, were not significantly different from those of Diet Controls at any concentration of 5-HT (Fig. 2B), despite the presence of physical dependence on ethanol. 3.2. Serotonin and ethanol effects on the sAHP

In addition to inducing membrane hyperpolarization, 5-HT also has been shown to reduce the magnitude of the

slow Ca2+-dependent sAHP following a series of action potentials by a mechanism proposed to involve 5-HT4 receptors [1]. Partial inhibition of the sAHP by 5-HT was confirmed in traces recorded from two CA1 neurons in Fig. 3A 1, A 2. Maximum inhibition of the sAHP was 42.3 _ 2.2% at 3 txM 5-HT (Fig. 4A) and was concentration-dependent. Buspirone (10 IxM) applied along with 5-HT (10 ixM) failed to reverse sAHP inhibition, consistent with the view that postsynaptic 5-HT1A receptors are not involved in sAHP inhibition (% initial baseline sAHP inhibition; 5-HT alone = 39.3 _+ 5.4% n = 10; 5-HT + buspirone = 44.6 _ 3.8% n = 4; P > 0.05). Surprisingly, cisapride (30 ixM for 30 min), a weak 5-HT4 receptor agonist [8], also did not reduce 5-HT-induced inhibition of the sAHP (% initial baseline inhibition; 5-HT alone = 45.2 _+ 6.3%, n = 9; 5-HT + cisapride = 51.1 + 5.4, n = 9; P > 0.05) as expected based on earlier reports [1]. Neither cisapride nor buspirone alone changed the magnitude of the sAHP (data not shown). Thus based on these limited preliminary studies, the role of 5-HT4 receptors in the

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Fig. 1. Effect of ethanol and buspirone on 5-HT-induced membrane hyperpolafization. A: traces from an ethanol naive 'Control' pyramidal neuron show changes from an initial resting membrane potential of - 5 5 mV induced by superfusion of increasing 'cumulative' concentrations of serotonin (1-100 txM). For all traces in this figure, vertical deflections represent changes in membrane potential due to constant current pulses (0.5 nA) applied for 300 ms every 5 s. Breaks between 5-HT concentrations represent brief periods when bridge circuit balance was adjusted. Membrane potential returned to resting level following removal of 5-HT from the superfusate during 'WASH'. B: traces from a cell (resting potential - 5 5 mV) exposed to 10 ixM serotonin alone and subsequently following a 20 min wash was retested with serotonin (1-10 t~M) combined with 10 ixM buspirone. 5-HT-induced hyperpolarization is blocked in the presence of buspirone which alone had no detectable effect on the resting membrane potential of this neuron. C: traces from a neuron (resting potential - 69 mV) exposed to 30 mM ethanol for 8 rain before application of increasing cumulative concentrations of serotonin (1-100 txM). As in 'A', maximum hyperpolarization is observed at 10 ixM serotonin and reversed during WASH of both ethanol and 5-HT.

A.H.L. Lau, G.D. Frye/Brain Research 731 (1996) 12-20

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this experiment that we initially dismissed was that ethanol might augment the sAHP during the continuous perfusions of up to 40 rain that were required for cumulative addition of increasingly higher concentrations of 5-HT. Earlier reports suggested that ethanol alone slowly increases the magnitude of the sAHP in CA1 hippocampal pyramidal neurons [10,36]. However, this action initially was considered a remote possibility since it was not consistently observed by others [34,39,42]. However, with the apparent enhancement of 5-HT inhibition of the sAHP (Fig. 4A), it became important to test the effects of ethanol alone. Fig. 3C shows traces from two cells where sAHPs were evoked 38 rain apart after continuous superfusion with or without 30 mM ethanol. Surprisingly, the sAHP recorded after 38 rain in ethanol was clearly increased. Fig. 5 shows the mean effects of ethanol over this same time interval expressed as the percentage increase in sAHP amplitude relative to initial baseline values. A significant increase in the magnitude of sAHPs for Ethanol relative to Control neurons was observed after exposure to ethanol for a period longer than 24 rain. Surprisingly, both Control and Ethanol groups showed a small but significant increase of

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Fig. 2. Concentration-dependent serotonin-induced hyperpolarization is inhibited by buspirone but not by acute or chronic ethanol. A: serotonin (1-100 IxM) alone (n = 15) or combined with 30 mM ethanol (n = 8) or 10 ~ M buspirone (n = 6) was applied cumulatively until stable responses were obtained, usually in 5 - 8 min. The magnitude of hyperpolarization (mV) of the membrane' potential from rest was measured and expressed as mean +S.E.M. Buspirone inhibited hyperpolarizing responses to serotonin (3=1001txM) :when compared to Control. B: serotonin (1-100 IxM) was similarly applied to cells from ethanol-dependent (n = 7) or diet-control (n = 7) animals. There were no significant differences between groups in the magnitude of 5-HT hyperpolarization. * P < 0.05 compared to comparabie Control serotonin concentration in A.

inhibition of the sAHP by 5-HT could not be confirmed, although 5-HT1A receptors did not appear to play a role. Next the acute effects of ethanol on 5-HT-mediated inhibition of the sAHP were examined. Traces from the neuron in Fig. 3B shows that 30 mM ethanol did not change the magnitude of the sAHP within 10 min after exposure, however, concentration-dependent inhibition by 5-HT (1-30 txM) appeared to be increased by ethanol. Fig. 4A shows that inhibition of the sAHP was significantly greater for 10 and 30 txM 5-HT in the presence of ethanol. In addition, a higher concentration of 5-HT (10-30 txM) was required for maximum inhibition of the sAHP (57.8 _+ 6.0%), although the ECs0 in the presence or absence of ethanol was similar ( ~ 2 ~M). One concern with

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Fig. 3. Effect of ethanol on serotonin-induced inhibition of the sAHP. A i: each trace from an ethanol naive 'Control' pyramidal neuron shows initial holding potential (adjusted with current injection to - 64 mV for all cells in the figure) followed by a burst of 5 - 6 action potentials induced by 300 ms depolarizing current step and a slowly decaying sAHP. Individual spikes are truncated and indistinguishable due to slow chart speed, sAHPs were inhibited by increasing 'cumulative' concentrations of serotonin (1-30 ~M; '5-HT'), but recovered after 10 rain without serotonin during 'WASH'. Individual traces were recorded when membrane hyperpolarizing actions of serotonin were shown to have stabilized 5 - 8 min. after applying each concentration. The holding potential was returned to - 6 4 mV from rest before each test. A2: shows comparable inhibition of the sAHP in a Control cell exposed only to 10 ixM serotonin. B: in another cell 30 m M ethanol (ETOH) initially (after 8 rain) had no effect on the sAHP compared to the preceding Control response. Cumulative serotonin concentrations applied in the continued presence of ethanol largely abolished the sAHP. C: traces from two cells tested at 0 or 38 rain after 30 mM ethanol (right) or continuous physiological solution alone (left) are shown. The sAHP of the ethanol exposed cell increased at 38 min relative to 0 rain, but not that of the control cell.

A.H.L. Lau, G.D. Frye~Brain Research 731 (1996) 12-20

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Fig. 5. Ethanol augments the sAHP over time. Pyramidal neurons were superfused with 30 mM ethanol (n = 7) or physiological solution (Control; n = 6) for a total 38 min and the sAHP examined at intervals approximately equivalent to those used when measurement of cumulative 5-HT treatment was made in Fig. 4A. Cells were held at a membrane potential of - 64 mV for all measurements of the AHP. The magnitude of sAHP augmentation is shown as the mean _+S.E.M. percentage of the initial response, either Control or Ethanol as appropriate, before drug application. * P < 0.05 when compared to the equivalent time for Control.

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Serotonin (~M) Fig. 4. Concentration-dependent 5-HT-induced inhibition of the sAHP is increased by acute ethanol but not by ethanol dependence. A: serotonin (1-30 IxM) alone (n = 10) or combined with 30 mM ethanol (n = 9) was applied cumulatively until stable responses were obtained, usually in 5-8 rain. Cells were held at a membrane potential of - 6 4 mV for all measurements of the sAHP. The magnitude of sAHP reduction is shown as the mean + S.E.M. percentage of the initial response either Control or Ethanol as appropriate, in the absence of serotonin. B: serotonin (1-30 txM) was similarly applied to hippocampal slices from Diet Control ( n = 12) or ethanol-dependent ( n = 11) rats. The magnitude of sAHP reduction is shown as the mean_+ S.E.M. percentage of the initial response in either diet-control or ethanol-dependent cells as appropriate, in the absence of 5-HT. There were no differences in the extent of 5-HT inhibition between groups. * P < 0.01 when compared to comparable Control serotonin concentration in A.

t h e s A H P o v e r the first 31 m i n r e l a t i v e to the initial b a s e l i n e s A H P (for e x a m p l e at 17 rain C o n t r o l = 110.7 42 . 4 % o f b a s e l i n e , n = 6, P < 0.05; E t h a n o l = 127.4 _+ 9.7%

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greater ethanol-induced enhancement of the sAHP was not f u l l y r e v e r s e d after r e m o v a l o f e t h a n o l for 15 rain (Fig. 5). T h u s t h e s e f i n d i n g s s u g g e s t e d t h a t the a p p a r e n t e n h a n c e m e n t o f 5 - H T i n h i b i t i o n o f the s A H P b y e t h a n o l m i g h t h a v e b e e n s e c o n d a r y to effects o f e t h a n o l to i n c r e a s e the m a g n i t u d e o f this m e a s u r e . T o clarify w h e t h e r e t h a n o l c o u l d i n c r e a s e 5 - H T i n h i b i tion o f t h e s A H P b y a direct r e c e p t o r action, i n the a b s e n c e

s A H P a u g m e n t a t i o n , cells w e r e s u p e r f u s e d o n l y o n c e w i t h a s i n g l e c o n c e n t r a t i o n o f 5 - H T ( 3 - 3 0 txM) a n d / o r 30 m M ethanol. These relatively brief 5-8 min experiments a v o i d e d t h e d e l a y e d effects o f e t h a n o l a n d a l l o w e d relat i v e l y i m m e d i a t e e t h a n o l - 5 - H T r e c e p t o r i n t e r a c t i o n to b e characterized. Under these conditions, ethanol enhanced i n h i b i t i o n o f the s A H P b y 10 p~M 5 - H T (Fig. 6, two-tailed). A d i f f e r e n c e also w a s f o u n d at 30 txM 5 - H T u s i n g o n e tailed test w h i c h c o u l d b e c o n s i d e r e d a p p r o p r i a t e h e r e

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Serotonin Fig. 6. Ethanol increases 5-HT inhibition of the sAHP. Pyramidal neurons were superfused briefly (5-8 rain) with a single concentration of 3, 10 or 30 txM serotonin either in physiological solution (Control) or with 30 mM ethanol (n = 10 for each group). Cells were held at a membrane potential of - 64 mV for all measurements of the sAHP. The magnitude of sAHP inhibition is shown as the mean 4-_S.E.M. percentage of the initial response, either Control or Ethanol as appropriate, before 5-HT application. * P < 0.05 when compared to the equivalent 5-HT concentration for Control using a two-tailed test. * * P < 0.05 when compared to the equivalent 5-HT concentration for Control using a one-tailed test.

18

A.H.L. Lau, G.D. Frye~Brain Research 731 (1996) 12-20

based on the finding in Fig. 4A where ethanol only increased 5-HT inhibition. These findings suggested that 5-HT-induced inhibition of the sAHP is weakly enhanced by ethanol, possibly by a direct action on the receptor. Finally, the impact of chronic ethanol treatment on 5-HT inhibition of the sAHP was evaluated to determine whether this caused any adaptive functional changes. 5-HT (1-30 p,M) inhibited sAHPs in cells from Diet Controls (Fig. 4B) in a concentration-dependent manner similar but not identical to that observed in slices from ethanol naive Controls (Fig. 4A). Diet Control responses, exhibited maximum inhibition of 50-55% with 10-30 IxM 5-HT which was slightly greater than that for naive Controls. However, ECs0s of 2-3 txM were similar for both control groups. The initial sAHP amplitude for Diet Controls (6.86 + 0.77 mV, n = 11) was not significantly different from that of ethanol-dependent neurons (6.03 _+ 0.39 mV, n = 10; P > 0.05). Inhibition of the sAHP by 5-HT in cells from ethanol-dependent rats, tested without ethanol in vitro, was not significantly different from that in Diet Controls at any concentration of 5-HT tested (Fig. 4B) suggesting that 5-HT sensitivity of the sAHP was unaffected by chronic ethanol dependence.

4. Discussion

5-HT receptors responsible for hyperpolarizing hippocampal CA1 pyramidal neurons are insensitive to direct effects of 30-100 mM ethanol, concentrations shown to have significant behavioral effects in intact rats [15]. In agreement with previous reports [3], 5-HT-induced hyperpolarization was largely blocked by buspirone, a 5-HT1A partial agonist with low potency and efficacy relative to 5-HT [4], which is consistent with mediation by the 5-HTIA receptor. On the other hand, inhibition of the sAHP by 5-HT was slightly increased by relatively short 5-8 rain exposure to ethanol 30 raM, but showed an even greater degree of inhibition of the sAHP once the sAHP was potentiated by ethanol treatment lasting as long as 24 min. While not blocked by cisapride in our hands, the receptor involved in sAHP inhibition is likely the 5-HT4 receptor (see below). Neither 5-HT-induced hyperpolarization or inhibition of the sAHP was modified in slices from ethanol-dependent rats. These findings suggest that 5-HT1A and 5-HT4 receptors on CA1 pyramidal neurons do not undergo functional neuroadaptation in association with development of physical dependence on ethanol or the withdrawal syndrome. The present findings are consistent with earlier reports that 5-HT inhibits the sAHP in CA1 pyramidal neurons [1,9,32]. Ethanol enhancement of 5-HT inhibition of the sAHP (Fig. 4A) appears to involve both a slight stimulation of 5-HT4 receptor activity by ethanol (Fig. 6), as well as a gradual increase in 5-HT sensitivity for sAHPs augmented by ethanol (Fig. 5). While a sensitizing action due

to cumulative application of 5-HT can not be ruled out, it would seem more likely that ethanol augmentation renders the K ÷ channel underlying the sAHP [9], more sensitive to 5-HT inhibition. This idea is consistent with evidence that 5-HT does not inhibit the Ca 2+ transients responsible for activating the sAHP [9], but interacts more directly with mechanisms regulating the sAHP K + channel. The nature of the 5-HT receptor responsible for this action, initially proposed to be a 5-HT4 receptor based on sensitivity to agonists [1], recently has been confirmed using antagonists [40]. The receptor is linked via a G-protein to adenylate cyclase and increases the formation of cAMP [19], leading to phosphorylation of the sAHP K + channel through protein kinase A [32]. Failure of buspirone to block 5-HT inhibition of the sAHP in our study is consistent with a lack of involvement of 5-HTIA receptors. Differences in the sensitivity of our slice preparation to 5-HT relative to 30 txM cisapride, a purported weak 5-HT4 agonist [8], may explain why we did not observe block of 10 txM 5-HT inhibition as previously reported with similar concentrations of these agents [1]. These authors found a wide range of sensitivity of individual pyramidal neurons to 5-HT inhibition of the sAHP. It is now clear that 5-HT4 receptors are widely distributed through out the mammalian brain [21], as well as the gut and represent an important transducer of 5-HT function [7]. This report represents one of the first to directly evaluate an interaction of ethanol with the purported 5-HT4 receptor and suggests as does another recent report, where a 5-HT4 receptor antagonist reduced ethanol preference [31], that these receptors may play an important role in mediating ethanol CNS pharmacology. The present findings support previous reports in CA1 pyramidal neurons [10,36] or dentate granule cells [36] that acute ethanol exposure for 20 min or more augments the sAHP (Fig. 6). The lack of change in sAHP amplitude in pyramidal neurons from chronic ethanol-treated animals was also a consistent finding [36]. For reasons that are not yet clear, acute ethanol augmentation of the sAHP in hippocampal pyramidal neurons has not been a consistent finding [34,39,42]. Ethanol (11-150 raM) had little net effect on the sAHP in either hippocampal CA1 or CA3 pyramidal neurons recorded in superfused slices [34,39]. However, sAHPs recorded in cultured hippocampal neurons derived from 20-day-old embryos were significantly inhibited by 14-44 mM ethanol [42]. Possibly, inability to clearly identify these neurons, culturing conditions or differences in maturity relative to adult neurons in hippocampal slices played a role in ethanol sensitivity. The mechanism responsible for acute sAHP augmentation by ethanol remains to be identified, but could follow ethanol-induced increases in basal intracellular free Ca 2+ levels [10,12]. Higher resting Ca 2+ would allow voltagegated Ca 2÷ transients to more rapidly reach threshold for sAHP activation and prolong an effective Ca 2÷ stimulus, thereby augmenting activation of the sAHP K ÷ channel

A.H.L. Lau, G.D. Frye/Brain Research 731 (1996) 12-20

which responds relatively slowly to Ca 2+ influx [9]. Increased resting Ca 2+ released from intracellular stores [12] rather than from enhanced voltage-gated Ca 2+ transients seems more likely to play a role in ethanol sAHP augmentation, since augmentation occurs in CaZ+-free media [10]. Also ethanol at the concentrations studied generally inhibits rather than stimulating Ca 2+ influx through voltageor ligand-gated channels [24,27,43]. However, the rather slow 20 min onset for sAHP augmentation also probably indicates additional mechanisms are involved, since ethanol increases resting Ca 2+ levels in as little as 10 s by releasing stores from inositol triphosphate insensitive endoplasmic reticulum stores in brain microsomes [11,12]. In the present study, chronic ethanol treatment did not down-regulate 5-HT1A receptor function as expected based on previous reports of reduced binding sites in the CA1 region o f the h i p p o c a m p u s for 8 - O H - 2 - ( d i - n propylamino)tetralin (8-OH-DPAT), a selective ligand for 5-HT1A receptors [13,29,41]. However, others have reported no change in 5-HT1A binding sites with chronic ethanol treatment [30] which would be consistent with our results. An apparent dissociation between receptor number and function after chronic ethanol treatment might be possible due to a large 5-HT1A receptor reserve that has been identified in hippocampus [14,18]. Dissociation of changes in receptor number and function based on an increased ratio of [3H]8-OH-DPAT to [3H]WAY-100635 binding in hippocampal tissues of ethanol-dependent animals has been interpreted as an increase in the proportion of functional, G-protein coupled 5-HT1A receptors, which was observed despite a reduction in total [3H]8-OH-DPAT labeled 5-HTtA binding sites [29]. It is also possible that ligand binding assays [29,41] identify a selective action of ethanol to reduce presynaptic 5-HT1A receptors, involved in regulating synaptic glutamate release onto pyramidal neurons [25]. A stable population of postsynaptic 5-HT1A receptors as suggested by our data would be difficult to distinguish from down-regulated presynaptic receptors using binding assays alone. However, functional down-regulation of presynaptic 5-HT1A receptors could be assessed by monitoring 5-HT inhibition of evoked field EPSPs in CA1 region, which show adaptive changes in rats made tolerant by repeated 8-OH-DPAT treatment [25]. Thus it would seem worthwhile to focus future studies on assessing potential ethanol-induced down-regulation of presynaptic 5-HT1A receptors. The lack of down-regulation of postsynaptic 5-HTtA receptors, as our data suggests (Fig. 2B), may not be too surprising, since acute ethanol exposure apparently does not provide a driving force for change by acutely enhancing 5-HTtA receptor function (Fig. 2A). However, we have previously shown that postsynaptic G A B A B receptors on CA1 pyramidal neurons undergo a small reduction in activity upon chronic ethanol treatment, despite lack of acute ethanol sensitivity [16]. A possible explanation might involve some form of indirect heterologous desensitization

19

such as occurs between G-protein coupled adenosine A 2 and prostaglandin E 1 receptors after chronic exposure to ethanol [28,35,44] where the function of these independent receptors is simultaneously diminished when a shared transducer is down-regulated. Postsynaptic 5-HT1A , G A B A B and adenosine A 1 receptors in CA1 pyramidal neurons all have been proposed to share a common effector mechanism, since each is coupled to K ÷ channels via a pertussis toxin sensitive G-protein [2,46]. A significant action of ethanol to down-regulate any one of these receptors could translate into functional down-regulation of the others as occurs for other G-protein coupled receptors [20]. However, recent evidence from cultured hippocampal neurons now suggests that the K ÷ channels activated by 5-HT and G A B A exhibit different conductances and kinetics and may be distinct channels [33]. Activation of distinct channels by postsynaptic 5-HTtA and GABA s receptors could rule out heterologous desensitization unless common Gproteins are involved. However, the lack of adaptive down-regulation of 5-HT1A receptors strongly suggests that G A B A B receptor function is not diminished due to heterologous desensitization involving 5-HT.

Acknowledgements This project was supported in part by Public Health Service Grants AA06322 and AA00101 (G.D.F.). The authors thank Ms. Annette Fincher for skillful technical and artistic assistance and Drs. Cathy Grover and William Griffith for their critical analysis of the manuscript.

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